(Springer) Cancer Immunology A Translational Medicine Context

Nima Rezaei
Editor
Cancer Immunology
A Translational
Medicine Context
123
Cancer Immunology
Nima Rezaei
Editor
Cancer Immunology
A Translational Medicine Context
Editor
Nima Rezaei, MD, MSc, PhD
Research Center for Immunodeciencies
Childrens Medical Center
Pediatrics Center of Excellence
Tehran University of Medical Sciences
Tehran
Iran
Department of Immunology
School of Medicine
and Molecular Immunology
Research Center
Tehran University of Medical Sciences
Tehran
Iran
ISBN 978-3-662-44005-6 ISBN 978-3-662-44006-3 (eBook)

DOI 10.1007/978-3-662-44006-3
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014952677
Springer-Verlag Berlin Heidelberg 2015

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This book would not have been possible without the continuous
encouragement by my parents and my wife Maryam.
I wish to dedicate it to my daughters Ariana and Arnika with
the hope that progress in diagnosis and treatment of these
diseases may result in improved survival and quality of life for
the next generations and at the same time that international
collaboration in research will happen without barriers.
Whatever I have learnt comes from my mentors. This book is
therefore dedicated also to all of them but most importantly to
the patients and their families, whose continuous support has
guided me during the years.
Foreword
Several empirical observations suggested a long time ago that established

human tumors could melt away in response to perturbations of the immune
system such as during acute infection. Such regressions of tumors occurred
most often but not exclusively when infection occurred at the tumor site and
sparked the interest of investigators in identifying the mechanism leading to
such occurrences based on the assumption that infection acted as an adjuvant
to boost existing but insufcient immune surveillance against neoplasms.
These anecdotal observations are reected not only in the scientic literature
such as the classic reports of William Cooley in the late 1800s but even dis-
cussed by classic authors such as the doctor-writer Anton Chekhov.
It took time, however, to elevate these concepts derived from empirical
observations to a science of molecular precision. Skepticism dominated the
scene for a long time including during the late 1980s, when the introduction of
systemic IL-2 therapy for the treatment of advanced melanoma and renal cell
carcinoma provided consistent and reproducible evidence that some advanced
cancers could regress and remain in long-term remission with a treatment that
vii
viii Foreword
had for sure no direct effect on cancer cells. Retrospectively, as too often
occurs in science, this skepticism was unwarranted, and the detractors of can-
cer immunotherapy made a disservice by slowing the progression of this bud-
ding discipline. Common criticisms were not directed against the observation
that cancers could regress but rather focused on denial about the overall effec-
tiveness of treatment, the sporadic nature of the regressions, and the relatively
high toxicity. In other words, the skeptics confused the clinical effectiveness
of a treatment with the value of a promising scientic observation.
I am emphasizing this because it is important to remember those difcult
moments now that books as sophisticated and comprehensive are presented on
a topic that was not even considered true science by most just a few decades
ago. Fortunately, several investigators did not give up, but focusing on the
value of an uncommon but reproducible observation carried the eld forward.
Thus this book! An achievement difcult to predict only two decades ago!
A book series that encompassed 77 chapters spanning biological aspects
of innate and adaptive immune responses to system biology approaches to
biomarker discovery, to portrays of clinical successes and discussion of regu-
latory processes that are about to revolutionize the development and licensing
of new investigational agents.
A signicant change occurred after the identication and molecular char-
acterization of antigens recognized by antibodies and/or T cells. Moreover,
the characterization of molecular mechanisms controlling the cross talks
between cancer and non-neoplastic somatic cells expanded the eld and
understanding of the mechanistic bases of immune-mediated tumor rejection.
These unarguable observations gave molecular precision to what was previ-
ously perceived as pointless practice. However, the true revolution came with
the clinical demonstration that some of the novel biological agents could sig-
nicantly improve the survival of patients, receiving, therefore, acceptance
and recognition as standard therapies through regulatory licensing.
Yet, challenges remain, and it is not the time to relax. Still, the benets,
though reproducible, are marginal both in terms of number of patients bene-
ting from the treatment and in the length of survival for those who benet.
Most importantly, the outcomes are capricious and unpredictable. Predictive
and surrogate biomarkers are missing in spite of novel technologies and strat-
egies that could help in the identication and stratication of patients. Still,
most clinical trials are designed to look at outcomes rather than comprehen-
sively learn in case of failures. Still, a gap exists between the potentials for
what we could do to better understand the biology of immune responsiveness
and what we actually do.
This book is written for those who want to move the eld forward both at the
clinical and the scientic level. Such a compendium can provide a contempo-
rary overlook at what has happened lately, which is remarkably logarithmic on
a time perspective. Yet, we wonder how elemental this edition may seem just
within a few years if the eld continues to evolve at the current pace. We hope
that a second edition will follow soon. Perhaps the editors should have asked
for a clairvoyants chapter. Hopefully, one of the young readers of this edition
may step forward and help dene the new frontiers of cancer immunotherapy.
Francesco M. Marincola, MD
Preface
The rapid ow of studies in the eld of cancer immunology during the last
decade has increased our understanding of the interactions between the
immune system and cancerous cells. In particular, it is now well known that
such interactions result in the induction of epigenetic changes in cancerous
cells and the selection of less immunogenic clones as well as alterations in
immune responses. Understanding the cross talk between nascent trans-
formed cells and cells of the immune system has led to the development of
combinatorial immunotherapeutic strategies to combat cancer.
Cancer Immunology Series, a three-volume book series, is intended as an
up-to-date, clinically relevant review of cancer immunology and immuno-
therapy. The book Cancer Immunology: A Translational Medicine Context, is
focused on the immunopathology of cancers. Cancer Immunology: Bench to
Bedside Immunotherapy of Cancers, is a translation text explaining novel
approaches in the immunotherapy of cancers. Finally, the book entitled
ix
x Preface
Cancer Immunology: Cancer Immunotherapy for Organ-Specic Tumors,

thoroughly addresses the immunopathology and immunotherapy of organ-
specic cancers.
In volume I, interactions between cancerous cells and various components
of the innate and adaptive immune system are fully described. Notably, the
principal focus is very much on clinical aspects, the aim being to educate
clinicians on the clinical implications of the most recent ndings and novel
developments in the eld. To meet this purpose, this volume consists of 26
chapters. After an overview on cancer immunology in Chap. 1, the role of
innate immunity in cancers is explained in Chaps. 2 and 3, followed by the
adaptive immunity, including B cells, T cells, T regulatory and Th17 cells,
cytokines, and chemokine receptors in Chaps. 4, 5, 6, 7, and 8, respectively.
CD95/CD95L signaling pathway, MHC class I molecules, and plasmacytoid
dendritic cells are separately described in Chaps. 9, 10, and 11, respectively.
Chapter 12 focuses on cancer immunoediting, while Chaps. 13 and 14 explain
apoptosis and autophagy in cancers. Subsequently, Chap. 15 presents the
prognostic value of innate and adaptive immunity in cancers. Epigenetics and
immunogenetics are explicated in Chaps. 16 and 17, respectively. In addition,
immunodeciencies (Chap. 18), immunosenescence (Chap. 19), nutrition
(Chap. 20), allergies (Chap. 21), and transmissible cancers (Chap. 22) are
individually described in the following chapters. Chapter 23 enlightens
systems biology in cancer immunology, while immunological diagnostic
tests, including ow cytometry for cancers, are mentioned in both Chaps. 24
and 25. Finally, by allocating the nal chapter to immunohistochemistry of
different cancers, volume I comes to its end.
The Cancer Immunology Series is the result of valuable contributions of
266 scientists from 91 well-known universities/institutes worldwide. I would
like to hereby acknowledge the expertise of all contributors for generously
devoting their time and considerable effort in preparing their respective chap-
ters. I would also like to express my gratitude to the Springer publication for
providing me the opportunity to publish the book.
Finally, I hope that this translational book will be comprehensible, cogent,
and of special value for researchers and clinicians who wish to extend their
knowledge on cancer immunology.
Nima Rezaei, MD, PhD

Acknowledgment
I would like to express my gratitude to the technical editor of this book,

Maryam Ebadi, MD. With no doubt, the book would not have been com-
pleted without her contribution.
Nima Rezaei, MD, PhD
xi
Contents
1 Introduction on Cancer Immunology

and Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nima Rezaei, Seyed Hossein Aalaei-Andabili,
and Howard L. Kaufman
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Cancer Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Cancer and Immune System Impairment . . . . . . . . . . . . . . . . 3
1.4 Immune System Reaction to Cancer . . . . . . . . . . . . . . . . . . . . 3
1.5 Genetic and Environmental Carcinogenesis . . . . . . . . . . . . . . 4
1.5.1 Cancer Cells Escape from Host
Immunosurveillance . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.5.2 Cancer Immunodiagnosis . . . . . . . . . . . . . . . . . . . . . . 4
1.6 Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6.1 Cancer Immunotherapy. . . . . . . . . . . . . . . . . . . . . . . . 5
1.6.2 Cancer Cell Switch . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 Inammatory and Innate Immune Cells in Cancer

Microenvironment and Progression . . . . . . . . . . . . . . . . . . . . . . 9
Patrick Brennecke, Paola Allavena, Ilaria Laface,
Alberto Mantovani, and Barbara Bottazzi
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Heterogeneity of Myeloid Cells in the Tumor
Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.1 Myeloid Subsets in the Tumor
Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.2 Recruitment of Myeloid Cells in Tumors . . . . . . . . . . 12
2.2.3 Tumor-Derived Factors Affecting Myeloid
Differentiation and Polarized Functions . . . . . . . . . . . 13
2.3 Pro-tumoral Functions of Tumor-Associated
Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.1 Tumor Proliferation and Survival . . . . . . . . . . . . . . . . 14
2.3.2 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
xiii
xiv Contents
2.3.3 Cancer Cell Dissemination . . . . . . . . . . . . . . . . . . . . . 16

2.3.4 Suppression of Adaptive Immunity. . . . . . . . . . . . . . . 18
2.4 Selected Aspects of Therapeutic
Targeting of TAMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3 Role of Innate Immunity in Cancers and

Antitumor Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Masahisa Jinushi and Muhammad Baghdadi
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2 Role of Innate Immune Cells in Cancer and
Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2.1 Natural Killer (NK) Cells . . . . . . . . . . . . . . . . . . . . . . 30
3.2.2 Natural Killer T (NKT) Cells . . . . . . . . . . . . . . . . . . . 31
3.2.3 -T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.4 Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.5 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2.6 Granulocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3 The Role of Innate Immune Receptors on Innate
Immune Cells in Cancer and Antitumor Immunity. . . . . . . . . 32
3.3.1 Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . 32
3.3.2 RIG-I-Like Helicases (RLHs) . . . . . . . . . . . . . . . . . . . 33
3.3.3 NOD-Like Receptors (NLRs) . . . . . . . . . . . . . . . . . . . 33
3.3.4 Phagocytosis Receptors. . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.5 C-Type Lectin-Like Receptors (CLRs) . . . . . . . . . . . . 34
3.3.6 NK Cell Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.7 B7 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4 The Role of Effectors Produced from Innate Immune
Cells in Cancer and Antitumor Immunity . . . . . . . . . . . . . . . . 37
3.4.1 Interferons (IFNs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4.2 Other Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.4.3 Chemokines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4 B Cells in Cancer Immunology: For or Against

Cancer Growth?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Qiao Li, Qin Pan, Huimin Tao, Xiao-Lian Zhang,
Shiang Huang, and Alfred E. Chang
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.2 CD40-Activated B (CD40-B) Cells . . . . . . . . . . . . . . . . . . . . 48
4.3 Tumor Killer B Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.4 Tumor-Inltrating B Cells (TIL-Bs) in Cancer. . . . . . . . . . . . 52
4.5 Resting B Cells and Regulatory B Cells in Cancer. . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Contents xv
5 The Role of Exhaustion in Tumor-Induced T Cell

Dysfunction in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Heriberto Prado-Garcia, Susana Romero-Garcia,
and Jose Sullivan Lopez-Gonzalez
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.2 T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.3 T Cell Anergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.3.1 T Cell Anergy in Cancer . . . . . . . . . . . . . . . . . . . . . . . 64
5.4 T Cell Exhaustion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.4.1 Mechanisms for Inducing T Cell Exhaustion . . . . . . . 65
5.4.2 Identication of Exhausted T Cells. . . . . . . . . . . . . . . 66
5.5 T Cell Exhaustion in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.5.1 A Particular Case: T Cell Exhaustion
in Lung Cancer Patients . . . . . . . . . . . . . . . . . . . . . . . 69
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
6 Regulatory T Cells and Th17 Cells in Cancer

Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Chang H. Kim
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6.2 Diversity of Tumor Microenvironments and
Tumor Tissue Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.3 Generation of Tregs and Th17 Cells . . . . . . . . . . . . . . . . . . . . 80
6.4 Impact of Tumor-Derived Factors on Regulation
of T-Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.5 Migration of Tregs and Th17 Cells into Tumors. . . . . . . . . . . 82
6.6 Impact of Tregs and Th17 Cells on
Antitumor Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . 84
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
7 Role of Cytokines in Tumor Immunity and Immune

Tolerance to Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Murugaiyan Gopal
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.2 Cytokine Regulation of the Antitumor Immune Response . . . 94
7.2.1 IL-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7.2.2 IL-27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
7.3 Cytokines in Immune Tolerance to Cancer . . . . . . . . . . . . . . . 101
7.3.1 TGF- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
7.3.2 IL-17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.3.3 IL-23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.3.4 IL-35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.3.5 IL-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
xvi Contents
8 Role of Chemokines and Chemokine Receptors in Cancer . . . 121

Mathieu Paul Rodero, Christophe Combadire,
and Alexandre Boissonnas
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.2 Chemokines and Chemokine Receptors . . . . . . . . . . . . . . . . . 123
8.3 Control of Tumor Cell Behavior . . . . . . . . . . . . . . . . . . . . . . . 125
8.3.1 Chemokines and Chemokine Receptor
Alterations During Neoplastic Transformation. . . . . . 125
8.3.2 Metastasis/Homing . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
8.3.3 Senescence, Proliferation, and Survival . . . . . . . . . . . 127
8.4 Control of Immune Cell Behaviors . . . . . . . . . . . . . . . . . . . . . 128
8.4.1 Chemokines Involved in T-Cell Antitumor
Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
8.4.2 Chemokines in Innate Immune Components . . . . . . . 130
8.4.3 Chemokine and Tumor-Induced Tolerance . . . . . . . . . 131
8.5 Alternative Tumor-Associated Physiological
Functions of Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
8.5.1 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
8.5.2 Fibrosis and Extracellular Matrix Remodeling . . . . . . 132
8.6 Clinical Aspect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.6.1 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
8.6.2 CC Chemokines/Chemokine Receptors . . . . . . . . . . . 133
8.6.3 CXC Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
8.6.4 CX3C Chemokine Receptors . . . . . . . . . . . . . . . . . . . 135
8.6.5 Chemokine Circulating Expression. . . . . . . . . . . . . . . 135
8.6.6 Therapeutic Strategies. . . . . . . . . . . . . . . . . . . . . . . . . 136
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
9 The CD95/CD95L Signaling Pathway: A Role

in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Amlie Fouqu and Patrick Legembre
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9.2 TNF Receptor Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
9.2.1 TNFR1 Signaling Pathways . . . . . . . . . . . . . . . . . . . . 144
9.2.2 TNF/TNFR: A Gold Mine for Therapeutic Tools . . . . 145
9.3 CD95: A Death Receptor?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
9.3.1 Structure/Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
9.3.2 Type I/II Signaling Pathways . . . . . . . . . . . . . . . . . . . 148
9.3.3 What Can We Learn from CD95 Mutations? . . . . . . . 148
9.3.4 Regulation of the Initial Steps of
CD95-Mediated Signaling . . . . . . . . . . . . . . . . . . . . . 150
9.3.5 Programmed Necrosis Also Known
as Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
9.3.6 CD95L, an Inammatory/Oncogenic Cytokine? . . . . 152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Contents xvii
10 MHC Class I Molecules and Cancer Progression:

Lessons Learned from Preclinical Mouse Models. . . . . . . . . . . 161
Irene Romero, Ignacio Algarra, and Angel M. Garcia-Lora
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
10.2 MHC-I Cell Surface Expression on Tumor Cells
and Primary Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . 162
10.2.1 Studies in GR9 Tumor Model: H-2 Antigen
Surface Expression and Tumorigenic Capacity . . . . . 164
10.3 MHC-I Expression and Metastatic Progression . . . . . . . . . . . 166
10.3.1 MHC Class I Expression on Primary
Tumor Cells May Determine Spontaneous
Metastatic Capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . 166
10.3.2 Different MHC-I Surface Expression on GR9
Tumor Clones Determines Their Spontaneous
Metastatic Capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . 167
10.4 Immunotherapy as a Treatment Against Cancers
Expressing Different MHC-I Surface Expression. . . . . . . . . . 169
10.4.1 Immunotherapy as a Treatment Against
Primary Tumors with Different Levels of
MHC-I Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
10.4.2 Immunotherapy as a Treatment Against
Metastatic Progression Derived from Primary
Tumors with Different MHC-I Expression . . . . . . . . . 170
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
11 Role of Plasmacytoid Dendritic Cells in Cancer . . . . . . . . . . . . 177

Michela Terlizzi, Aldo Pinto, and Rosalinda Sorrentino
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
11.2 Localization and Trafcking Patterns of Plasmacytoid
Dendritic Cells (pDCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
11.3 Plasmacytoid Dendritic Cells (pDCs) Phenotype . . . . . . . . . . 179
11.4 Activation of pDCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
11.5 pDCs: Bridging the Gap Between Innate
and Adaptive Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
11.6 pDCs and Human Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
11.6.1 Role of pDCs in Human Infections. . . . . . . . . . . . . . . 184
11.6.2 Role of pDCs in Autoimmune Diseases . . . . . . . . . . . 185
11.6.3 Role of pDCs in Cancer . . . . . . . . . . . . . . . . . . . . . . . 186
11.7 Potential Therapies: Clinical Signicance . . . . . . . . . . . . . . . 189
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
xviii Contents
12 Cancer Immunoediting: Immunosurveillance,

Immune Equilibrium, and Immune Escape . . . . . . . . . . . . . . . 195
Alka Bhatia and Yashwant Kumar
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
12.2 Cancer Immunoediting with Its Three Es: Reection
of the Dual Role of Immunity in Cancer. . . . . . . . . . . . . . . . . 196
12.2.1 Immune Elimination: Evidences For and Against . . . 197
12.2.2 The Equilibrium Phase: The Most Controversial
and the Least Understood Phase . . . . . . . . . . . . . . . . . 200
12.2.3 Immune Escape: The Best Studied Phase . . . . . . . . . . 201
12.3 Tumor Antigens and Cancer Immunoediting . . . . . . . . . . . . . 203
12.4 The Tumor Microenvironment During
Cancer Immunoediting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
12.5 Clinical Relevance of the Immunoediting
Process in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
13 Apoptosis and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Mei Lan Tan, Heng Kean Tan,
and Tengku Sifzizul Tengku Muhammad
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
13.2 Mechanisms of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
13.2.1 Extrinsic Apoptosis Pathway. . . . . . . . . . . . . . . . . . . . 212
13.2.2 Intrinsic Apoptosis Pathway . . . . . . . . . . . . . . . . . . . . 213
13.3 Apoptosis and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
13.4 Apoptosis Signaling Pathways and Therapeutic
Targets in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
13.4.1 TRAIL (TRAIL Ligands, Monoclonal
Antibodies Against TRAIL-R1 and TRAIL-R2) . . . . 220
13.4.2 Bcl-2 Family Proteins (BH3 Mimetics
and Bcl-2 Antisense) . . . . . . . . . . . . . . . . . . . . . . . . . . 225
13.4.3 Proteasome Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 227
13.4.4 Inhibitor of Apoptosis Protein (IAP) Antagonists. . . . 229
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
14 Autophagy and Necroptosis in Cancer. . . . . . . . . . . . . . . . . . . . 243

Mei Lan Tan, Heng Kean Tan, Ahmed Ismail Hassan Moad,
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
14.2 Autophagy and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
14.3 Autophagy Signaling Pathways and Therapeutic
Strategies in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
14.3.1 mTOR Signaling Pathway Inhibitors . . . . . . . . . . . . . 249
14.3.2 Pro-autophagics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
14.3.3 Autophagy Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 251
14.4 Mechanisms of Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Contents xix
14.5 Necroptosis and Possible Therapeutic

Targets in Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
14.6 Crosstalk in Apoptosis, Autophagy, and Necroptosis . . . . . . . 261
14.7 Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
15 Prognostic Value of Innate and Adaptive Immunity

in Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Fabio Grizzi, Giuseppe Di Caro, Federica Marchesi,
and Luigi Laghi
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
15.2 Immune Inltration as a Major Player of the
Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
15.3 Cellular Players of the Innate Immunity in Cancer. . . . . . . . . 277
15.3.1 Tumor-Associated Macrophages (TAM). . . . . . . . . . . 277
15.3.2 Tumor-Associated Neutrophils (TAN) . . . . . . . . . . . . 278
15.4 Cellular Players of the Adaptive Immunity in Cancer . . . . . . 278
15.5 Prognostic Value of Innate and Adaptive Cells
of the Immune System in Cancer . . . . . . . . . . . . . . . . . . . . . . 279
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
16 Epigenetics and microRNAs in Cancer . . . . . . . . . . . . . . . . . . . 285

Petra M. Wise, Kishore B. Challagundla, and Muller Fabbri
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
16.2 MiRNAs Regulate Effectors of the Epigenetic Machinery. . . 286
16.3 MiRNAs Are Epigenetically Regulated in
Several Types of Human Cancers . . . . . . . . . . . . . . . . . . . . . . 289
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
17 Immunogenetics of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Armin Hirbod-Mobarakeh, Ali Akbar Amirzargar,
Behrouz Nikbin, Mohammad Hossein Nicknam,
Anton Kutikhin, and Nima Rezaei
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
17.2 Cancers: Why Are There Different Faces? . . . . . . . . . . . . . . . 296
17.3 Immune Polymorphism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
17.4 Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.4.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.4.2 Immunogenetic Tools . . . . . . . . . . . . . . . . . . . . . . . . . 298
17.5 Immunogenetics: A Champion in Fighting
the Losing Battle Against Cancer . . . . . . . . . . . . . . . . . . . . . . 303
17.6 Human Leukocyte Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.2 Genes Behind HLA . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
17.6.3 From Polymorphisms to Clinic . . . . . . . . . . . . . . . . . . 306
xx Contents
17.6.4 HLA Typing and HLA Association Studies:

Lessons from the Past . . . . . . . . . . . . . . . . . . . . . . . . . 308
17.6.5 Typing Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
17.6.6 Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . 311
17.6.7 Linkage Disequilibrium . . . . . . . . . . . . . . . . . . . . . . . 311
17.7 The Cytokine Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
17.7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
17.7.2 Interleukin-1 Superfamily . . . . . . . . . . . . . . . . . . . . . 313
17.7.3 Interleukin-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
17.7.4 Interleukin-6 (IL-6) . . . . . . . . . . . . . . . . . . . . . . . . . . 317
17.7.5 Interleukin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
17.7.6 Interleukin-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
17.7.7 Interleukin-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
17.7.8 Tumor Necrosis Factor- and Lymphotoxin- . . . . . 324
17.7.9 Interferon Gamma (IFN-) . . . . . . . . . . . . . . . . . . . . 330
17.7.10 Transforming Growth Factor- (TGF-). . . . . . . . . . 330
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
18 Primary Immunodeciencies and Cancers . . . . . . . . . . . . . . . . 343

Mona Hedayat, Waleed Al-Herz, Asghar Aghamohammadi,
Kim E. Nichols, and Nima Rezaei
18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
18.2 Primary Antibody Deciencies . . . . . . . . . . . . . . . . . . . . . . . . 344
18.2.1 Common Variable Immunodeciency. . . . . . . . . . . . . 344
18.2.2 X-Linked Agammaglobulinemia. . . . . . . . . . . . . . . . . 345
18.2.3 Selective IgA Deciency. . . . . . . . . . . . . . . . . . . . . . . 346
18.3 Combined Immunodeciencies. . . . . . . . . . . . . . . . . . . . . . . . 346
18.3.1 IL-2-Inducible T-Cell Kinase Deciency . . . . . . . . . . 346
18.3.2 Purine Nucleoside Phosphorylase Deciency. . . . . . . 347
18.3.3 Dedicator of Cytokinesis 8 Deciency . . . . . . . . . . . . 348
18.3.4 RHOH Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
18.3.5 MAGT1 Deciency. . . . . . . . . . . . . . . . . . . . . . . . . . . 350
18.4 Phagocyte Defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
18.4.1 Severe Congenital Neutropenia
(Kostmann Syndrome) . . . . . . . . . . . . . . . . . . . . . . . . 351
18.4.2 ShwachmanDiamond Syndrome. . . . . . . . . . . . . . . . 352
18.4.3 GATA2 Deciency . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
18.5 Defects in Innate Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . 353
18.5.1 Epidermodysplasia Verruciformis . . . . . . . . . . . . . . . . 353
18.5.2 Warts, Hypogammaglobulinemia, Infections,
and Myelokathexis Syndrome . . . . . . . . . . . . . . . . . . . . . . 354
18.6 Diseases of Immune Dysregulation . . . . . . . . . . . . . . . . . . . . 354
18.6.1 X-Linked Lymphoproliferative Disease . . . . . . . . . . . 354
18.7 Syndromes with Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . 355
18.7.1 Autoimmune Lymphoproliferative Syndrome . . . . . . 355
18.7.2 Autoimmune Polyendocrinopathy with
Candidiasis and Ectodermal Dystrophy . . . . . . . . . . . 356
Contents xxi
18.8 Other Well-Dened Immunodeciencies . . . . . . . . . . . . . . . . 356

18.8.1 DNA Repair Defects . . . . . . . . . . . . . . . . . . . . . . . . . . 356
18.8.2 Signal Transducer and Activator of
Transcription 3 Deciency . . . . . . . . . . . . . . . . . . . . . 357
18.8.3 WiskottAldrich Syndrome. . . . . . . . . . . . . . . . . . . . . 360
18.8.4 Chromosome 22q11.2 Deletion Syndrome . . . . . . . . . 361
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
19 Immunosenescence, Oxidative Stress, and Cancers . . . . . . . . . 377

Tamas Fulop, Graham Pawelec, Gilles Dupuis,
Rami Kotb, Bertrand Friguet, and Anis Larbi
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
19.2 Immune System and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 378
19.2.1 Immunosenescence or Immune Aging . . . . . . . . . . . . 378
19.2.2 Innate Immune System . . . . . . . . . . . . . . . . . . . . . . . . 379
19.2.3 Adaptive Immune System . . . . . . . . . . . . . . . . . . . . . . 383
19.2.4 Interaction Between Innate and Adaptive
Immune Responses: Effect of Aging. . . . . . . . . . . . . . 384
19.3 Inammation Aging and Oxidative Stress . . . . . . . . . . . . . . . 385
19.4 Immunosenescence and Cancer . . . . . . . . . . . . . . . . . . . . . . . 387
19.5 Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
20 Nutrition, Immunity, and Cancers . . . . . . . . . . . . . . . . . . . . . . . 395

Hassan Abolhassani, Niyaz Mohammadzadeh Honarvar,
Terezie T. Mosby, and Maryam Mahmoudi
20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
20.2 Role of Nutrition in Predisposition
of Cancer from an Immunologic View . . . . . . . . . . . . . . . . . . . 396
20.2.1 Protein-Calorie Balance . . . . . . . . . . . . . . . . . . . . . . . 396
20.2.2 Essential Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.3 Antioxidants (Selenium, Vitamin E,
and Vitamin C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.4 Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.5 Vitamin B6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.6 Folate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.2.7 Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
20.3 Aging as a Confounder of the Triangle of Nutrition,
Immunity, and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
20.4 Role of Cancer in Predisposition to Malnutrition
from an Immunologic View. . . . . . . . . . . . . . . . . . . . . . . . . . . 398
20.5 Role of Nutritional Support in Immune
Restoration of Cancer Patients . . . . . . . . . . . . . . . . . . . . . . . . 399
20.5.1 Arginine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.2 Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.3 Branched Chain Amino Acids. . . . . . . . . . . . . . . . . . . 400
xxii Contents
20.5.4 Nucleotides, Long-Chain Omega-3 Polyunsaturated

Fatty Acids, and Eicosapentaenoic Acid . . . . . . . . . . . 400
20.5.5 Fructooligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.6 Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.7 Vitamins C and E . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
20.5.8 Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
21 Allergies and Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Delia Rittmeyer and Axel Lorentz
21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
21.2 Molecular Mechanisms of Allergy . . . . . . . . . . . . . . . . . . . . . 408
21.3 Types of Allergic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
21.4 Molecular Basics of Carcinogenesis . . . . . . . . . . . . . . . . . . . . 409
21.5 Types of Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
21.6 Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
21.7 Relationship Between Allergies and Cancers in General . . . . 411
21.7.1 Cancers Positively Correlated with Allergies . . . . . . . 411
21.7.2 Tumor-Promoting Effects of Allergies . . . . . . . . . . . . 412
21.7.3 Cancers Negatively Correlated with Allergies . . . . . . 413
21.8 Tumor-Protecting Effects of Allergies . . . . . . . . . . . . . . . . . . 414
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
22 Cancer Immunology of Transmissible Cancers . . . . . . . . . . . . 419

Katrina Marie Morris and Katherine Belov
22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
22.2 Canine Transmissible Venereal Tumor . . . . . . . . . . . . . . . . . . 420
22.2.1 Prevalence and Transmission . . . . . . . . . . . . . . . . . . . 420
22.2.2 Histology and Clonality . . . . . . . . . . . . . . . . . . . . . . . 420
22.2.3 Disease Progression . . . . . . . . . . . . . . . . . . . . . . . . . . 421
22.2.4 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
22.3 Devil Facial Tumor Disease . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.1 Prevalence and Appearance. . . . . . . . . . . . . . . . . . . . . 422
22.3.2 Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.3 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
22.3.4 Do Devils Have an Impaired Immune System? . . . . . 423
22.3.5 Devils Have Low MHC Diversity . . . . . . . . . . . . . . . . 423
22.3.6 Expression of Immunosuppressive Cytokines . . . . . . 423
22.3.7 Regulation of Cell Surface MHC . . . . . . . . . . . . . . . . 423
22.4 Comparison of DFTD and CTVT . . . . . . . . . . . . . . . . . . . . . . 424
22.5 Evolution of Transmissible Cancers . . . . . . . . . . . . . . . . . . . . 424
22.6 Transmissible Tumors as a Cancer Model . . . . . . . . . . . . . . . 425
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
Contents xxiii
23 Envisioning the Application of Systems Biology

in Cancer Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Julio Vera, Shailendra K. Gupta, Olaf Wolkenhauer,
and Gerold Schuler
23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
23.1.1 The Omics Paradigm and the Use of
Statistical Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
23.1.2 Mathematical Modeling and Systems
Theory: Dissecting the Complexity
Emerging Out of the Structure
of Biochemical Networks . . . . . . . . . . . . . . . . . . . . . . 431
23.1.3 Bridging Biological Scales Through the
Integration of Biological Data in Multi-scale
Models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
23.2 One Step Further: Integrating the Different
Perspectives of Systems Biology into
a Unied Framework. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
23.3 Does Cancer Immunology Need a Systems
Biology Approach? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
23.4 A Quick View on Current Results . . . . . . . . . . . . . . . . . . . . . . 434
23.4.1 Computational Biology, Bioinformatics, and
High-Throughput Data Analysis Used in the
Design of Immune Therapies for Cancer . . . . . . . . . . 434
23.4.2 Mathematical Models Used in Basic
Oncology Research . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
24 Principles of Immunological Diagnostic Tests for Cancers . . . 451

Amber C. Donahue and Yen-lin Peng
24.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
24.2 Overview of Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
24.2.1 Monoclonal vs. Polyclonal Antibodies . . . . . . . . . . . 452
24.2.2 Antibody Fragments . . . . . . . . . . . . . . . . . . . . . . . . . 453
24.2.3 Reporter Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
24.2.4 Primary and Secondary Antibodies. . . . . . . . . . . . . . 454
24.3 Immunoprecipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
24.4 Immunoblotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
24.5 Radioimmunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
24.6 Enzymatic Immunoassays. . . . . . . . . . . . . . . . . . . . . . . . . . . 457
24.7 Immunocytochemical and Immunohistochemical Assays . . 460
24.8 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
24.9 Bead-Based Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
24.10 Antibody Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
24.11 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
xxiv Contents
25 Flow Cytometry in Cancer Immunotherapy:

Applications, Quality Assurance, and Future . . . . . . . . . . . . . . 471
Ccile Gouttefangeas, Steffen Walter, Marij J.P. Welters,
Christian Ottensmeier, Sjoerd H. van der Burg,
Cedrik M. Britten, and Cliburn Chan
25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
25.2 Main Flow Cytometry Assays in Cancer
Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
25.3 Panel Development and Quality Assurance . . . . . . . . . . . . . . 474
25.4 Prociency Programs Addressing Flow
Cytometry Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
25.5 Structured Reporting of Immune Assay Experiments. . . . . . . 478
25.6 Organization of Immune Monitoring in
Multicenter Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
25.7 Towards Automated Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 480
25.8 New Methods and Technologies . . . . . . . . . . . . . . . . . . . . . . . 482
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
26 Immunohistochemistry of Cancers . . . . . . . . . . . . . . . . . . . . . . 491

Alireza Ghanadan, Issa Jahanzad, and Ata Abbasi
26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
26.2 Immunohistochemistry of Skin Tumors . . . . . . . . . . . . . . . . . 492
26.2.1 Markers of Normal Skin . . . . . . . . . . . . . . . . . . . . . . . 492
26.2.2 Epithelial Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
26.2.3 Sweat Gland Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 495
26.2.4 Trichogenic Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 495
26.2.5 Sebaceous Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
26.2.6 Melanocytic Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . 496
26.2.7 Prognostic Markers of Melanoma. . . . . . . . . . . . . . . . 497
26.2.8 Specic Mesenchymal Tumors of the Skin. . . . . . . . . 497
26.3 Immunohistochemistry of Head and Neck Tumors . . . . . . . . 499
26.3.1 Tumors of the Nasal Cavity and
Paranasal Sinuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
26.3.2 Tumors of the Larynx, Nasopharynx,
and Oropharynx. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
26.3.3 Tumors of the Salivary Glands . . . . . . . . . . . . . . . . . . 501
26.3.4 Immunohistochemistry of Salivary
Gland Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
26.3.5 Tumors of Thyroid and Parathyroid Glands . . . . . . . . 505
26.4 Immunohistochemistry of Lung Tumors. . . . . . . . . . . . . . . . . 505
26.4.1 Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
26.4.2 Mesothelioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
26.5 Immunohistochemistry of Gastrointestinal Tumors . . . . . . . . 507
26.5.1 Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
26.5.2 Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
26.5.3 Stomach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
Contents xxv
26.5.4 Small Intestine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

26.5.5 Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.6 Anal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.7 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.8 Pancreas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
26.5.9 Gastrointestinal Stromal Tumor . . . . . . . . . . . . . . . . 513
26.5.10 Neuroendocrine Carcinomas. . . . . . . . . . . . . . . . . . . 513
26.6 Immunohistochemistry of the Urinary Tract. . . . . . . . . . . . . . 513
26.6.1 Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
26.6.2 Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
26.7 Immunohistochemistry of Female
and Male Genital Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.1 Uterine Cervix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.2 Vulva and Vagina. . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.3 Uterine Corpus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
26.7.4 Ovary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
26.7.5 Breast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
26.7.6 Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
26.7.7 Testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
26.8 Immunohistochemistry of Lymphoma . . . . . . . . . . . . . . . . . . 521
26.9 Immunohistochemistry of Soft Tissue
and Bone Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
26.9.1 Epithelial Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . 523
26.9.2 Myogenic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . 526
26.9.3 Nerve and Schwann Cell Markers. . . . . . . . . . . . . . . 530
26.9.4 Endothelial Markers . . . . . . . . . . . . . . . . . . . . . . . . . 530
26.9.5 Fibrohistiocytic Markers . . . . . . . . . . . . . . . . . . . . . . 531
26.9.6 Lipocytic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.7 Chondrocyte Markers . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.8 Osteogenic Markers . . . . . . . . . . . . . . . . . . . . . . . . . 533
26.9.9 Unknown-Origin Soft Tissue Tumors . . . . . . . . . . . . 534
26.10 Immunohistochemistry of the Nervous System . . . . . . . . . . . 534
26.10.1 Neuroepithelial Tumors. . . . . . . . . . . . . . . . . . . . . . . 535
26.10.2 Non-neuroepithelial Tumors . . . . . . . . . . . . . . . . . . . 536
26.10.3 Undifferentiated Tumors . . . . . . . . . . . . . . . . . . . . . . 538
26.10.4 Proliferative Markers. . . . . . . . . . . . . . . . . . . . . . . . . 538
26.11 Immunohistochemistry of Pediatric Tumors. . . . . . . . . . . . . . 538
26.12 Immunosurveillance, Immune Editing,
Immune Constant of Rejection, Immune Contexture,
and Immune Scoring of Cancers . . . . . . . . . . . . . . . . . . . . . . . 541
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
Contributors
Seyed Hossein Aalaei-Andabili, MD Thoracic and Cardiovascular

Surgery, Department of Surgery, College of Medicine, University of Florida,
Gainesville, Florida, USA
Research Center for Immunodeciencies, Childrens Medical Center,
Tehran University of Medical Sciences, Tehran, Iran
Ata Abbasi, MD, MPH Department of Pathology, Cancer Institute,
Imam Khomeini Complex Hospital, School of Medicine,
Hassan Abolhassani, MD, MPH Research Center for Immunodeciencies,
Childrens Medical Center Hospital, Tehran University of Medical Sciences,
Tehran, Iran
Division of Clinical Immunology, Department of Laboratory Medicine,
Karolinska Institutet at Karolinska University Hospital Huddinge,
Stockholm, Sweden
Asghar Aghamohammadi, MD, PhD Research Center for
Immunodeciencies, Department of Allergy and Immunology,
Childrens Medical Center, Pediatrics Center of Excellence,
Ignacio Algarra, PhD Departamento de Ciencias de la Salud, Facultad de
Ciencias Experimentales, Universidad de Jaen, Jaen, Spain
Waleed Al-Herz, MD Department of Pediatrics, Faculty of Medicine,
Kuwait University, Safat, Kuwait
Paola Allavena, MD Laboratory of Cellular Immunology, Humanitas
Clinical and Research Center, Milan, Rozzano, Italy
Ali Akbar Amirzargar, PhD Molecular Immunology Research Center,
and Department of Immunology, School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Muhammad Baghdadi, MD, PhD Division of Immunobiology,
Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
xxvii
xxviii Contributors
Katherine Belov, BSc, PhD Faculty of Veterinary Science,

University of Sydney, Sydney, NSW, Australia
Alka Bhatia, MD Department of Experimental Medicine and
Biotechnology, Post Graduate Institute of Medical Education and Research,
Chandigarh, India
Alexandre Boissonnas, PhD Centre dImmunologie et des Maladies
Infectieuses CIMI-Paris, U1135, Institut National de la Sant et de la
Recherche Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Universitaire de Cancrologie (UPMC-IUC), CHU Piti-Salptrire,
Paris, France
Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7, INSERM,
U1135, CNRS, ERL 8255, Centre dImmunologie et des Maladies
Infectieuses (CIMI), Paris, France
Barbara Bottazzi, PhD Laboratory of Immunopharmacology,
Humanitas Clinical and Research Center, Milan, Rozzano, Italy
Patrick Brennecke, MSc, PhD Laboratory of Immunopharmacology,
Cedrik M. Britten, MD TRON, Translational Oncology at the University
Medical Center of the Johannes-Gutenberg University gGmbH and
Association for Cancer Immunotherapy (CIMT), Mainz, Germany
Sjoerd H. van der Burg, PhD Experimental Cancer Immunology and
Therapy, Department of Clinical Oncology (K1-P), Leiden University
Medical Center, Leiden, The Netherlands
Giuseppe Di Caro, PhD Laboratory of Molecular Gastroenterology,
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
Kishore B. Challagundla, PhD Department of Pediatric Hematology/
Oncology, Childrens Center for Cancer and Blood Diseases,
Norris Comprehensive Cancer Center, University of Southern, California,
Childrens Hospital Los Angeles, Los Angeles, CA, USA
Department of Pediatric Hematology/Oncology, Childrens Hospital Los
Angeles, Los Angeles, CA, USA
Cliburn Chan, MBBS, PhD Department of Biostatistics and
Bioinformatics, Duke University Medical Center, Durham, NC, USA
Alfred E. Chang, MD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Division of Surgical Oncology, Department of Surgery, University of
Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA
Christophe Combadiere, PhD Centre dImmunologie et des Maladies
Infectieuses (CIMI), U1135, Institut National de la Sant et de la Recherche
Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Paris, France
Contributors xxix
Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7,

INSERM, U1135, CNRS, ERL 8255, Centre dImmunologie et des
Maladies Infectieuses (CIMI), Paris, France
Amber C. Donahue, PhD Department of Hematology/Oncology
Research and Development, Quest Diagnostics-Nichols Institute,
San Juan Capistrano, CA, USA
Gilles Dupuis, PhD Biochemistry Department and Graduate Program in
Immunology, University of Sherbrooke, Sherbrooke, QC, Canada
Muller Fabbri, MD, PhD Department of Pediatric Hematology/Oncology,
Childrens Center for Cancer and Blood Diseases, Norris Comprehensive
Cancer Center, University of Southern, California, Childrens Hospital Los
Department of Pediatric Hematology/Oncology and Molecular
Microbiology and Immunology, Childrens Hospital Los Angeles,
Los Angeles, CA, USA
Amlie Fouqu, PhD Universit Rennes-1, Rennes, France
INSERM U1085, IRSET, Equipe Labellise Ligue Contre Le Cancer
Death Receptors and Tumor Escape, Rennes, France
Bertrand Friguet, PhD Biological Adaptation and Ageing UMR
UPMC-CNRS 8256 ERL INSERM U1164, Unit de vieillissement stress,
inammation UR 4, Universite Pierre et Marie Curie-Paris 6, Jussieu,
Paris, France
Tamas Fulop, MD, PhD Geriatrics Division, Department of Medicine,
Research Center on Aging, University of Sherbrooke, Sherbrooke, QC,
Canada
Angel M. Garcia-Lora, PhD Servicio de Analisis Clinicos and
Inmunologia, UGC Laboratorio Clinico, Hospital Universitario Virgen de
las Nieves, Granada, Spain
Alireza Ghanadan, MD Department of Pathology, Cancer Institute,
Imam Khomeini Complex Hospital, School of Medicine,
Department of Dermatopathology, Razi Dermatology Hospital,
Tehran, Iran
Murugaiyan Gopal, PhD Department of Neurology, Center for
Neurologic Diseases, Brigham and Womens Hospital, Harvard Medical
School, Harvard Institutes of Medicine, Boston, MA, USA
Ccile Gouttefangeas, PhD Department of Immunology,
Institute for Cell Biology, University of Tbingen,
Tbingen, Germany
Fabio Grizzi, PhD Laboratory of Molecular Gastroenterology,
xxx Contributors
Shailendra K. Gupta, PhD Department of Systems Biology and

Bioinformatics, Institute of Computer Science, University of Rostock,
Rostock, Germany
Department of Bioinformatics, CSIR-Indian Institute of Toxicology
Research, Lucknow, India
Mona Hedayat, MD Division of Immunology, Boston Childrens Hospital,
Harvard Medical School, Boston, MA, USA
Armin Hirbod-Mobarakeh, MD Molecular Immunology Research Center,
School of Medicine, Childrens Medical Center, Tehran University of
Niyaz Mohammadzadeh Honarvar, PhD School of Nutrition and
Dietetics, Tehran University of Medical Sciences, Tehran, Iran
Shiang Huang, MD Hubei Province Stem Cell Research and Appling
Center, Wuhan Union Hospital, Wuhan, China
Department of Hematology, Wuhan Union Hospital, Wuhan, Hubei, China
Issa Jahanzad, MD Department of Pathology, Cancer Institute, Imam
Khomeini Complex Hospital, School of Medicine, Tehran University of
Masahisa Jinushi, MD, PhD Research Center for Infection-Associated
Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
Howard L. Kaufman, MD Department of General Surgery and
Immunology and Microbiology, Rush University Cancer Center,
Rush University Medical Center, Chicago, IL, USA
Chang H. Kim, PhD Laboratory of Immunology and Hematopoiesis,
Department of Comparative Pathobiology, College of Veterinary Medicine,
Weldon School of Biomedical Engineering, Center for Cancer Research,
Purdue University, West Lafayette, IN, USA
Rami Kotb, MD, FRCPC Department of Medicine,
BCCA Victoria, British Columbia Cancer Center and The University
of British Columbia, Victoria, BC, Canada
Yashwant Kumar, MD, DNB Department of Immunopathology, Post
Graduate Institute of Medical Education and Research, Chandigarh, India
Anton Kutikhin, MD, PhD Department of Epidemiology,
Kemerovo State Medical Academy, Kemerovo, Russian Federation
Ilaria Laface, PhD Laboratory of Immunopharmacology,
Luigi Laghi, MD Department of Biotechnologies and Translational
Medicine, Humanitas Clinical and Research Center, University of Milan,
Rozzano, Milan, Italy
Anis Larbi, PhD Singapore Immunology Network (SIgN), Biopolis, Agency
for Science Technology and Research (A*STAR), Singapore, Singapore
Contributors xxxi
Patrick Legembre, PhD Universit Rennes-1, Rennes, France

INSERM U1085, IRSET, Equipe Labellise Ligue Contre Le Cancer
Death Receptors and Tumor Escape, Rennes, France
CRLCC Centre Eugne Marquis, Avenue bataille Flandres Dunkerque,
Rennes, France
Qiao Li, PhD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Jose Sullivan Lopez-Gonzalez, PhD Departamento de Enfermedades
Cronico-Degenerativas, Instituto Nacional de Enfermedades Respiratorias
Ismael Coso Villegas, Mexico City, Distrito Federal, Mexico
Axel Lorentz, PhD Department of Nutritional Medicine, University
of Hohenheim, Stuttgart, Germany
Maryam Mahmoudi, MD, PhD School of Nutrition and Dietetics,
Alberto Mantovani, MD Department of Biotechnologies and
Translational Medicine, University of Milan, Milan, Italy
Federica Marchesi, PhD Department of Immunology and Inammation,
Department of Biotechnologies and Translational Medicine,
Humanitas Clinical and Research Center, University of Milan,
Rozzano, Milan, Italy
Ahmed Ismail Hassan Moad, PhD Department of Medical Laboratories,
College of Medicine and Health Sciences, Hodeidah University, Hodeidah,
Yemen
Katrina Marie Morris, BAnVetBioSc, PhD Faculty of Veterinary
Science, University of Sydney, Sydney, NSW, Australia
Terezie T. Mosby, MSc, RD, LDN Department of Nutrition,
St. Jude Childrens Research Hospital, Memphis, TN, USA
Tengku Sifzizul Tengku Muhammad, PhD Institute of Marine
Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu,
Terengganu, Malaysia
Kim E. Nichols, MD Department of Oncology, St. Jude Childrens
Research Hospital, Memphis, Tennessee, TN, USA
Mohammad Hossein Nicknam, MD, PhD Molecular Immunology
Research Center, and Department of Immunology, School of Medicine,
Behrouz Nikbin, MD, PhD Molecular Immunology Research Center, and
Department of Immunology, School of Medicine, Tehran University of
xxxii Contributors
Christian Ottensmeier, MD, PhD, FRCP Faculty of Medicine,

University of Southampton, Southampton General Hospital,
Southampton, UK
Qin Pan, PhD Surgery Department, University of Michigan,
Ann Arbor, MI, USA
State Key Laboratory of Virology, Department of Immunology,
Hubei Province Key Laboratory of Allergy and Immunology,
Wuhan University School of Medicine, Wuhan, Hubei, China
Department of Immunology, Wuhan University School of Medicine,
Wuhan, Hubei, China
Graham Pawelec, MA, PhD Tbingen Ageing and Tumor Immunology
Group, Second Department of Internal Medicine, Center for Medical
Research, University of Tuebingen, Tbingen, Germany
Gilles Dupuis, MA, PhD Biochemistry Department and Graduate Program
in Immunology, Faculty of Medicine and Health Sciences, University of
Sherbrooke, Sherbrooke, Canada
Yen-lin Peng, MSc Department of Hematology/Oncology Research
and Development, Quest Diagnostics-Nichols Institute,
San Juan Capistrano, CA, USA
Aldo Pinto Department of Pharmacy (DIFARMA), University of Salerno,
Fisciano, Salerno, Italy
Heriberto Prado-Garcia, PhD Departamento de Enfermedades
Nima Rezaei, MD, MSc, PhD Research Center for Immunodeciencies,
Childrens Medical Center, Pediatrics Center of Excellence,
Department of Immunology, School of Medicine, and Molecular Immunology
Research Center, Tehran University of Medical Sciences, Tehran, Iran
Delia Rittmeyer, MSc Department of Nutritional Medicine,
University of Hohenheim, Stuttgart, Germany
Matihieu P. Rodero, PhD Centre dImmunologie et des Maladies
Infectieuses (CIMI), U1135, Institut National de la Sant et de la Recherche
Mdicale (INSERM), Universit Pierre et Marie Curie Institut
Paris, France
Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7,
INSERM, U1135, CNRS, ERL 8255, Centre dImmunologie et des
Maladies Infectieuses (CIMI), Paris, France
Irene Romero, PhD Servicio de Analisis Clinicos and Inmunologia,
UGC Laboratorio Clinico, Hospital Universitario Virgen de las Nieves,
Granada, Spain
Contributors xxxiii
Susana Romero-Garcia, PhD Departamento de Enfermedades

Gerold Schuler, PhD Department of Dermatology, Faculty of Medicine,
Friedrich Alexander Universitt, University of Erlangen-Nurnberg,
Erlangen, Germany
Rosalinda Sorrentino, PhD Department of Pharmacy (DIFARMA),
University of Salerno, Fisciano, Salerno, Italy
Heng Kean Tan, BSc (Hons) Malaysian Institute
of Pharmaceuticals and Nutraceuticals, Ministry of Science,
Technology and Innovation (MOSTI), Minden, Pulau Pinang, Malaysia
Mei Lan Tan, PhD Advanced Medical and Dental Institute,
Universiti Sains Malaysia, Bandar Putra Bertam, Kepala Batas,
Pulau Pinang, Malaysia
Malaysian Institute of Pharmaceuticals and Nutraceuticals, Ministry
of Science, Technology and Innovation (MOSTI), Minden,
Pulau Pinang, Malaysia
Huimin Tao, MSc Surgery Department, University of Michigan,
Ann Arbor, MI, USA
Department of Hematology, Wuhan Union Hospital, Wuhan, Hubei, China
Michela Terlizzi, PhD Department of Pharmacy (DIFARMA),
University of Salerno, Fisciano, Salerno, Italy
Julio Vera, PhD Department of Dermatology, Faculty of Medicine,
Friedrich Alexander Universitt, University of Erlangen-Nurnberg,
Erlangen, Germany
Laboratory of Systems Tumor Immunology, Department of Dermatology,
University Hospital Erlangen, Erlangen, Germany
Steffen Walter, PhD Immatics Biotechnologies GmbH,
Tbingen, Germany
Marij J. P. Welters, PhD Experimental Cancer Immunology and Therapy,
Department of Clinical Oncology (K1-P), Leiden University Medical
Center, Leiden, The Netherlands
Petra M. Wise, PhD Department of Pediatric Hematology/Oncology,
Childrens Center for Cancer and Blood Diseases, Norris Comprehensive
Cancer Center, University of Southern, California, Childrens Hospital
Los Angeles, Los Angeles, CA, USA
Department of Pediatric Hematology/Oncology, Childrens Hospital Los
Olaf Wolkenhauer, PhD Department of Systems Biology and
Bioinformatics, Institute of Computer Science, University of Rostock,
Rostock, Germany
xxxiv Contributors
Xiao-Lian Zhang, PhD State Key Laboratory of Virology,

Department of Immunology, Hubei Province Key Laboratory
of Allergy and Immunology, Wuhan University School
of Medicine, Wuhan, Hubei, China
Department of Immunology, Wuhan University School of Medicine,
Wuhan, Hubei, China
Abbreviations
3-UTR 3-untranslated region

3D Three-dimensional
3-MA 3-Methyladenine
4-OHT 4-Hydroxytamoxifen
5AC 5-Azacytidine
Ab Antibody
ABC Adenosine triphosphate-binding cassette
Abs Antibodies
AC Adenocarcinoma
ACC Acinar cell carcinoma
ACC Adenoid cystic carcinoma
Ad5 Adenovirus serotype 5
ADCC Antibody-dependent cellular cytotoxicity
ADCP Antibody-dependent cellular phagocytosis
ADP Anti-adipophilin
Ag Antigen
AHR Aryl hydrocarbon receptor
AIA Ag-induced arthritis
AICD Activation-induced cell death
AIDS Acquired immune deciency syndrome
AIF Aapoptosis-inducing factor
AILT Angioimmunoblastic T-cell lymphoma
AIRC Italian Association for Cancer Research
AIRE Autoimmune regulator
ALK Anaplastic large cell lymphoma kinase
ALL Acute lymphoblastic leukemia
ALP Alkaline phosphatase
alphaGalCer Alpha-galactosylceramide
ALPS Autoimmune lymphoproliferative syndrome
AML Acute myeloid leukemia
ANCs Absolute neutrophil counts
ANN Articial neural network
ANT Adenine nucleotide translocase
APC Antigen-presenting cells
APCP Adenosine 5-(, -methylene) diphosphate
APCs Antigen-presenting cells
xxxv
xxxvi Abbreviations
APECED Autoimmune polyendocrinopathy with candidiasis and ecto-

dermal dystrophy
APL Acute promyelocytic leukemia
APM Antigen presentation machinery
APS-1 Autoimmune polyendocrine syndrome type I
ARB Average relative binding
ARDS Acute respiratory distress syndrome
ASCs Adult stem cells
ASM Acid sphingomyelinase
ASPS Alveolar soft part sarcoma
ATCL Anaplastic large cell lymphoma
ATLL Adult T-cell lymphoma/leukemia
ATM Ataxia telangiectasia mutated
ATO Arsenic trioxide
ATP Adenosine triphosphate
ATR Ataxia telangiectasia/Rad3-related kinase
ATRA All-trans retinoic acid
B SLL/CLL B-cell small lymphocytic lymphoma/chronic lymphocytic
lymphoma
BAFF B-cell activating factor
BALs Bronchoalveolar lavage
BCA Basal cell adenocarcinoma
BCC Basal cell carcinoma
BCG Bacillus Calmette-Gurin
BCR B-cell antigen receptor
BER Base excision repair
bFGF Basic broblast growth factor
BLI Bioluminescence imaging
Bregs Regulatory B cells
BSO Buthionine sulfoximine
BTK Brutons tyrosine kinase
BTLA B- and T-lymphocyte attenuator
C/EBPb CCAT/enhancer-binding protein b
CAFs Cancer-associated broblasts
CaP Prostate cancer
CARD Caspase-recruitment domain
CBA Cytometric bead array
CBR Clinical benet response
CC Choriocarcinoma
CC Chromophobe carcinoma
CCS Clear cell sarcoma
CD Clusters of differentiation
CD40-B CD40-activated B
CD40L CD40 ligand
CDC Complement-dependent cytotoxicity
c-FLIP Cellular FLICE-inhibitory protein
CFSE Carboxyuorescein diacetate succinimidyl ester
CGN Chromogranin
Abbreviations xxxvii
CHL Classic Hodgkin lymphoma

CHS Contact hypersensitivity
CIA Collagen-induced arthritis
CIC/CRI Cancer Immunotherapy Consortium of the Cancer Research
Institute in the USA
CIHR Canadian Institutes of Health Research
CIMT Cancer Immunotherapy
CIP CIMT Immunoguiding Program
CK Cytokeratin
CLA Cutaneous lymphocyte-associated antigen
CLEC9A C-type lectin domain family 9A
CLL Chronic lymphocytic leukemia
CLRs C-type lectin and lectin-like receptors
CLRs C-type lectin receptors
CMA Chaperone-mediated autophagy
CMC Chronic mucocutaneous candidiasis
CML Chronic myeloid leukemia
CNS Central nervous system
Con Concanavalin
CP Core particle
CpG-A ODN CpG-A oligodeoxynucleotide
CpG-ODN CpG oligodeoxynucleotide
CPS Cancer Prevention Study
CQ Chloroquine
CR Complete remission
CRC Colorectal cancer
CRCC Clear RCC
CRDs Cysteine-rich domains
CrmA Cytokine response modier A
CRP C-reactive protein
CRT Calreticulin
CS Classic seminoma
CS&T Cytometer setup and tracking
CSC Cancer stem cell
CSF-1 Colony-stimulating factor
CSF-1R CSF-1 receptor
CSF3R Colony-stimulating factor 3 receptor
CSR Class switch recombination
c-state Cytosolic state
CTC Circulating tumor cells
CTL Cytotoxic T lymphocyte
CTS Cathepsins
CTVT Canine transmissible venereal tumor
CVID Common variable immunodeciency
Cyt Cytochrome
DAMP Damage-associated molecular pattern
DC Dendritic cells
DCC Deleted in colorectal cancer
xxxviii Abbreviations
DC-SIGN Dendritic cell-specic ICAM-3 grabbing non-integrin

DD Death domain
DDP Diamindichloridoplatin
DED Death effector domain
DES Desmin
DFTD Devil facial tumor disease
DHh Desert hedgehog homolog
DISC Death-inducing signaling complex
DKO Double knockout
DLBCL Diffuse large B-cell lymphoma
DNAM DNAX-accessory molecule
DNMTs DNA methyltransferases
DNR Dominant-negative TGF- type II receptor
DNT Double-negative T
DR Death receptor
DRMs Detergent-resistant microdomains
DSB Double-strand break
DSRCT Desmoplastic small round cell tumor
DSS Dextran sulfate sodium
DT Diphtheria toxin
DTE Desmoplastic trichoepithelioma
DTH Delayed-type hypersensitivity
DTR Diphtheria toxin receptor
DUBs Deubiquitinases
EAE Experimental autoimmune encephalomyelitis
EBNA Epstein-Barr virus nuclear antigen
EBV Epstein-Barr virus
EC Embryonal carcinoma
ECL Electrochemiluminescent
ECM Extracellular matrix
ECP Eosinophil cationic protein
EGF Epidermal growth factor
EGFR EGF receptor
ELISA Enzyme-linked immunosorbent assay
EM Effector memory
EMC Epithelial-myoepithelial carcinoma
EMSA Electrophoretic mobility shift assay
EMT Epithelialmesenchymal transition
EndoG Endonuclease G
ER Endoplasmic reticulum
ER Estrogen receptor protein
ER+ Estrogen receptor-positive
ERK Extracellular signal-regulated kinase
ES Embryonic stem
ES/PNET Ewing sarcoma/peripheral neuroectodemal tumor
EV Epidermodysplasia verruciformis
FADD Fas-associating protein with a death domain
FAK Focal adhesion kinase
Abbreviations xxxix
FasL Fas ligand

FcRII Fc receptor II
FDA Food and Drug Administration
FL Follicular lymphoma
FLIP FLICE-inhibitory protein
Flt3L FMS like tyrosine kinase 3 ligand
Fluc Firey luciferase
FRB FKBP12-rapamycin-binding domain
FSC Forward scatter light
FZD Frizzled
GAP GTPase-activating protein
GBM Glioblastoma multiforme
GC Germinal center
GCLP Good clinical laboratory practice
GEFs Guanine nucleotide exchange factors
GEM Genetically engineered mouse
GEMM Genetically engineered mouse models
GFI1 Growth factor-independent 1
GFP Green uorescent protein
GI Gastrointestinal
GITR Glucocorticoid-induced tumor necrosis factor receptor-related
protein
Gld Generalized lymphoproliferative disease
Gli Gli transcription factors
Gln Glutamine
Glu Glutamate
GLUD1 Glutamate dehydrogenase 1
GLUL Glutamate-ammonia ligase
GM-CSF Granulocyte macrophage colony-stimulating factor
G-MDSC Granulocytic MDSC
GMP Good manufacturing practice
GPU Graphical processing units
GRAFT Genetically transplantable tumor model systems
GrB Granzyme B
GSIs Gamma secretase inhibitors
GSK-3 Glycogen synthase kinase-3
GVDH Graft-versus-host-disease
GWAS Genome-wide association studies
HAX1 HS-1-associated protein X
HBE Human bronchial epithelial
HBV Hepatitis B virus
HCC Hepatocellular carcinoma
HCL Hairy cell leukemia
HCV Hepatitis C virus
HD Healthy donors
HDAC Histone deacetylase
HDACi Histone deacetylase inhibitors
HDACs Histone deacetylases
xl Abbreviations
HEV High endothelial venules

HGF Hepatocyte growth factor
HGPIN High-grade prostate intraepithelial neoplasia
HGS Human Genome Sciences
Hh Hedgehog
HIES Hyper-IgE syndrome
HIF2 Hypoxia-inducible factor 2-
HIV Human immunodeciency virus
HL Hodgkins lymphoma
HLA Human leukocyte antigen
HLH Hemophagocytic lymphohistiocytosis
HNC Head and neck cancer
HP Human papilloma
HPC Hematopoietic progenitor cells
HPV Human papilloma virus
HRG Histidine-rich glycoprotein
HRP Horseradish peroxidase
HRR Homologous recombination repair
HS Herpes simplex
HSC Hematopoietic stem cells
HSCT Hematopoietic stem-cell transplantation
HSP Heat shock proteins
HVEM Herpesvirus entry mediator
IAP Inhibitor of apoptosis protein
IB Immunoblotting
IBCC Inltrating basal cell carcinoma
ICAD Inhibitor of caspase-activated DNase
ICAM Intercellular adhesion molecule
ICAM-3 Intercellular adhesion molecule 3
ICC Immunocytochemistry
ICOS Inducible costimulator
ICOS-L Inducible costimulator ligand
ICS Intracellular cytokine staining
IDC Invasive ductal carcinoma
IDO Indoleamine 2, 3-dioxygenase
IELs Intraepithelial lymphocytes
IFN Interferon
IFN Interferon gamma
IFN- Interferon
Ig Immunoglobulin
IgAD IgA deciency
IgE Immunoglobulin E
IHC Immunohistochemistry
IHC/ICC Immunohistochemistry and immunocytochemistry
IHh Indian hedgehog
IkB Inhibitor of kB
IKK IB kinases
IL Interleukin
Abbreviations xli
IL-10 Interleukin-10
IL-1Ra Interleukin-1Ra
IL-1 Interleukin-1
IL-2R Interleukin-2 receptor-
ILC Invasive lobular carcinoma
IM Inner mitochondrial membrane
IMPT Intensity-modulated proton therapy
IMRT Intensity-modulated radiotherapy
IMS Intermembrane space
INF Interferons
iNOS inducible nitric oxide synthase
IP Immunoprecipitation
iPS Induced pluripotent stem
IRF Transcription factor
ISPC In silico planning comparative
ITAM Immunoreceptor tyrosine-based activation motif
ITIM Immunoreceptor tyrosine-based inhibition motif
ITK T-cell kinase
IVD In vitro diagnostic
JAK Janus kinase
JNK Jun N-terminal kinase
KARs Killer activation receptors
KGF Keratinocyte growth factor
KIRs Killer cell immunoglobulin-like receptors
KIRs Killer inhibitory receptors
KSHV Kaposi sarcoma-associated herpesvirus
LAT Linker of activation in T-cell
LC Luminal cells
LCA Leukocyte common antigen
LCMV Lymphocytic choriomeningitis virus
LCs Langerhans cells
LCT Leydig cell tumor
LD Linkage disequilibrium
LIR LC3 interacting region
LMP-1 Latent membrane protein-1
LNA Locked nucleic acid
LNs Lymph nodes
LOH Loss of heterozygosity
LOX Lysyl oxidase
LPL Lymphoplasmacytic lymphoma
Lpr Lymphoproliferation
LPS Lipopolysaccharide
LTA Lymphotoxin-
LUBAC Linear ubiquitin chain assembly complex
mAb Monoclonal antibody
Mac Macrophages
MAC Microcystic adnexal carcinoma
MALT Mucosa-associated lymphoid tissue
xlii Abbreviations
MAMP Microbe-associated molecular pattern

MAPK Mitogen-activated protein kinase
MC Molluscum contagiosum
MC Myoepithelial carcinoma
MCA Methylcholanthrene
MCC Merkel cell carcinoma
MCMV Mouse cytomegalovirus
M-CSF Macrophage colony-stimulating factor
mDCs Myeloid-derived dendritic cells
MDS Myelodysplasia
MDSC Myeloid-derived suppressor cells
MEC Mucoepidermoid carcinoma
MEXT Ministry of Education, Culture, Sports, Science and Technology
MF Mycosis fungoides
MFI Mean uorescence intensity
MGMT Methylguanine methyltransferase
MGUS Gammopathy of unknown signicance
MHC Major histocompatibility complex
MIACA Minimal information on reported results including reporting
information on cellular assays
MIAME Minimal information about microarray experiments
MIATA Minimal information about T-cell assays
MIBBI Minimal information on biological and biomedical
investigations
MIC-A MHC class I chain-related A
MIF Macrophage inhibitory factor
MIG Monokine induced by interferon-
miRNAs MicroRNAs
MISC Motility-inducing signaling complex
MKPs MAP kinase phosphatases
ML-IAP Melanoma inhibitor of apoptosis protein
MM Multiple myeloma
M-MDSC Monocytic MDSC
MMP Metalloproteases
MMR Mismatch repair
MnO Manganese oxide
MOMP Membrane permeabilization
MPSC Metastatic pulmonary small cell carcinoma
MSA Muscle-specic antigen
MSCs Mesenchymal stem cells
MSF Migration-stimulating factor
MSI Microsatellite instability
m-state Matrix state
mTOR Mammalian target of rapamycin
MVD Microvascular density
MYG Myogenin
MZL Marginal zone lymphoma
NADPH Nicotinamide adenine dinucleotide phosphate oxidases
Abbreviations xliii
NAIP Neuronal apoptosis inhibitory protein

NCCD Nomenclature Committee on Cell Death
NCR Natural cytotoxicity receptor
ncRNAs noncoding RNAs
NEC Neuroendocrine carcinoma
NER Nucleotide excision repair
NF Nuclear factor
NFAT Nuclear factor of activated T cells
NF-B Nuclear factor-kappa B
NHANES National Health and Nutrition Examination Survey
NHEJ Nonhomologous end-joining
NHL Non-Hodgkin lymphoma
Ni Nickel
NiS Nickel sulde
NK Natural killer
NKG2D Natural killer group two member D
NKT Natural killer T
NLPHL Nodular lymphocyte predominant Hodgkin lymphoma
NLRs NOD-like receptors
NLRs Nucleotide-binding domain and leucine-rich-repeat-containing
proteins
NMC NUT midline carcinoma
NOD Nucleotide-binding oligomerization domain
NP Normal prostate
NPC Nasopharyngeal carcinoma
NPY Neuropeptide Y
NSCLC Non-small cell lung cancer
NSCLC Non-small cell lung carcinoma
Nt Nucleotides
NTKs Neurothekeoma
NUT Nuclear protein in testis
OARs Organs at risk
OC Oncocytoma
ODEs Ordinary differential equations
ONB Olfactory neuroblastoma
OPN Osteopontin
OPRCC Oncocytic papillary RCC
PAC Prostate adenocarcinoma
PAC Pulmonary adenocarcinoma
PAGE Polyacrylamide gel, and separated by electrophoresis
PAK p21-activated kinase
PAMPs Pathogen-associated molecular patterns
PARP Poly ADP-ribose polymerase
PAX Paired box
PB Peripheral blood
PBMC Peripheral blood mononuclear cell
PBMCs Blood mononuclear cells
PCD Programmed cell death
xliv Abbreviations
PCG Protein coding gene

PD Paget disease
PDAC Pancreatic ductal adenocarcinoma
pDCs Plasmacytoid dendritic cells
PDGF Platelet-derived growth factor
PD-L1 Programmed cell death-1 ligand
PE Phosphatidylethanolamine
PE Pleural effusion
PEMCs Pleural effusion mononuclear cells
PET Positron emission tomography
PFS Progression-free survival
PH Pleckstrin homology
PHA Phytohemagglutinin
PI3K Phosphatidylinositol 3-kinase
PIDs Primary immunodeciencies
PIP3 Phosphatidylinositol-3,4,5-triphosphate
PKB Protein kinase B
PKC Protein kinase C
PLAD Pre-ligand binding assembly domain
PLGC Polymorphous low-grade adenocarcinoma
PlGF Placental growth factor
PMA Phorbol myristate acetate
PMNs Polymorphonuclear leukocytes
PMT Photomultiplier tube
PNET/ES Peripheral neuroectodermal tumor/extraskeletal Ewing sarcoma
PNP Purine nucleoside phosphorylase
PR Progesterone receptor
PRC Polycomb Repressive Complex
PRCC Papillary RCC
pre-pDCs Precursor of pDCs
PROTOR Protein observed with Rictor
PRRs Pattern recognition receptors
PS Phosphatidylserine
PSSM Position-specic scoring matrix
Ptc Patched dependence receptor
PTCH1 Patched receptor
PTM Posttranslational modication
PTPC Permeability transition pore complex
PVDF Polyvinylidene uoride
PYGL Glycogen phosphorylase
QDs Quantum dots
QoL Quality of life
RA Rheumatoid arthritis
RAGE Receptor for advanced glycation end products
Raptor Regulatory-associated protein of mTOR
Rb Retinoblastoma protein
RCC Renal cell carcinoma
RFK Riboavin kinase
RFLPs Restriction fragment length polymorphisms
Abbreviations xlv
RHIM RIP homotypic interaction motif

RHOH Ras homolog family member H
RHOH Rho GTPase
RIA Radioimmunoassay
RICD Reactivation-induced cell death
Rictor Rapamycin-insensitive companion of mTOR
RIG-1 Retinoic acid-inducible gene I
RIP Receptor interacting protein
RISC RNA-induced silencing complex
RLHs RIG-I-like helicases
RMS Rhabdomyosarcoma
ROS Reactive oxygen species
RS Reference samples
SA Sebaceous adenoma
SAP Signaling associated protein
SBDS ShwachmanBodianDiamond syndrome
SC Sebaceous carcinoma
SCC Squamous cell carcinoma
SCCHN Squamous cell carcinoma of the head and neck
SCF Stem cell factor
SCID Severe combined immune-decient
SCLCL Small cell lung cancer
SCM Small cell melanoma
SCN Severe congenital neutropenia
SCNP Single-cell network proling
SCs Stem cells
SCT Sertoli cell tumor
SDC Salivary duct carcinoma
SDS ShwachmanDiamond syndrome
SDS Sodium dodecyl sulfate
SEC Small cell eccrine carcinoma
SED Subepithelial cell dome
SFB Segmented lamentous bacteria
Shh Sonic hedgehog
SHh Sonic hedgehog homolog
SHM Somatic hypermutation
siRNA Small interfering RNA
SIRP- Signal-regulatory protein-
SLAM Signaling lymphocytic activation molecule
SLE Systemic lupus erythematosus
SMC Skeletal muscle cells
SMM Stabilized matrix method
Smo Smoothened
SNEC Small cell neuroendocrine carcinoma
SNP Single nucleotide polymorphisms
SNUC Sinonasal undifferentiated carcinoma
SOBP Spreadout Bragg peak
SOCE Store-operated Ca2+ entry
SOPs Standard operating procedures
xlvi Abbreviations
SP Side population
SP-A Surfactant protein A
SPECT Single-photon emission computed tomography
SPIO Superparamagnetic iron oxide
SPN Solid pseudopapillary neoplasm
SS Sjgren syndrome
SS Spermatocytic seminoma
SSC Side-scattered light
SSCC Small cell squamous carcinoma
SSO Sequence-specic probes
SSP Sequence-specic primers
SSPCs Salivary gland stem/progenitor cells
STAT Signal transducer activator of transcription
STAT1 Signal transducer and activator of transcription-1
STIM Stromal interaction molecule
SVZ Subventricular zone
SYN Synaptophysin
T1D Type 1 diabetes
T2 Transitional 2 immature
TAA Tumor-associated antigens
TACI Transmembrane activator and calcium modulator and
cyclophilin ligand interactor
TADC Tumor-associated dendritic cells
TAM Tumor-associated macrophages
TAMC Tumor-associated myeloid cells
TAN Tumor-associated neutrophils
TAP Transporter associated with Ag presentation
TAP Transporter associated with Ag processing
TApDCs Tumor-associated pDCs
TAPs Peptide transporters
TAS Trait-associated SNP
TAs Tumor antigens
TB Tuberculosis
TBI Total body irradiation
tBID Truncated BID
TC/HRBCL T-cell/histiocyte-rich B-cell lymphoma
TCF-4 T cell factor
TCL T-cell lymphoma
TCR T cell receptor
TDLN Tumor-draining lymph node
TEM Tie2-expressing monocytes
TEM Transmission electron microscopy
TEMRA Terminally differentiated effector memory
TFBSs Transcription factor binding sites
TFH T follicular helper
TGB Thyroglobulin
TGF- Transforming growth factor
Th T helper
TIL Tumor-inltrating lymphocytes
TIL-Bs Tumor-inltrating B cells
Abbreviations xlvii
TLR Toll-like receptor

TLT Tertiary lymphoid tissue
TME Tumor microenvironment
TNC Tenascin C
TNF Tumor necrosis factor
TNF-R Tumor necrosis factor receptor
TNF Tumor necrosis factor alpha
TNF- Tumor necrosis factor-
TNM Tumor-node-metastasis
TRADD TNF-receptor-associated death domain
TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
Tregs Regulatory T cells
TSC Tuberous sclerosis complex
TSGs Tumor suppressor genes
TSH Thyroid-stimulating hormone
TSLP Thymic stromal lymphopoietin
TTP Time to progression
U1snRNP U1 small nuclear ribonucleoprotein
UADT Upper aerodigestive tract
UC Urothelial carcinoma
UCH Ubiquitin C-terminal hydrolases
ULBPs Unique long 16 binding proteins
Unfrac Unfractionated
UNPC Undifferentiated nasopharyngeal carcinoma
uPA Urokinase plasminogen activator
UPP Ubiquitin-proteasome pathway
UPS Ubiquitin-proteasome system
USP Ubiquitin-specic proteases
USPIO Ultrasmall superparamagnetic iron oxide nanoparticles
UV Ultraviolet
UVRAG Ultraviolet radiation resistance-associated gene
VEGF-A Vascular endothelial growth factor-A
VIM Vimentin
VINIII Vulvar intraepithelial neoplasia grade III
VNTR Variable number tandem repeat
VZ Varicella zoster
WAS WiskottAldrich syndrome
WASp WAS protein
WASP WiskottAldrich syndrome protein
WGS Whole genome sequencing
WHIM Warts, hypogammaglobulinemia, infections,
and myelokathexis
WM Waldenstrom macroglobulinemia
WT Wild-type
X-IAP X-linked inhibitor of apoptosis protein
XLA X-linked agammaglobulinemia
XLN X-linked neutropenia
XLP X-linked lymphoproliferative disease
XLT X-linked thrombocytopenia
YST Yolk sac tumor
Introduction on Cancer
Immunology and Immunotherapy 1
Nima Rezaei, Seyed Hossein Aalaei-Andabili,
and Howard L. Kaufman
Contents 1.5 Genetic and Environmental

Carcinogenesis............................................. 4
1.1 Introduction................................................. 1 1.5.1 Cancer Cells Escape from Host
1.2 Cancer Immunity ........................................ 2 Immunosurveillance ..................................... 4
1.5.2 Cancer Immunodiagnosis ............................. 4
1.3 Cancer and Immune
System Impairment .................................... 3 1.6 Cancer Treatment ....................................... 5
1.6.1 Cancer Immunotherapy ................................ 5
1.4 Immune System 1.6.2 Cancer Cell Switch.................................... 6
Reaction to Cancer ..................................... 3
1.7 Concluding Remarks .................................. 6
References ............................................................... 7
N. Rezaei, MD, MSc, PhD (*)

Department of Immunology, Research Center for
Immunodeciencies, Childrens Medical Center, 1.1 Introduction
Pediatrics Center of Excellence, Tehran University
of Medical Sciences, Dr Qarib St, Keshavarz Blvd,
Tehran 14194, Iran
Cancer is a life-threatening disease, which can
involve all human organs and tissues. It is the
Department of Immunology, School of Medicine, and
Molecular Immunology Research Center,
second leading cause of death and is responsible
Tehran University of Medical Sciences, for 25 % of all deaths in the United States. In
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran 2012, more than 1.6 million new cases (848,170
e-mail: rezaei_nima@tums.ac.ir men and 790,740 women) of invasive cancers
S.H. Aalaei-Andabili, MD were diagnosed in the United States alone [1].
Thoracic and Cardiovascular Surgery, The major cancers in adults include lung, breast,
Department of Surgery, College of Medicine,
University of Florida, Gainesville,
prostate, and colorectal cancer. In addition,
Florida 100129, USA 60,824 adolescents and young adults aged
Research Center for Immunodeciencies,
1529 years old were diagnosed with invasive
Childrens Medical Center, Tehran University cancers between 1975 and 2000 [2]. Among all
of Medical Sciences, Tehran, Iran invasive cancers, lymphoma was the most com-
e-mail: dr.alaei@yahoo.com mon cancer (20 %), followed by invasive skin
H.L. Kaufman, MD cancer (15 %), male genital system cancer
Department of General Surgery and Immunology (11 %), and endocrine system cancer (11 %) [2].
and Microbiology, Rush University Medical Center,
Rush University Cancer Center, Chicago,
Although cancer incidence has increased among
IL 60612, USA people younger than 45 years old during 1975
e-mail: howard_kaufman@rush.edu 2000, overall cancer incidence has decreased in
N. Rezaei (ed.), Cancer Immunology: A Translational Medicine Context, 1

DOI 10.1007/978-3-662-44006-3_1, Springer-Verlag Berlin Heidelberg 2015
2 N. Rezaei et al.
men by 0.6 % per year during 20042008. patient survival. Total mortality rates vary from
Remarkably, the rate remained stable among 6 % in thyroid cancer to 97 % in pancreatic
females due to the high rate of breast cancer [3]. cancer [6].
Many cancer predisposing factors have been
recognized; it has been found that cancer inci-
dence is signicantly associated with age from 1.2 Cancer Immunity
10 to 60 years. Additionally, male gender is at
higher risk of developing cancer compared to Cancer immunology has been studied for a long
females [2]. Race is another important factor for time; however, the molecular and cellular basis of
cancer development; before 40 years of age, non- tumor immunity is not completely understood.
Hispanic whites and, after 40 years of age, Advances in understanding the basis of immuno-
African-Americans/blacks have the highest inci- surveillance and progress in the treatment of
dence [4]. Other risks factors include life style infectious disease have had a major impact on the
choices such as tobacco use, obesity, and lack of development of tumor immunotherapy. The mod-
exercise and environmental factors such as expo- ern era of tumor immunology began in the 1950s
sure to excessive sun, radiation during childhood, when the role of T cell responses in tissue
human papilloma virus (HPV), human immuno- allograft rejection was initially identied. Since
deciency virus, and Epstein-Barr virus (EBV) then, it has been conrmed that tumors occur in
infection [4]. association with impaired function of T cells,
Cancer can be a life-threatening health prob- indicating the importance of the immune system
lem, especially when the tumor has metastasized in the development and progression of cancer [7].
to other organs. It is estimated that 577,190 The identication of tumor-associated antigens,
patients (including 301,820 men and 275,370 knowledge of effector T cell responses, and the
women) died from cancer in the United States in role of regulatory and suppressor T cell popula-
2012. Four cancers lung and bronchus, prostate, tions are now shaping the use of the immune sys-
and colorectal in men and lung and bronchus, tem to treat cancer.
breast, and colorectal in women are responsible In addition to an improved understanding of the
for approximately 50 % of cancer-related deaths. immune system, signicant advances in under-
Fortunately, the overall cancer-related mortality standing the molecular basis of neoplasia have
has been decreasing in recent years. The death occurred. Precise control of cellular activity and
rate decreased by 1.8 % per year among men and metabolism is crucial for proper physiologic func-
1.6 % per year among women. The highest mor- tion. Notably, cell division is an important process
tality reduction has been found among African- that requires precise regulation. The main differ-
Americans (2.4 % per year), followed by ence between tumor cells and normal cells is lack
Hispanics (2.3 % per year); however, American of growth control during the cell division process.
Indians/Alaska natives were an exception, and the This uncontrolled cell division can originate from
rate remained unchanged in this population [1]. various factors, such as chemical agents, viral
Cancer survival signicantly impacts infections, and mutations that lead to escape of
patients quality of life. Five-year mortality cells from the checkpoints which properly control
rates depend on several factors; survival is cell division. According to the type of tumor and
worse among males over 30 years of age, and proliferation rate, cancers can be benign or malig-
the survival gets worse for patients over nant [8]. It has been found that some tumors are
45 years old in both males and females. Non- caused by oncogenic viruses that induce malig-
Hispanic whites have the best survival rate and nant transformation. These oncogenic viruses can
African-Americans have the worst survival be both RNA and DNA viruses. Also, viral infec-
with survival differences as great as 20 % at tion may lead to leukopenia and immunode-
5 years after cancer diagnosis [5]. Furthermore, ciency, increasing the risk of malignancy.
the type of cancer is another risk factor for Therefore, prophylactic immunization against
1 Introduction on Cancer Immunology and Immunotherapy 3
oncogenic viruses (such as EBV, HPV, and HBV) immunosurveillance [12, 13]. In addition, tumors
might be a logical strategy for prevention of malig- produce soluble factors which downregulate the
nancy [9]. Indeed, a vaccine against the human interleukin-2 receptor- (IL-2R), leading to sup-
papilloma virus has shown signicant impact on pression of T cell function. Furthermore, estab-
preventing cervical intraepithelial neoplasia and lished tumors may result in severe protein
may prevent development of cervical carcinoma. expenditures in hosts, contributing to impairment
of immune system function [14].
1.3 Cancer and Immune

System Impairment 1.4 Immune System
Reaction to Cancer
It has been reported that impaired immune
response can induce tumor growth and prevent A critical question is whether cancer cells are
effective antitumor suppression, possibly through sufciently different from their normal cellular
a process of sneaking through which allows counterparts, and can thus be recognized by the
improved growth of small tumors rather than immune system. The immune system also pro-
large tumors [10]. Tumors may also produce duces a group of complementary markers with
immunosuppressive factors, such as interleukin- protective effects against cancer and other immu-
10 (IL-10), transforming growth factor- nologic or inammatory stresses. These markers
(TGF-), and alpha-fetoprotein, which suppress include proteins released by T cells and are gen-
innate immune responses against cancer. This has erally classied as cytokines. Cytokines
led to investigations using neutralizing antibodies include interleukins, interferons, tumor-necrosis
against these immunosuppressive factors [7]. factors (TNF), and lymphocyte-derived growth
In contrast, tumor-specic cytotoxic T lympho- factors. The production of tumor-specic anti-
cytes (CTLs) can be genetically altered to become bodies and/or activation of tumor antigen-specic
resistant to the TGF- inhibitory effect by trans- T cells target tumor-associated antigens typically
gene expression of a mutant dominant-negative found on the cell membrane. Studies have sug-
TGF- type II receptor (DNR). In addition, spe- gested that vaccination in the presence of com-
cic T cells genetically manipulated to produce plements can lead to tumor lysis. While
IL-12 can overcome the inhibitory effects of incompletely dened, several soluble and cellular
IL-10. On the other hand, tumors may express mediators of tumor rejection have been described,
FasL and stimulate apoptosis of tumor-inltrating including complement factors, active macro-
effector T cells. Small interfering RNA (siRNA) phages, T cells, and NK cells. While T cells
can be used to knock down the Fas receptor in require antigen specicity, the soluble and cellu-
tumor-specic CTL, leading to a signicant lar mechanisms of the innate immune response
decrease in their susceptibility to Fas-/FasL- can recognize the malignant phenotype in the
mediated apoptosis [11]. absence of antigen specicity [15].
The interaction between the immune system Since most tumor-associated antigens are self-
and established cancers is complex, because in proteins, the immune response is largely weak
addition to increasing carcinogenesis by various and patients may develop immune tolerance to
carcinogens among compromised subjects, cancer tumor-associated antigens. Furthermore, the cells
cells themselves can lead to severe immunosup- of the immune system may not adequately pene-
pression. It has been reported that patients involved trate to the internal tumor microenvironment,
with primary immunodeciency syndromes have resulting in slower immune-mediated tumor
higher risk of cancer development. In a report by elimination. However, it is possible that the
Kersey et al., subjects that had an inherited abnor- immune system may be more effective in control-
mal lymphoid system were susceptible to malig- ling tumor growth rate rather than tumor regres-
nant transformation and impairment of tumor sion [10]. Recently, it has been found that
4 N. Rezaei et al.
nutrition also plays a crucial role in protection costimulatory molecules, which mediate the
against human cancer, and normal levels of zinc activation of T cells. Another strategy resulting
are required for protection against the detrimen- in failure of tumor immunosurveillance could be
tal effects of various immunosuppressive cyto- the expression of very low levels of antigens,
kines [16]. unable to stimulate an immune response. Under
some circumstances, such as failure of the
immune response to induce a rapid response,
1.5 Genetic and Environmental cancer cells may proliferate rapidly. Further
Carcinogenesis strategies for escape of tumor cells from immu-
nosurveillance are based on inhibitory tumor-
It has been found that genetic factors are as mediated signaling by CTLs, as occurs through
important as environmental carcinogens. Trials changes in cell death receptor signaling. Other
have tested carcinogenesis of retrovirus infection strategies which allow tumor cells to evade the
between different breeds of animals. A unique immune system are the secretion immunosup-
carcinogen resulted in disparate outcomes among pressive molecules dampening tumor-reactive
different breeds, indicating the importance of effector T cells and the induction of regulatory
genetic background in the progression of cancer. and/or suppressor cells [19].
Environmental factors may also suppress immune To date, most direct evidence on tumor immu-
responses and dysregulate immunosurveillance nosurveillance originates from experimental
mechanisms [17]. studies in animal models. These models have sup-
ported the potential for antitumor immunity via
vaccination, as, for example, by administration of
1.5.1 Cancer Cells Escape from Host inactivated cancer cells, or through removal of a
Immunosurveillance primary tumor. In addition, antitumor immunity
can be adoptively transferred through administra-
Antigens that distinguish tumor cells from nor- tion of tumor-reactive T lymphocytes. The com-
mal cells depend on the histologic origin of the plexities of immunotherapy are evident as nearly
tumor. Tumor-associated antigens may be viral in all immune system components can inuence
origin, represent mutated self-antigens, be tumor growth and progression. Although there is
cancer-testis antigens which are expressed only evidence for antitumor immunity in humans and
by tumor cells and normal testes, or be normal several new agents have gained regulatory
differentiation antigens. Thus, tumor cells may approval for cancer therapy, further investigation
express similar antigens to normal cells, allowing is warranted to increase the impact of tumor
tumor cells to escape immune system attack immunotherapy for more cancer patients [20].
through induction of innate and/or peripheral tol-
erance. A corollary to this is that immunotherapy
or stimulation of immune responses to some 1.5.2 Cancer Immunodiagnosis
tumor-associated antigens may lead to damage of
normal tissues and organs, as exemplied by the Nowadays, new immunomolecular diagnostic
development of autoimmunity induced by approaches have been suggested for tumor detec-
anti-CTLA-4 or anti-PD1 monoclonal antibody tion. Monoclonal antibodies marked with radio-
(mAb) treatment [18]. isotopes have been used for in vivo diagnosis of
A number of complex mechanisms have been small tumor foci. In addition, monoclonal anti-
suggested for the escape of cancer cells from bodies have been used for in vitro recognition of
host immunosurveillance. Tumors alter their the cell of origin for tumors with poor differentia-
characteristics by decreased expression of tion. Immunodiagnostics have also been used to
immunogenic tumor-associated antigens, MHC determine the extent of metastatic disease, espe-
class I molecules, beta2-microglobulin, and cially metastasis to the bone marrow [21].
1.6 Cancer Treatment has been injected to the tumor mass and has had
benecial effects in the treatment of melanoma
Systemic cancer treatment is based on four and head and neck squamous cell carcinoma
general therapeutic approaches: (1) chemother- [26]. Although vaccine-based therapy has not
apy, which contains a wide group of cytotoxic been effective in some types of cancer, there are
drugs that interfere with cell division and DNA studies that have shown an overall survival bene-
synthesis; (2) hormonal therapy, which contains t compared to placebo therapy [27].
drugs that interfere with growth signaling via Another immune-targeted approach is mAbs
tumor cell hormone receptors; (3) targeted ther- which block T cell checkpoints functioning to
apy, which involves a novel group of antibodies suppress T cell responses. Cytotoxic T
and small-molecule kinase suppressors that prin- lymphocyte-associated antigen 4 (CTLA4) is a
cipally target proteins crucial in cancer cell member of a large family of molecules regulating
growth signaling pathways; and (4) immunother- T cell immune responses. CTLA4 is expressed
apy, which targets the induction or expansion of on CD4+ and CD8+ T cells, as well as on FOXP3+
antitumor immune responses [22]. regulatory T cells [28]. Administration of mAbs
targeting human CTLA4 leads to the rejection of
established tumors in a small cohort of patients
1.6.1 Cancer Immunotherapy with metastatic melanoma and demonstrated
improved overall survival in patients with meta-
Tumor immunotherapy is a novel therapeutic static melanoma, resulting in US FDA approval
approach for cancer treatment, with increasing for the treatment of metastatic melanoma [29].
clinical benets. Tumor immunotherapy is based Monoclonal antibodies which block other T
on strategies which improve the cancer-related cell checkpoints, such as the programmed cell
immune response through either promoting com- death protein 1 (PDCD1/PD1), programmed cell
ponents of the immune system that mediate an death ligand 1 (PDL1/CD274), CD276 (B7H3)
effective immune response or via suppressing antigen, V-set domain-containing T cell function
components that inhibit the immune response. inhibitor 1 (B7x), and B and T lymphocyte atten-
Two current approaches commonly used for uator, have also entered clinical trials. In addi-
immunotherapy are allogeneic bone marrow tion, early phase studies have demonstrated
transplantation and mAbs targeting cancer cells signicant therapeutic activity in several types of
or T cell checkpoints [23]. Recently, various cancer, including melanoma, renal cell carci-
other approaches have been tested for cancer noma, non-small cell lung carcinoma, and ovar-
immunotherapy, and some are undergoing further ian cancer [30]. It has been reported that PDL1
clinical evaluation. expression by tumor cells is associated with poor
Initially, anticancer vaccines were considered clinical outcome and may be associated with
for prevention and treatment of various tumors clinical response to anti-PD1 and anti-PDL1
[23]. It is estimated that more than 15 % of human therapy. Also, ligation of PDL1 leads to inactiva-
cancers are caused by viral infection [24]. tion of tumor-inltrating cells [31]. On the other
Vaccine-based immunotherapy may, thus, be hand, regulatory T cells have an immunosuppres-
most useful for virus-induced cancers. Consistent sive role in the tumor microenvironment. Studies
with this hypothesis, a 50 % complete remission of anti-PD1 and anti-PDL1 are in progress.
(CR) of HPV-associated vulvar intraepithelial Moreover, the combination of these agents with
neoplasia grade III (VINIII) has been reported anti-CTLA4 and other immunotherapy strategies
[25]. An attenuated, oncolytic herpes simplex has yielded promising results.
type 1, which is genetically engineered to secrete The combination of antitumor vaccines with
granulocyte-macrophage colony-stimulating fac- agents targeting the IL-12 receptor resulted in
tor (GM-CSF), has been developed for cancer conicting results. This may be due to the
therapy. This oncolytic immunotherapeutic agent upregulation of IL-12 receptor by both activated
6 N. Rezaei et al.
T effector cells and regulatory T cells [32]. Thus, During rapid proliferation of cancer cells, pre-
new approaches focused on more specic cise orchestrated enzyme formation needed for
targeting of regulatory T cells which reduce their suitable metabolism of its different compo-
suppressive effects on the immune system are nents might get unbalanced, and products
necessary. Adoptive T cell therapy has been which are not observed in normal dividing cells
described as an effective therapeutic approach for are produced [38]. Recently, it has been
cancer immunotherapy in early phase clinical tri- reported that these biochemical switches
als. In this method, a large number of tumor- lead to uncontrolled multiplication of cancer
specic T cells derived from peripheral blood, or cells. One switch has been found for a type of
preferably from the tumor microenvironment leukemia. It has been suggested that targeting
(with or without genetic manipulation to express tumor switches can make treatment of cancers
a high-afnity antigen-specic T cell receptor very simple [20]. Nonetheless, it is unclear
(TCR)), are adoptively transferred to patients how this may be used to optimize tumor
with established tumors [33]. Recently, CD19 immunotherapy.
which is expressed by mature B cells and a Since cancer immunology is a highly com-
majority of non-Hodgkin lymphoma (NHL) cells plex process, further research is needed to more
has been used as another novel promising thera- completely understand how the immune system
peutic target [34]. Chemotherapy-mediated cell recognizes and eradicates cancer. In this book,
death leads to immune responses in a drug- we will describe a variety of novel mechanisms
induced biochemical cell death cascade- currently under investigation for mediating
dependent manner, suggesting benecial effects aspects of tumor immunology with a particular
of chemotherapy and immunotherapy in combi- focus on promising therapeutic approaches, pro-
nation [35]. It seems that future goals of tumor ducing a complete comprehensive up-to-date
immunotherapy are headed towards chemoim- textbook.
munotherapy. Potential candidates for this com-
bination approach include antitumor vaccines,
Toll-like receptor (TLR) signaling pathway ago- 1.7 Concluding Remarks
nists/antagonists, cytokines, and mAbs targeting
T cell checkpoints, such as CTLA4, PD1, or Cancer is a life-threatening health problem which
PDL1/2 [36]. Also, it seems that radiation and is related to several genetic and environmental
radiofrequency ablation are future candidates for risk factors that manipulate immune system func-
combination therapy with immunotherapy [37]. tion. Cancers themselves produce immunosup-
Although immunotherapy and its combination pressor factors to impair cells division check
with other therapeutic approaches such as radio- points, leading to uncontrolled proliferation of
immunotherapy may be benecial for tumor cancer cells. Importantly, tumor cells have learned
treatment, there are several limitations that need how to escape from immune system attack via
to be addressed; dening the optimal target presenting of similar antigens to normal cells and
patient, optimal biological dose, and schedule, expression of very low levels of antigens.
the need for better trial designs incorporating Therefore, diagnosis of tumors and their progres-
appropriate clinical endpoints, and the identica- sion is not easy. Recently, immunodiagnostic
tion and validation of predictive biomarkers are methods are shown to be helpful in the diagnosis
just a few to point to [23]. of cancers and determining the extent of metasta-
sis. On the other hand, classic treatment of cancers
led to unsatisfactory results, and intelligent immu-
1.6.2 Cancer Cell Switch nological approaches, such as regulatory T-cell
targeting, adoptive T-cell administration, and
Cancer cells can switch on genes mostly related combination of immunotherapy and chemotherapy
to the earlier embryonic stages of development. are addressed. Results of antitumor vaccines,
Toll-like receptor (TLR) signaling pathway ago- 13. Kersey JH, Spector BD, Good RA. Primary immuno-
deciency diseases and cancer: the immunodeciency-
nists/antagonists, cytokines, and mAbs targeting
cancer registry. Int J Cancer. 1973;12:33347.
T-cell checkpoints, such as CTLA4, PD1, or 14. Sheu BC, Hsu SM, Ho HN, Lien HC, Huang SC,
PDL1/2 are promising. However, due to the Lin RH. A novel role of metalloproteinase in cancer-
highly complexity of the cancer immunology, mediated immunosuppression. Cancer Res. 2001;
61(1):23742.
still a lot of gaps exist in this eld that indicate the
15. Schlager SI, Ohanian SH, Borsos T. Correlation
necessity of further researches for complete between the ability of tumor cells to resist humoral
understanding of cancers immunological behav- immune attack and their ability to synthesize lipid. J
iors and emerging of more novel immunothera- Immunol. 1978;120(2):46371.
16. Lin YS, Caffrey JL, Lin JW, Bayliss D, Faramawi MF,
peutic strategies.
Bateson TF, et al. Increased risk of cancer mortality
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Inammatory and Innate Immune
Cells in Cancer Microenvironment 2
and Progression
Patrick Brennecke, Paola Allavena, Ilaria Laface,

Alberto Mantovani, and Barbara Bottazzi
Contents 2.1 Introduction

2.1 Introduction ................................................ 9
Inammation is a consistent feature of the tumor
2.2 Heterogeneity of Myeloid Cells
in the Tumor Microenvironment .............. 10
microenvironment and has been considered the
2.2.1 Myeloid Subsets in the Tumor seventh hallmark of cancer [16]. As suggested
Microenvironment........................................ 10 by current estimates, 25 % of cancers are asso-
2.2.2 Recruitment of Myeloid Cells in Tumors .... 12 ciated with chronic inammation sustained by
2.2.3 Tumor-Derived Factors Affecting
Myeloid Differentiation
infections (e.g., hepatitis) or inammatory con-
and Polarized Functions ............................... 13 ditions of diverse origin (e.g., prostatitis) [6].
In addition, even tumors not directly connected
2.3 Pro-tumoral Functions
of Tumor-Associated Myeloid Cells.......... 13 to inammation are characterized by the pres-
2.3.1 Tumor Proliferation and Survival ................ 14 ence of cells and mediators of the inammatory
2.3.2 Angiogenesis................................................ 15 response [7].
2.3.3 Cancer Cell Dissemination .......................... 16
Apart from malignant cells, host cells inl-
2.3.4 Suppression of Adaptive Immunity ............. 18
trate tumors, including leukocytes, broblasts,
2.4 Selected Aspects of Therapeutic and endothelial cells. Leukocytes, and in particu-
Targeting of TAMC .................................... 19
lar myeloid cells, are the most consistent cellular
2.5 Concluding Remarks ................................. 20 component of solid tumors. Tumor-associated
References ............................................................... 21 myeloid cells (TAMC) mainly support tumor
growth and progression, thereby contrasting the
P. Brennecke, MSc, PhD P. Allavena, MD T-cell inltrate, which mainly has antitumoral
I. Laface, PhD B. Bottazzi, PhD (*) activity. TAMC all arise from hematopoietic stem
Laboratory of Immunopharmacology, Humanitas cells (HSC) within the bone marrow (Fig. 2.1) and
Clinical and Research Center,
Via Manzoni 113, Milan, Rozzano 20089, Italy further differentiate into macrophage/granulo-
e-mail: drpbrennecke@gmail.com; cyte progenitors. The tumor inltrate comprising
paola.allavena@humanitasreserach.it; the myeloid populations skews tumor-mediated
Ilaria.laface@humanitasreserach.it; immunosuppression, tissue remodeling, tumor
barbara.bottazzi@humanitasresearch.it
progression and metastasis [8, 9]. TAMC dem-
A. Mantovani, MD onstrated high plasticity, resulting in two extreme
Department of Biotechnologies and Translational
Medicine, University of Milan, 20122 Milan, Italy polarized macrophage (M1 and M2) and neutro-
phil (N1 and N2) phenotypes [10, 11]. Cross talk
Humanitas Clinical and Research Center,
Via Manzoni 56, Milan, Rozzano 20089, Italy between the different cellular components was
e-mail: alberto.mantovani@humanitasreserach.it demonstrated, resulting in tuning of the adaptive

10 P. Brennecke et al.
Bone marrow Blood/spleen Tumor
TEM TEM
M1 TAM
Monocyte TAM
M-MDSC M-MDSC
MDSC
M2 TAM
HSC CMP IMC G-MDSC G-MDSC
N1 TAN
Neutrophil TAN
iDC TADC
N2 TAN
Fig. 2.1 Differentiation pathways of tumor-associated and N1N2 for neutrophils. CMP common myeloid pro-
myeloid cells. Myeloid cells originate from hematopoietic genitors, IMC immature myeloid cells, TEM Tie2-
stem cells (HSC) in the bone marrow. Here the networks expressing monocytes, MDSC myeloid-derived suppressor
that give rise to the various myeloid cell lineages in diverse cells, M-MDSC myeloid MDSC, G-MDSC granulocytic
compartments (bone marrow, blood/spleen, and tumor) and MDSC, TAM tumor-associated macrophages, TAN tumor-
their precursors are illustrated. In the tumor tissue, macro- associated neutrophils, iDC immature dendritic cells,
phages and neutrophils display a gradient of differently TADC tumor-associated dendritic cells
polarized phenotypes whose extreme are M1M2 for TAM
immune response, promotion of angiogenesis, approaches based on targeting of tumor-inltrating

and tissue remodeling [8]. myeloid cells and/or soluble mediators will be
Results obtained so far clearly indicate that discussed.
TAMC are major players in the connection
between inammation and cancer. Ongoing
efforts, which led to a better understanding of 2.2 Heterogeneity of Myeloid
their biological properties, indicated that myeloid Cells in the Tumor
cell-inltrating growing tumor could have a Microenvironment
prognostic value, thus representing an attractive
target for novel biological therapies of tumors. 2.2.1 Myeloid Subsets in the Tumor
In this chapter, we will mainly focus on Microenvironment
myeloid cells inltrating tumors and mention
soluble mediators involved in their recruitment or Solid tumors are characterized by the presence
released by TAMC, which affect tumor progres- of a leukocyte inltrate including lymphocytes
sion and dissemination (cytokines, chemokines, and myeloid cells from early stages. Growing
and proteases). Furthermore, new therapeutic evidence indicated that the leukocyte inltrate
2 Inammatory and Innate Immune Cells in Cancer Microenvironment and Progression 11
has a prognostic value. For instance, it has been responses [22, 14, 23, 11, 24]. Thus, macro-
described that inltrating T lymphocytes are phages are a very heterogeneous cell population,
associated with a favorable prognosis in colorec- able to display different functions depending on
tal cancer, melanoma, ovarian cancer, and breast the context. Macrophages can be either immuno-
cancer [12, 13]. In contrast, myeloid cells are stimulatory at the beginning of the inammatory
most frequently associated with a poor progno- response or immunosuppressive which dampen
sis [14]. TAMC (Fig. 2.1) comprise ve distinct inammation [25, 18, 14, 26, 27].
myeloid populations, namely, tumor-associ- A similar dichotomy with polarization towards
ated macrophages (TAM), monocytes express- two extreme phenotypes (N1 and N2) has been
ing the angiopoietin-2 (Ang-2) receptor Tie2 also described for neutrophils [28]. Besides
(known as Tie2-expressing monocytes or TEM), exerting antibacterial functions, neutrophils can
myeloid-derived suppressor cells (MDSC), inltrate tumors playing a major role as key
tumor-associated neutrophils (TAN), and tumor- mediators in malignant transformation, tumor
associated dendritic cells (TADC). progression, and regulation of antitumor activity
Tumor-associated macrophages belong to the [29]. Tumor-associated neutrophils (TAN) have
early inltrating leukocyte populations within received interest only recently, mainly due to
tumors, thus preceding lymphocytes, and are their short life span and the observation that
usually the most abundant immune population in tumor microenvironment can sustain and prolong
the tumor microenvironment [6, 15]. They derive the survival of polymorphonuclear leukocytes
from blood monocytes actively recruited from (PMN) [30, 31].
the circulation into tumor tissues. Early studies A particular small subset of TAMC is repre-
demonstrated that appropriately stimulated mac- sented by Tie2-expressing monocytes (TEM):
rophages are able to kill tumor cells in vitro; they express several monocyte/macrophage mark-
however, TAM, conditioned by the tumor micro- ers, along with the angiopoetin-2 receptor, Tie2,
environment, loose the cytotoxic capability and and are endowed with proangiogenic properties
rather exert several pro-tumoral functions, medi- [3235]. Tie2-expressing monocytes can be dis-
ating cancer-related inammation, angiogenesis, tinguished from the majority of TAM by their sur-
immunosuppression, tissue remodeling, and face marker prole (Tie2+, CD11b+) and their
metastasis [16, 17, 6]. preferential localization to areas of angiogenesis
The heterogeneous behavior of TAM is a [33], while they are largely missing in nonneo-
hallmark of myeloid cells and is oversimplied plastic area adjacent to tumors [35]. Indeed, Tie2
in a polarization concept with two extreme M1 is constitutively expressed at low levels by a sub-
and M2 phenotypes [1820] with distinct and stantial fraction (20 %) of circulating monocytes
somehow opposite functions. M1 macrophages and is overexpressed upon monocyte homing into
are classically activated by bacterial products growing tumors or regenerating tissues [33, 36].
and Th1 cytokines (e.g., LPS/interferon-). Following Ang-2 stimulation, Tie2+ monocytes
They are potent producers of inammatory and acquire an M2-like phenotype, with increased
immunostimulating cytokines, trigger adaptive expression of IL-10, CCL17, arginase 1 (Arg-1),
responses, secrete reactive oxygen species (ROS) and scavenger and mannose receptors and low
and nitrogen intermediates, and have cytotoxic expression of proinammatory molecules such as
effect towards transformed cells. On the other IL-12 and TNF- [37, 38].
hand, M2 macrophages or alternatively activated Myeloid-derived suppressor cells (MDSC) are
macrophages differentiate in response to Th2 a heterogeneous population of immature myeloid
cytokines (e.g., interleukin (IL)-4, IL-13) [21]. cells, having the ability to suppress T-cell func-
In contrast to their M1 counterpart, M2 macro- tions [39, 40]. They are derived from myeloid
phages produce growth factors, leading to tissue progenitors in bone marrows which do not differ-
repair and angiogenesis activation, have high entiate into mature granulocytes, macrophages,
scavenging activity, and inhibit adaptive immune or dendritic cells. MDSC have been isolated from
blood, spleen, and bone marrow of tumor-bearing immature phenotype (iDC) [5557]. The immature
mice and inltrate the tumor tissue, where local stage of TADC is responsible for the tolerogenic
tumor-associated factors promote their activa- response of adaptive immunity against tumors and
tion [41]. In tumor-bearing mice, two main strongly contributes to tumor immune evasion [58].
subsets of MDSC were identied: monocytic
MDSC (M-MDSC), characterized by CD11b+,
Ly6G, and Ly6Chigh, and granulocytic MDSC 2.2.2 Recruitment of Myeloid Cells
(G-MDSC), characterized by CD11b+, Ly6Ghigh, in Tumors
and Ly6C [42]. M-MDSC were shown to gov-
ern the ability of differentiating into monocytes TAMC derive from monocytes and granulocytes,
(macrophages) and (DC), whereas G-MDSC do extravasated from the circulation and inltrating
not possess this potential [43]. These subsets the tumor mass. Recruitment of blood cells into
are functionally different: M-MDSC-mediated tumors is mediated by chemoattractants released
immunosuppression is based on upregulation of by tumor and stromal cells. CC chemokine 2
inducible nitric oxide synthase (iNOS), expres- (CCL2), originally known as monocyte chemotac-
sion of Arg-1, and production of suppressive tic protein 1 (MCP1), was the rst relevant tumor-
cytokines, whereas G-MDSC-mediated immuno- derived chemotactic factor described [59, 60].
suppression is characterized by antigen-specic Several other chemokines attracting myeloid cells
responses (including ROS release requiring pro- have been identied, including CCL5, CCL7,
longed MDSC and T-cell contacts) [44]. Tumor- CCL8, and CXC chemokine 1 (CXCL1) and
associated MDSC generally exhibit an M2-like CXCL12 [6163]. Furthermore, urokinase plas-
phenotype, while M1 and M2 phenotypes could minogen activator (uPA); growth factors such as
coexist in some mouse tumor models [45, 46]. colony-stimulating factor (CSF)-1, transforming
Human MDSC are still poorly dened [47], growth factor (TGF-), basic broblast growth
even if they have been isolated from blood of factor (bFGF, also known as FGF-2), and vascular
patients with glioblastoma, colon cancer, breast endothelial growth factor (VEGF); and antimicro-
cancer, lung cancer, or kidney cancer [4852]. bial peptides (e.g., human beta-defensin-3) were
Recent studies have proposed that human MDSC shown to be involved in myeloid recruitment into
have a characteristic CD34+, CD33+, CD11b+, and neoplastic tissues [64, 9, 6567].
HLA-DR prole [42]. Similarly to the murine The prototypic chemoattractant for neutro-
counterpart, human MDSC are divided into two phils, CXCL8, is mainly responsible for the
main subsets: monocytic MDSC (M-MDSC), recruitment of TAN; other related chemokines of
characterized by the expression of CD14, and the CXC subfamily are also involved, including
granulocytic MDSC (G-MDSC), identied by CXCL1, CXCL2, and CXCL6 [68, 69]. Moreover,
positivity for CD15. tumor-derived TGF- can promote neutrophil
A small number of dendritic (DC) are found migration [70].
in most human and murine neoplasms. Similarly CC chemokine receptor 2 (CCR2), CCL2 recep-
to macrophages and neutrophils, plasticity is a tor, CXCL12, CXCL5, and stem cell factor (SCF,
main feature of these cells. DC are differentially also known as KIT ligand) play a pivotal role in the
localized in tumors; for example, in breast cancer recruitment of MDSC into tumors [7173]; in addi-
immature langerin+ DC are interspersed within tion, Bv8, also known as prokineticine 2 (PROK2),
the tumor mass, whereas more mature CD83+, might be essential for MDSC recruitment [74, 75].
DC-LAMP+ DC are conned to the peritumoral Finally, the proinammatory proteins S-100A9 and
area [53]. In contrast to TAM, tumor-associated S-100A8, produced by MDSC, are implicated in an
dendritic cells (TADC) were found in the invasive autocrine loop promoting accumulation of suppres-
front of papillary thyroid carcinoma [54]. Growing sor cells into tumors [76, 77].
evidences demonstrate that the majority of TADC TEM do not express CCR2 and are therefore
found within the tumor microenvironment have an recruited towards tumors by different mechanisms
[35, 78, 79]. Other CC chemokines, such as is driven by cytokines and other signals released
CCL3, CCL5, and CCL8, are produced by tumor in the tumor microenvironment [92]. Among
cells and could play a role in TEM recruitment these IL-10, IL-6, CCL2, CSF-1, and prostaglan-
[80]. Ang-2, overexpressed by tumor cells and din E2 (PGE2) were reported to promote M2-like
inamed tissues, has been shown to exert a che- polarization [93, 94]. TGF- is overexpressed by
motactic effect on Tie2-expressing blood mono- tumor cells and plays a crucial role in promoting
cytes in vitro, suggesting that the Ang-2/Tie2 axis an immunosuppressive phenotype, in addition to
might be involved in recruiting TEM into tumors driving N2 polarization of TAN [31].
[81, 32, 35, 34, 82]. In addition, recent data sug- Many tumor-derived factors were implicated
gest the involvement of the CXCL12-CXCR4 in MDSC expansion such as GM-CSF, M-CSF,
homing axis for TEM inltration [82]. IL-6, IL-1, VEGF, and PGE2 [44, 95]. In addi-
In recent years, it has been shown that tumor- tion, Bronte and coworkers recently found that
derived factors such as VEGF, CXCL12, CXCL8, cytokine-mediated induction of MDSC was com-
-defensins, and hepatocyte growth factor (HGF) pletely dependent on the transcription factor
are secreted into the bloodstream and are believed CCAT/enhancer-binding protein b (C/EBPb),
to attract iDC into the tumor bed [8386]. shown to function as a master regulator in this
Moreover, CCL20, CCL7, as well as the receptors process [96]. Further it was proposed that a com-
CCR5 and CCR6 were demonstrated to be impor- bination of at least two signals is necessary for
tant for TADC recruitment towards the tumor [87]. MDSC functionality and expansion, for example,
Proliferation can also contribute to sustaining GM-CSF, inhibiting maturation of myeloid cells,
TAMC levels in solid tumors. A paracrine loop and a proinammatory molecule such as
has been evidenced for TAM, with production of interferon- (INF-) [41].
colony-stimulating factor 1 (CSF-1) by murine Soluble factors released by tumor cells (i.e.,
brosarcoma cells acting on TAM-expressing IL-10, VEGF, TGF-, etc.) contribute to keep
CSF-1 receptor (CSF-1R) [88]. A nding con- DC in an immature pro-tumorigenic phenotype.
rmed more recently by Condeelis and Pollard Furthermore, in preclinical studies of breast
[89] showed the effect of epidermal growth factor cancer, it was shown that tumor-derived factors
(EGF) produced by TAM and tumor-derived altered DC maturation by secretion of thymic stro-
CSF-1 on recruitment and survival of macro- mal lymphopoietin (TSLP), which in turn induces
phages during tumor growth. Indeed, macro- the expression and secretion of the OX40 ligand,
phage proliferation has been demonstrated to a molecule that contributes to sustain the M2-like
occur during type II inammation [90]. phenotype of TAM.
2.2.3 Tumor-Derived Factors 2.3 Pro-tumoral Functions

Affecting Myeloid of Tumor-Associated
Differentiation and Polarized Myeloid Cells
Functions
Myeloid cells exposed to the tumor microenviron-
Upon arrival in the tumor, monocytes differenti- ment most frequently promote tumor progression.
ate to macrophages primarily in response to They can secrete soluble factors which support
CSF-1 produced by tumor cells. Although coex- proliferation and invasion of tumor cells, activate
istence of diverse TAM subpopulations with dis- angiogenesis, and promote resistance to therapies
tinct functions depending on tumor stage and (Fig. 2.2). High TAM or TAN inltration generally
geographical localization within the same tumor correlates with poor patient outcome [97, 6, 16,
has been proposed, they mostly have an M2-like 11, 98101], but few exceptions to this nding are
phenotype [91]. Many different studies demon- also reported. For instance, in colorectal cancer
strated that M2 (pro-tumoral) TAM polarization (CRC) contrasting results reported that TAM
TAM Th1; Th2; Treg

lymphocytes
TAN
Tumor
TADC
TEM
Tumor cell
MDSC
Immunosuppression Matrix degradation Angiogenesis

remodeling
Tumor survival Local invasion Resistance to

proliferation metastasis therapies
Fig. 2.2 Pro-tumoral functions of tumor-associated the release of soluble factors (i.e., cytokines, growth and
myeloid cells. TAMC exposed to the tumor microenviron- proangiogenic factors, proteolytic enzymes, etc.) and result
ment exert several pro-tumoral functions, including promo- in higher tumor survival and proliferation, local invasion
tion of angiogenesis, matrix degradation, and suppression and dissemination, resistance to therapies
of adaptive immunity. These effects are mediated through
density is associated with positive or negative and CD86) was associated with better patient sur-
patient outcome [102, 103105]. On the same line, vival in CRC, whereas the same cell population
TAN inltrate is associated with a favorable prog- within the tumors did not have any predictive
nosis in patients with gastric carcinomas [106], but value [109, 110]. Thus, TAMC exert complex
also with more aggressive pancreatic tumors roles on growing tumors affecting different aspects
[107]. Macrophage subsets might have distinct of tumor progression, i.e. tumor cell proliferation
roles, as observed in lung adenocarcinoma were and survival, angiogenesis, tumor dissemination,
the number of CD204+ TAM showed a strong and resistance to therapies.
association with poor patient outcome, while the
CD68+ TAM population did not [108]. The con-
cept that not only the number and the presence of 2.3.1 Tumor Proliferation
specic cell subsets but also the localization of and Survival
inltrating cells might have specic functions and
predictive values is increasingly emerging. TAM were shown to have the ability to promote
Accordingly, peritumoral TAM density with high tumor growth directly through the production of
expression of co-stimulatory molecules (CD80 trophic and activating factors for stromal and
cancer cells (EGF, bFGF, VEGF, platelet-derived The prototypic myeloid cell with angiogenic
growth factor [PDGF], TGF-) [111, 112, 6, properties is the Tie2 monocyte [32, 35]. TEM
113] in response to stimuli from the tumor micro- can be found in close proximity to nascent blood
environment. For example, IL-13 and IL-4 pro- vessels within solid tumors. In addition, TEM
duced by CD4+ T-cell-inltrating tumors, such as depletion completely prevented neovasculariza-
breast cancer, led to the production and secretion tion in preclinical models (spontaneous pancre-
of EGF by TAM [114]. Moreover, production of atic adenocarcinoma, human glioma grown
proinammatory cytokines, including TNF- orthotopically in the mouse) [33]. Interestingly,
and IL-6, by TAM and other cells of the tumor TEM ablation did not affect the number of inl-
microenvironment (e.g., epithelial cells), sustains trating TAM or TAN, suggesting that TEM are an
tumor growth and inhibits apoptosis [115119]. entity on their own and not just precursors of
Several lines of evidence suggest that TAN are TAM [35]. How TEM stimulate angiogenesis has
required for the rapid growth of tumor cells and not been claried yet, but preliminary indications
their depletion inhibits tumor development [120, in murine tumor models point to the fact that
28]. Proteins stored within neutrophil granules perivascular TEM secrete bFGF. It is believed
(e.g., elastase) may have a role in tumor initiation that release of such factors in close proximity to
[121]. In addition, neutrophil-derived ROS have vessels could directly stimulate angiogenesis or
been associated with DNA damage [122]. TAN MMP9 secretion, which in turn would release
were shown to be able to produce soluble factors growth factors entrapped within the extracellular
(cytokines and chemokines, HGF, oncostatin M), matrix (ECM).
driving processes like angiogenesis, wound heal- TAM have also a profound inuence on the
ing, and hematopoiesis and thus exerting a role in regulation of tumor angiogenesis [129]. It was
tumor promotion and growth [123125, 121, demonstrated in several preclinical studies that
101]. For instance, HGF released by neutrophils TAM positively correlated with microvascular
enhances the invasiveness of human cholangio- density (MVD) [130133]. Lin and coworkers
cellular and hepatocellular carcinoma cells were the rst to describe the direct role of TAM
in vitro, and HGF levels in bronchoalveolar in driving the angiogenic switch in a spontane-
lavage uids were found to correlate with neutro- ous mammary carcinoma mouse model [134].
phil number in patients with bronchoalveolar car- Likewise, depletion of monocytes by clodronate
cinomas, which further correlates with poor treatment in a preclinical model with Lewis lung
patient prognosis [101]. carcinoma led to lower TAM inltration and
angiogenesis, further underlining the importance
and the involvement of macrophages in tumor
2.3.2 Angiogenesis angiogenesis [135].
TAM express various molecules modulating
To sustain the increased metabolic demand of angiogenesis, such as VEGF, bFGF, TNF-,
growing tumors, the development of a tumor vas- IL-1, CXCL8, cyclooxygenase 2 (COX2, also
culature is required. VEGF is the primary, but not known as PTGS2), plasminogen activator, uPA,
the only, angiogenic factor released by tumor cells PDGF-, MMP7, MMP9, and MMP12 [136].
and is involved in the angiogenic switch that Hypoxia exerts a crucial role in the upregulation
can occur at various stages of tumor progression, of gene transcription in TAM, promoting VEGF
depending on the tumor type and the microenvi- expression [137141]. Other recent studies
ronment. Other factors are involved, including showed a direct involvement of TAM in tumor
PDGF-, bFGF, angiopoietins, and CXCL12 angiogenesis and neovascularization via transdif-
(SDF-1) [126]. Tumor-associated myeloid cells ferentiation into endothelial cells when stimu-
were shown to contribute to tumor angiogenesis lated by angiogenic factors [142, 143].
by production of growth factors, cytokines, and More recent studies have shown that MDSC
proteases [80] such as VEGFA, Bv8, and metal- can contribute to tumor angiogenesis. In a pre-
loproteases (MMP) [10, 127, 65, 128]. clinical model for colon cancer, MDSC positively
correlated with tumor growth rate and blood ves- DC maturation and further promote angiogenesis
sel density [144]. Moreover, tumor angiogenesis via this autocrine loop [153, 151].
was signicantly lowered by blocking Bv8 with a
neutralizing antibody, a treatment that signi-
cantly reduced the number of MDSC [74]. 2.3.3 Cancer Cell Dissemination
Metalloproteases, particularly MMP9, MMP2,
MMP13, and MMP14, produced by MDSC, were The major cause of death in cancer results from
shown to enhance VEGF bioavailability by mobi- therapy-resistant metastases. Stephen Pagets
lization from the ECM [144, 145]. Increased conclusion in the late nineteenth century that the
recruitment of MDSC has also been demonstrated metastatic process depends on cross talk between
in the presence of hypoxia, possibly stimulating selected cancer cells (the seeds) and a specic
tumor angiogenesis [126, 74]. Parallel to TEM, organ microenvironment (the soil) is still valid
MDSC were also observed to be localized in the and is experimentally conrmed [154, 155].
vicinity of blood vessels. Under certain condi- Tumor metastasis is a complex multistep process,
tions, some MDSC acquire endothelial cell shape, during which malignant cells spread from the pri-
start to express endothelial markers including mary tumor site to secondary distant organs. The
CD31 and VEGFR2, and are eventually incorpo- different steps of cancer cell dissemination can be
rated into the tumor endothelium [144]. subdivided into local invasion, entry into the
TAN were shown to rapidly release VEGF bloodstream (intravasation), survival in the blood-
from internal storage compartments, leading to stream, extravasation, and colonization [156].
endothelial proliferation and tubule formation Mesenchymal, endothelial, and immune cells are
[146, 147]. In addition, TNF- and GM-CSF required to form an appropriate microenviron-
secreted by tumor cells were shown to trigger the ment for tumor progression [157]. Immune cells,
release of proangiogenic chemokines by particularly macrophages, neutrophils, T lympho-
TAN. The number of TAN in myxobrosarcoma cytes, and natural killer (NK) cells, are major
positively correlated with tumor MVD [148]. sources of proteases that degrade the host tissue,
Furthermore, in a xenograft mouse model of allowing cancer cells to disseminate.
human melanoma where cancer cells were engi- The set of proteolytic enzymes found in tumor
neered to constitutively produce CXCL6, it was microenvironment comprises matrix metallopro-
found that the number of TAN as well as angio- teases, serine proteases, and cysteine proteases
genesis was markedly increased [149]. Studies in (i.e., cathepsin) [158162]. Matrix proteases exert
the RIP1-TAG2 mouse model for pancreatic car- essential functions in physiological conditions as
cinogenesis revealed formation of dysplastic, active regulators of postnatal tissue development
neutrophil-bearing, angiogenic islets upon malig- and remodeling. In addition, they are important
nant transformation. In the abovementioned for tissue repair in response to injury and regulate
model, neutrophil depletion of the islets led to cancer progression modulating the tumor micro-
dramatically lowered angiogenesis [150]. environment, particularly the leukocyte inltrate
In recent years, it has become more and more [163]. MMP were shown to activate TGF-,
apparent that iDC make a profound contribution which is an important regulator of T-cell and TAN
to tumor angiogenesis [85]. TNF- and CXCL8 functions [164]. Proteases also produce specic
produced by iDC from ovarian cancer ascites cleavage fragments of target chemokines with
triggered the release of various growth factors independent biological activity, ranging from
from EC [85, 151]. Moreover, iDC were shown anergic products (CXCL7, CXCL4, CXCL1),
to release osteopontin which promotes monocyte antagonists (CCL7), or more potent chemoattrac-
secretion of the proangiogenic IL-1 [152]. tants (CXCL8), thereby modulating the leukocyte
Finally, it was recently observed that iDC pro- composition within a tumor [165167].
duced high levels of VEGF and CXCL8 under Besides their inuence on the tumor inltrate,
hypoxic conditions, which, in turn, might inhibit proteases were shown to promote cancer cell
invasion and intravasation. The cleavage of cell- It has been suggested that tumor cells can
adhesion molecules like E-cadherin induces the inuence the microenvironment of secondary
disruption of cell-cell junctions leading to loos- organs promoting the formation of a pre-
ening of cell-cell contacts which, together with metastatic niche [174, 175]. Tumor-derived fac-
ECM protein turnover, facilitated cancer cell tors and HSC are crucial components of the
migration and invasion into the surrounding tis- pre-metastatic niche. VEGF derived from tumor
sue and vasculature. Tight regulation of the sin- cells promote recruitment to the secondary organs
gle proteases within the tumor microenvironment of VEGFR1-expressing HSC that induce bro-
allows the control of tumor cell invasion [168]. nectin and MMP9 expression by resident bro-
After invasion to the surrounding tissues, can- blasts, creating favorable conditions for
cer cells enter the blood circulatory system directly settlement of future metastases [176]. Other solu-
or indirectly via the lymphatic system. Since the ble factors released by tumor cells can promote
majority of circulating tumor cells (CTC) are the formation of pre-metastatic niche. In a murine
eliminated by NK cells [169], only about 0.01 % model of breast cancer, tumor cells were found to
of CTC survive in the bloodstream [157]. Platelets induce production of CCL17 and CCL22 in the
play a key role in hematogenous metastasis and lung; both attracting CCR4+ tumor and immune
contribute to the survival of CTC in the blood- cells which establish a microenvironment for
stream by both thrombin-dependent and thrombin- metastases settlement at secondary organs [177].
independent mechanisms [170]. After a passage Moreover, it was demonstrated that the proto-
into the bloodstream, CTC adhere to vessel walls typic hypoxia-induced protein lysyl oxidase
for extravasation when they are in the vicinity of (LOX), often found in tumors, leads to cross-
secondary metastatic organs. Circulating tumor linking of collagen IV in basement membranes,
cells take advantage of the capability of neutro- in addition to recruitment of CD11b+ myeloid
phils and platelets to produce and secrete adhesion cells which adhere to the abovementioned colla-
molecules, such as integrins and selectins which gen meshwork. The captured CD11b+ myeloid
all aid the nearby CTC to adhere and ultimately cells were shown to secrete MMP2, which facili-
extravasate [170, 171]. tated invasion and recruitment of metastasizing
The arrest of cancer cells to specic organs tumor cells [178].
seems to be primarily mechanical [172]. TAMC, TAM and MDSC in particular, are
However, chemokines and chemokine receptors important players of tumor progression and met-
are also involved in organ-specic colonization, astatic colonization through the cross talk with
which nally drive cells along tissue-specic tumor cells. For instance, macrophages play a
chemokine gradients. Furthermore, a non- crucial role in conferring an invasive phenotype
chemokine pathway also exists, in which immune to epidermal keratinocytes from Snail transgenic
cells support organ-specic cancer cell dissemi- mice [179]. TAM contribute to cancer cell dis-
nation. One example is represented by the two semination by releasing enzymes involved in
inammatory mediators S100-A8 and S100-A9, degradation of the ECM (i.e., MMP and cathep-
which were shown to promote metastasis through sin) [168, 161, 180, 76], or motility factors.
serum amyloid A 3 (SAA-3) [173]. Recently we found that tumor-derived soluble
The subsequent growth of arrested tumor cells factors, particularly CSF-1, activate a transcrip-
will depend on the molecular interactions tion program in macrophages resulting in upreg-
between cancer cells and the microenvironment ulation of a series of genes, especially
of the new organ. Although cancer cells are migration-stimulating factor (MSF). MSF is a
sometimes said to home to specic organs truncated isoform of human bronectin 1, physi-
(e.g., breast tumors metastasizing to bone), it is ologically expressed during fetal life and upregu-
more likely that this organ specicity is due to lated in M2-like macrophages [181, 182]. MSF
efcient organ-specic growth rather than prefer- exerts a chemotactic effect on tumor cells, indi-
ential homing of cells to a particular organ. cating that macrophage products released in the
tumor microenvironment can support the pro- sive phenotype, with production of high levels of
invasive phenotype of tumor cells [181]. An the immunosuppressive cytokines IL-10 and
example of the cross talk between TAM and TGF- and reduced expressions of IL-12 [19,
tumor cells involved in metastatic colonization is 186, 92, 187, 188]. In addition, they have reduced
shown in breast cancer, where EGF secreted by tumoricidal activity and are poor in antigen pre-
TAM increases migration and invasion of neigh- sentation [189]. Furthermore, TAM secret che-
boring breast cancer cells which express high mokines, such as CCL17 or CCL22, that
levels of EGF receptor (EGFR). On the other preferentially attract Th1, Th2, and T regulatory
hand, cancer cells secrete high levels of CSF-1, a (Treg) lymphocytes with defective cytotoxic
main chemoattractant for TAM which expresses functions, or such as CCL18, that recruit nave
the cognate receptor CSF-R1. Therapies aiming T cells which become anergic in contact with
at inhibiting this cross talk by blocking CSF-R1 M2 macrophages and iDC [8, 190192].
and/or EGFR were shown to be successful [183, MDSC play a prominent role in the inhibition
184]. Macrophages and their reciprocal cross talk of tumor-specic immune responses. MDSC
with tumor cells are mandatory for tumor cell localized within the tumor microenvironment has
migration, regardless of the factor inducing cell an M2-like phenotype and mediate immunosup-
invasion (i.e., SDF-1). pression through multiple pathways, that is, pro-
A myeloid cell population involved in tumor duction of Arg-1 [193], iNOS [194, 195], ROI,
progression, including invasion, is represented by and suppressive cytokines including IL-10 and
MDSC. A direct role for MDSC in tumor metasta- TGF- [196], or via the activation and recruitment
sis has not been demonstrated; however, a connec- of Treg [196, 197]. MDSC inhibit homing to
tion was suggested by the study on mice decient lymph nodes of CD4+ and CD8+ T cells and sup-
for the TGF- receptor type 2 (TGF--R2), in which press their activation [198, 199]. It was found that
MDSC were concentrated on the invasive margin. cysteine uptake by MDSC limited its availability
In addition, it is possible to reduce lung metastases for uptake by T cells, which in turn disables their
by antagonizing CXCR2 and CXCR4, two recep- activation and renders them nonfunctional.
tors involved in homing of MDSC [145]. As previ- Furthermore, it was shown that posttranslational
ously mentioned, PGE2 and the proinammatory T-cell receptor modications mediated via gener-
molecule S100A9 have been identied as main ation of peroxynitrite species led to anergy of
effectors of MDSC accumulation and function. effector CD8+ T cells [196]. MDSC can also
Accordingly, S100A9 decient mice rejected impair innate immunity through cross talk with
implantation of colorectal cancer, while administra- macrophages which led to decreased production
tion of wild-type MDSC reverted the phenotype of IL-12 by macrophages and increased produc-
and colorectal cancer cells could successfully tion of IL-10 by MDSC, thus driving a polariza-
engraft [76]. In addition, TGF- was demonstrated tion towards an M2-like phenotype [200].
to be instrumental in MDSC homing, mediated via In addition to the above described mecha-
CXCL12-CXCR4 and CXCL5-CXCR2 axis in a nisms in TAM and MDSC, TADC were found to
preclinical mammary cancer model [145]. be involved in suppression of adaptive immunity.
One mechanism leading to the induction of
tumor-specic T-cell tolerance was via upregula-
2.3.4 Suppression of Adaptive tion of inhibitory molecules such as B7-H1 [201]
Immunity or by inducing the expression of Arg-1 [202].
Moreover, it was shown that the induction of
Besides the effect on tumor growth and dissemi- oxygen-dependent pathways led to the downreg-
nation, TAMC have also the potential to suppress ulation of CD3 epsilon and T-cell apoptosis
the adaptive immune response, leading to cancer [203]. Furthermore, Muller and coworkers dem-
immune evasion [185]. onstrated that upregulation of indoleamine
M2-like polarized tumor-inltrating macro- 2,3-dioxygenase (IDO) in TADC contributed to
phages are characterized by an immunosuppres- immunosuppression [204].
2.4 Selected Aspects approach to affect TAM specically is to try to

of Therapeutic Targeting reeducate them to become tumoricidal or, in
of TAMC terms of polarization, to try to repolarize them
towards an M1 phenotype. Several successful tri-
The above summarized data describing the pro- als using CpG-oligodeoxynucleotide (TLR9 ago-
tumoral role of the myeloid inltrate of tumors nists) were performed in combination with
make clear that TAMC are reasonable targets for anti-IL-10 receptor or anti-CD40 antibodies,
novel therapeutic approaches. As illustrated which reverted pro-tumoral M2-like TAM to M1
above, TAMC can directly promote tumor cell macrophages displaying antitumor activity [216
growth releasing growth factors and proangio- 218]. Rolny et al. recently demonstrated that
genic molecules, in addition to suppression of skewing of M2 TAM towards M1 leads to effec-
tumor-specic immune responses. Strategies tive antitumoral activity of host histidine-rich
explored in the last years are focused on the stop- glycoprotein (HRG), which in consequence leads
page of the mechanisms leading to suppression to inhibition of angiogenesis and promoted anti-
of lymphocyte activity and, on the other side, on tumor immune responses [219]. Gazzaniga and
the reduction of recruitment of myeloid cells and coworkers reported promising results using the
repolarization of M2-like pro-tumoral cells to molecule legumain, which targets M2 polarized
proinammatory M1 macrophages. There is a TAM specically, and was able to induce a robust
wide range of preclinical and clinical research CD8+ T-cell answer leading to reduced tumor
aimed at eliminating or reprogramming TAMC growth and inhibition of tumor angiogenesis
[39]: here we only mention some examples of the [220]. Furthermore, it was shown that zoledronic
results obtained so far in this growing eld of acid was able to revert M2 towards M1 TAM and
anticancer research. inhibit breast carcinogenesis by targeting the
Many studies have shown that targeting TAM mevalonate pathway [221]. Moreover, it was
might be a successful strategy to limit tumor demonstrated that direct reeducation of TAM
growth and metastasization and to achieve bet- using the prototypical M1 polarizing cytokine
ter therapeutic responses [32, 44, 59, 82, 189, INF- [222] is successful in promoting antitumor
205, 206, 207]. One example is represented by activity in minimal residual disease [8]. In line
bisphosphonates [208] traditionally used in the with the abovementioned results are the ndings
clinic to treat osteoporosis, which were shown to that inhibition of M2 polarization led to restora-
be very effective in depleting TAM and inhibit- tion of M1 proinammatory phenotype and inhi-
ing angiogenesis as well as metastatic spread bition of tumor growth in several preclinical
in preclinical animal models for breast cancer animal models [92, 223, 224].
[209, 210]. Furthermore, Germano and cowork- To counteract the pro-tumoral activities of
ers recently showed that specic targeting of MDSC, two general strategies can be envisaged;
macrophages with the marine antitumor agent the rst consists of transforming these immature
trabectedin was very successful in four different cells into mature cells devoid of suppressive
preclinical tumor animal models [211]. activity, and the second is focused on blocking
An alternative strategy is to target circulating MDSC suppressive functions. Depletion of
monocytes known as precursors of TAM. Two MDSC producing high levels of TGF- (in an
candidate molecules are the M-CSF receptor IL-13-dependent manner) led to the restoration
(solely expressed by monocyte-macrophages) of T-cell-mediated immunosurveillance in a pre-
and the chemokine CCL2, involved in monocyte clinical mouse model for brosarcoma [225].
recruitment within tumors. Since preclinical Several studies have shown that metabolites of
studies on prostate and colon cancer [212215] all-trans-retinoic acid are able to differentiate
identied CCR2+Ly6C+ cells as targets involved MDSC into DC and macrophages, reducing
in cancer progression and metastasis, CCL2 anti- MDSC accumulation [226, 227]. This effect was
bodies are currently investigated for therapeutic demonstrated to be benecial for patients suffer-
applications in human cancer treatment. Another ing from metastasizing renal cancer, since in
these patient less circulating MDSC were was demonstrated that delivery of regulatory
detected in the bloodstream [228]. Furthermore, miRNA, particularly miRNA 155 in a nanoparti-
one of the benecial effects of the anticancer cle formulation, leads to reprogramming of
drug gemcitabine is its potential to eliminate immunosuppressive TADC to highly active anti-
MDSC without affecting T, B, NK cells, or mac- tumoral TADC which provoked regression of
rophages [229]. established ovarian tumors [236].
The second possibility to counteract MDSC In light of the recent results, tumor therapy
function is to block their inhibitory function, for with drugs targeting the inammatory tumor
example, by using COX2 inhibitors, phosphodi- microenvironment in combination with treatment
esterase (PDE5), and nonsteroidal anti- aimed at defeating TAM, TAN, and other myeloid
inammatory drugs releasing NO [44]. Blocking cells holds promise for the future.
of IL-1 inhibits cancer progression and metasta-
sis [230] and decreases MDSC accumulation and
suppressive activity [42]. Moreover, the proan- 2.5 Concluding Remarks
giogenic chemokine Bv8 was shown to be impor-
tant for mobilization and homing of MDSC to In recent years, it has become clear that inam-
tumor sites and therefore qualies as an interest- mation has an essential role in tumor promotion
ing therapeutic target [74]. [16]. The inammatory tumor microenviron-
Complete neutrophil depletion in already ment, mainly consisting of soluble factors and
immunocompromised patients is not desirable; host cells, has a predominant role in all aspects
therefore, the strategy of choice concerning TAN of the disease (progression, angiogenesis,
might be to disturb their tumor homing ability, in immune surveillance). In particular, a heteroge-
other words to interfere with their ability to neous group of myeloid cells is the most consis-
migrate. To this purpose, preclinical experiments tent host cell component of solid tumor [8, 9].
using anti-CXCR2 antibodies were performed TAM, TEM, MDSC, TAN, and TADC display
and were shown to be successful [231]. distinct specialized functions, as well as overlap-
Furthermore, considering the well-documented ping activities (e.g. angiogenesis). Tumor and
key role of TGF- in skewing TAN towards a N2 stromal cells release different chemoattractants
phenotype, this cytokine keeps promising poten- involved in the recruitment of myeloid cells from
tial for treatment [70, 31]. the blood into the growing tumor. Cytokines and
Some studies indicate that blocking IL-10 other soluble factors released in the tumor micro-
together with the administration of CpG oligo- environment can contribute to induce a protu-
nucleotides are able to unblock the functionally moral phenotype, promoting M2 polarization of
paralyzed TADCs and to reactivate antitumor TAM [92], N2 polarization of TAN [31], MDSC
responses [232]. Another strategy enhancing expansion [41], or preventing maturation of
immunotherapy might be targeting of soluble DCs. Thus the different TAMC populations
factors like VEGF, IL-10, TGF-, gangliosides, potentially represent a target for new therapeutic
and others, which are all tumor secreted factors approaches aimed at breaking the protumoral
leading to abnormal differentiation of DC, often networks established by cancer-associated
leaving them in an immature state [233]. Other myeloid cells.
and more recent strategies make use of siRNA
nano-complexes which lead to reprogramming of Acknowledgments The authors would like to gratefully
TADC from an immunosuppressive to an acti- acknowledge the nancial supports of the European
vated anticancer phenotype [234]. Furthermore, Research Council (ERC project HIIS), the European
it was shown that in situ stimulated CD40 and Commission (FP7-HEALTH-2011-ADITEC-280873),
the Italian Association for Cancer Research, the Italian
toll-like receptor 3 (TLR3) TADC were success-
Ministry of Health and University, Fondazione CARIPLO
fully transformed from immunosuppressive to (project 20092582), and Regione Lombardia (project
immunostimulatory cells [235]. More recently it Metadistretti SEPSIS).
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Role of Innate Immunity in Cancers
and Antitumor Response 3
Masahisa Jinushi and Muhammad Baghdadi
Contents 3.4 The Role of Effectors Produced

from Innate Immune Cells
3.1 Introduction ................................................ 29 in Cancer and Antitumor Immunity ........ 37
3.2 Role of Innate Immune Cells 3.4.1 Interferons (IFNs)......................................... 37
in Cancer and Antitumor Immunity ........ 30 3.4.2 Other Cytokines ........................................... 38
3.2.1 Natural Killer (NK) Cells ............................. 30 3.4.3 Chemokines .................................................. 39
3.2.2 Natural Killer T (NKT) Cells ....................... 31 3.5 Concluding Remarks ................................. 40
3.2.3 -T Cells ..................................................... 31
3.2.4 Macrophages ................................................ 31 References ............................................................... 40
3.2.5 Dendritic Cells ............................................. 32
3.2.6 Granulocytes................................................. 32
3.3 The Role of Innate Immune Receptors
on Innate Immune Cells in Cancer 3.1 Introduction
and Antitumor Immunity .......................... 32
3.3.1 Toll-Like Receptors (TLRs) ......................... 32 Cellular components of the innate immune system
3.3.2 RIG-I-Like Helicases (RLHs) ...................... 33
serve as a rst line of defense against tumori-
3.3.3 NOD-Like Receptors (NLRs) ...................... 33
3.3.4 Phagocytosis Receptors ................................ 33 genic cells. Recognition of transformed cells by
3.3.5 C-Type Lectin-Like Receptors (CLRs) ........ 34 pattern-recognition receptor (PRRs) on the innate
3.3.6 NK Cell Receptors ....................................... 34 immune cells activates specialized inammatory
3.3.7 B7 Family ..................................................... 37
signaling cascades, including transcription factor
nuclear factor-kappa B (NF-B) and interferon
regulatory transcription factor (IRF), which lead
to the release of various cytokines and chemo-
kines attracting and activating effector lympho-
cytes at the tumor site. In addition, effector cells
kill transformed cells through the activation of
perforin or death receptor-mediated pathways,
M. Jinushi, MD, PhD (*)
Research Center for Infection-Associated Cancer, as well as secretion of cytokines necessary
Institute for Genetic Medicine, Hokkaido University, for the initiation of immune responses against
Kita-ku, Kita 15, Nihi 7, Sapporo 060-0815, Japan transformed cells [1, 2]. However, some tumor
e-mail: Jinushi@igm.hokudai.ac.jp cells escape from the innate immune machinery,
M. Baghdadi, MD, PhD which leads to the dysfunction of innate immune
Division of Immunobiology, compartment, signaling pathways, and effector
Institute for Genetic Medicine, Hokkaido University,
Kita 15, Nishi 7, Sapporo 060-0815, Japan functions. This manipulation of innate immune
e-mail: drmhb@igm.hokudai.ac.jp systems by tumor microenvironments includes

30 M. Jinushi and M. Baghdadi
impairment of antigen processing and presen- NK cells have the ability to distinguish trans-
tation by antigen-presenting cells (APCs) [3], formed cells from normal cells by recognizing a
inhibition of innate immune signaling pathways variety of cell surface receptors, including killer
[4, 5], and anti-inammatory cytokines such as activation receptors (KARs), killer inhibitory
IL-10 and transforming growth factor- (TGF- receptors (KIRs), natural killer group two mem-
) [6, 7]. Moreover, tumors manipulate innate ber D (NKG2D), DNAX-accessory molecule
immune systems to create protumorigenic envi- (DNAM), etc., which will be discussed later in
ronments, which lead to further tumor progres- this chapter. For example, KIRs on NK cells has a
sion and metastasis. Therefore, it is critical high afnity to the specic alleles in HLA class I
to clarify the molecular mechanisms through molecules, transducing an inhibitory signal to the
which the interaction between tumors and innate NK cells and preventing it from eliminating non-
immune systems is modied during different transformed cells. However, deletion of a single
phases of tumorigenesis. allele in HLA class I and/or induction of activat-
In this chapter, we describe the general func- ing receptors such as NKG2D ligands, which fre-
tions of innate immunity in cancer and antitumor quently occurs on transformed cells, triggers
host response. In addition, an overview is pro- effector functions of NK cells against tumor cells
vided on the mechanism through which coor- [10, 16]. Recent studies have focused on licens-
dinated actions of innate immune signals and ing NK cells to become functionally competent
their downstream effectors have an impact on through the interaction with self-MHC mole-
the immunosurveillance and immune subversion cules. Ly49C is an inhibitory receptor expressed
within the tumor microenvironment. on a subset of NK cells, which interact with self-
MHC molecules on target cells, and plays an
unexpected role in enabling immature NK cells to
3.2 Role of Innate Immune Cells develop into functioning, mature cells. On the
in Cancer and Antitumor other hand, Ly49C-negative NK cells are consid-
Immunity ered as non-licensed and remain at an imma-
ture stage [17]. These evolutionary processes of
3.2.1 Natural Killer (NK) Cells NK cell development and activation may help
explain why donor NK cells administrated to leu-
NK cells are important effector cells for protec- kemia patients during bone marrow transplanta-
tion against viruses and some tumors, since NK tion do not always show antitumor effects [18].
cell-depleted mice were more susceptible to The NK cell-mediated cytotoxic activities medi-
3-methylcholanthrene (MCA)-induced tumors ate the release of granule contents (perforin and
[8]. Chemokines such as CXCL12 and granzyme) onto the surface of the tumor cell [19].
CXCL3L1 are key factors for NK migration to The interaction between NK cells and dendritic
tumor sites [9], where they play an important cells (DCs) is crucial for the amplication of
role in the tumor immunosurveillance [10]. NK innate responses and the induction of potent adap-
cells recognize and eliminate transformed cells tive immunity. Immature DCs are susceptible to
by releasing perforin or death signal-associated NK cell-mediated cytolysis [20], while mature
receptors such as FAS and TRAIL (tumor DCs are activated by NK cells through cytokines
necrosis factor-related apoptosis-inducing (TNF- and IFN-) and receptor (NKp30 and
ligand) [1113]. NK cells secrete interferon NKG2D)-mediated mechanisms [21, 22]. On the
gamma (IFN-) which helps to activate T-cell- other hand, activated DCs trigger effector activi-
mediated immunity and suppress tumor angio- ties of NK cells, such as IFN- production, prolif-
genesis [14, 15]. Moreover, various innate eration, and cytotoxic activities [23]. In addition,
immune networks such as cytokines and PRR treatment with TLR3 agonist polyinosinic-poly-
recognition systems play an important role in cytidylic acid (Poly (I: C)) triggers DCs to activate
stimulating effector functions of NK cells as dis- antitumor activities of NK cells [24, 25]. Thus, the
cussed later. reciprocal interaction between NK and DC
3 Role of Innate Immunity in Cancers and Antitumor Response 31
regulates the direction and quality of antitumor 3.2.3 -T Cells

immunity, which is important for the development
of effective cancer immunotherapy. Although -T cells represent a small popula-
tion among T lymphocytes, they share several
features with innate immune cells. -T cells
3.2.2 Natural Killer T (NKT) Cells show high frequencies in intraepithelial lym-
phocytes (IELs) in the skin and gut mucosa and
NKT cells are innate lymphocytes which share possess a distinct T-cell receptor on their sur-
features of both NK cells and T cells. NKT cells face with limited diversity, which may serve as
express particular NK cell markers such as a pattern-recognition receptor [35]. Moreover,
CD161 or NKR-P1, in addition to an invariant -T cells lack CD4 and CD8 expressed by
T-cell receptor alpha chain (V14-J18 in mice -T cells and express a number of molecules
and V24-J18 in humans) [26]. The invariant shared with NK cells or APCs, such as Fc
T-cell receptor alpha chain is specic for glyco- gamma RIII/CD16 and PRRs. -T cells also
lipid antigens presented by CD1d, which is an recognize lipid-derived antigens and function
MHC class I-related molecule expressed on as professional phagocytes which recognize
antigen-presenting cells and also found in some and ingest apoptotic tumor cells and may inu-
tumor cells. NKT cells were shown to play a role ence antitumor immune responses [36, 37].
in the tumor immunosurveillance, since J18/ Mice lacking -T cells showed increased
mice showed increased susceptibility to chemi- incidence of chemically induced sarcoma and
cally induced tumors and experimentally induced spindle cell carcinoma, indicating the importance
metastases [27]. Moreover, the administration of of these cells in tumor immunosurveillance [38].
-galactosylceramide, a natural lipid isolated In addition, -T cells express NKG2D receptors
from marine sponges which efciently binds to and interact with their ligands on transformed
CD1d and thus activates NKT cells, induces anti- cells, leading to enhanced cytotoxic activities and
tumor immune responses against established effector cytokine production [39, 40]. The acti-
murine tumors [28]. The antitumor activities of vated -T cells then serve as the major early
NKT cells are mediated by IFN- production, source of IFN-, which contribute to maturation
which also activates NK and CD8+ T cells. NKT of APCs and prime -T cells, and mediate cyto-
cell activities are also important for granulocyte toxicity against tumor cells [40, 41].
macrophage colony-stimulating factor (GM-CSF)
and IL-12-based cytokine strategies [29, 30].
Recent reports have identied subpopulations of 3.2.4 Macrophages
NKT cells which secrete TH1 or TH2 cytokines
and thus play different roles in the pathogenesis Macrophages serve as a rst line of defense
of many diseases. For example, CD4 NKT cells against tumorigenesis by directly killing tumor
serve as potent effectors for triggering tumor cells and producing various antitumor mediators
rejection in various murine tumor models, while [42]. On the other hand, macrophages render
CD4+ NKT cells contribute to the pathogenesis of tumor cells with the ability to acquire invasive
allergic diseases and tumors by promoting the and metastatic activities [43]. Macrophages are
release of IL-4, IL-5, and IL-13 [31, 32]. Indeed, differentiated from immature myeloid precursors
IL-13 released from NKT cells antagonizes or circulating monocytes released from the bone
tumor immunosurveillance by promoting TGF- marrow [44]. In particular, the inammatory
secretion from Gr-1+ myeloid suppressor cells monocytes expressing Ly6C are preferentially
[33, 34]. Thus, the identication of factors inu- attracted from the circulation into the tumor site
encing the differentiation of specic NKT cell by tumor-derived chemokines, such as CCL2
subsets during tumor development is important in (MCP1-1) and CCL5 (RANTES) and CXCL12
order to optimize the therapeutic interventions (SDF1) [4547]. Immature monocytes are then
which utilize NKT cell functions against tumors. differentiated into either M1 or M2 macrophages
by distinct sets of cytokines when entered into immune response cascades against tumors [60].
distinct tumor microenvironments [48]. M1 mac- Granulocytes induce tumor destruction through
rophages may induce antitumor response by pro- the release of cathepsin G, azurocidin, reactive
ducing IFN- and IL-12 and triggering cytotoxic oxygen species, and inammatory cytokines.
activities [49, 50]. In contrast, tumor microen- Moreover, granulocytes, along with macrophages
vironments adopt multiple strategies to tip a and T cells, are main effectors that elicit antitu-
balance in the favor of differentiating M2-type mor responses by DNA vaccines in murine tumor
macrophages through complex network of cyto- models [61]. In addition, dense inltration of gran-
kines, chemokines, and growth factors [43, 51]. ulocytes in tumor tissues is associated with clini-
Taken together, macrophages have a dual role cal responses of GM-CSF-secreting cancer cells
in modulating tumorigenesis and antitumor host and Bacillus Calmette-Gurin (BCG) in patients
responses. Thus, detailed characterization of with advanced melanoma and bladder carcinoma,
molecular machineries which govern macro- respectively [62, 63]. On the other hand, granu-
phage polarization in tumors seems necessary for locytes contribute to tumor angiogenesis and
a thorough understanding of pharmacological metastasis by promoting secretion of proteinases,
targeting of macrophages and their derivatives. ROS, and cytokines that may acts as antitumor
effectors in different conditions [64]. Therefore,
granulocytes have both pro- and antitumor activi-
3.2.5 Dendritic Cells ties depending on distinct environments.
DCs are professional APCs contributing to the

induction of both innate and adaptive immune 3.3 The Role of Innate Immune
responses against pathogens as well as tumors. Receptors on Innate Immune
DCs express Toll-like receptors (TLRs) and co- Cells in Cancer
stimulatory molecules necessary for the activation and Antitumor Immunity
of various effectors [52]. Due to the potent immu-
nogenicity of DC, tumor microenvironments adopt 3.3.1 Toll-Like Receptors (TLRs)
multiple tactics to subvert DC functions. In addi-
tion, tumor-inltrating DCs can both induce tumor Toll-like receptors (TLRs) are innate immune recep-
growth and metastasis by regulating angiogenesis, tors mainly expressed on APCs, such as macro-
host immunity, and tumor metastasis [5356]. phages and dendritic cells. They play an important
Moreover, indoleamine 2, 3-dioxygenase (IDO)- role in host defense against pathogens by recogniz-
producing DCs cause poor tumor immunogenicity ing pathogen-associated molecular patterns
via generating Foxp3-positive regulatory T cells (PAMPs) and damage-associated molecular pattern
[57] and interacting with other innate lymphocytes molecules (DAMPs). The recognition of PAMPs
such as -T cells [58] and NKT cells [59]. and DAMPs by PRRs activates inammatory path-
In summary, tumor-inltrating DCs represent a ways such as NF-B and IRF-mediated signals,
double-edged sword which can induce an immune leading to antitumor mediators like type I interfer-
response against tumors or tolerize the immune ons, as well as cell survival and proliferation [65].
system against tumors and contribute to tumor Various sets of TLR ligands induce the upregula-
growth and metastasis. Thus, a deep understand- tion of co-stimulatory molecules and proinamma-
ing about DC biology at tumor microenvironment tory cytokine production by APCs, thus breaking
is critical to optimize anticancer therapies and the tolerogenic status to various tumor antigens
improve the clinical output of DC vaccines. and inducing antigen-specic antitumor immune
responses [6668]. In addition, TLR4 on DCs could
interact with high mobility group box 1 (HMGB1)
3.2.6 Granulocytes and facilitate antigen cross-presentation to anti-
tumor T lymphocytes [69]. Thus, TLRs agonists
Granulocytes, the key mediators of inamma- serve as effective adjuvants in harnessing potent
tion, have a potential role in the initiation of antitumor immune response and clinical responses.
In contrast, tumor cells license TLRs on NLR-mediated innate immune systems play an
myeloid cells to acquire invasive and metastatic important role in both antitumor immunity and
activities by promoting the secretion of vari- tumorigenicity. For example, nucleotide-binding
ous protumorigenic mediators, such as TNF- oligomerization domain-containing protein 1
and S100A8 [70, 71]. Thus, the careful opti- (NOD1) has a protective role against tumors, and
mization of suitable TLRs ligands for cancer the knockdown of NOD1 promotes tumor growth
immunotherapy is critical in order to avoid proin breast cancer model in vivo [78, 79]. NOD-
tumorigenic inammation caused by the TLRs like receptor family pyrin domain containing 3
expressed on innate immune cells in tumor (NLRP3) serves as a sensor for activating the
microenvironments. inammasome pathway which regulates pro-cas-
pase-1 cleavage and subsequent IL-1 activation
[80]. NLRP3 is a negative regulator of chemical
3.3.2 RIG-I-Like Helicases (RLHs) colon carcinogenesis. In a dextran sulfate sodium
(DSS) and azoxymethane-induced colon cancer
RIG-I-like helicases (RLHs) are specic fami- model, NLRP3/ mice showed increased colitis
lies of pattern-recognition receptors bear- and colitis-associated cancer, which was corre-
ing caspase-recruitment domain (CARD) at lated with attenuated levels of IL-1 and IL-18
N-terminus and helicase domains, which are at the tumor site [81]. However, in other models,
responsible for detecting intracellular dou- NLRP3 may also have a role in the promotion
ble-strand RNA and inducing innate immune of tumors as in inammation-induced skin can-
responses. RLHs include retinoic acid-inducible cers through the enhancement of inammatory
gene-I (RIG-I), myeloid differentiation anti- environment [82], which suggest a dual role for
gen-5 (MDA5), and laboratory of genetics and NLLRP3 in the regulation of host immunity for
physiology-2 (LGP2 or DHX58), which are pro- or antitumor responses. ATP released by
expressed constitutively in both immune and dying tumor cells serves as a nd-me signal
nonimmune cells. RLHs recruit specic intra- and recruits phagocytes to facilitate the engulf-
cellular adaptors to initiate NF-B- and IRF- ment of apoptotic cells [83]. Thus, ATP serves as
mediated inammatory signaling pathways that an agonist for NLRP3 whose activation triggers
lead to the synthesis of type I interferons (IFNs) IL-1 production and cross-priming of antitumor
and other proinammatory cytokines [72, 73]. CD8+ T cells [84].
The utilization of RLHs ligands as adjuvants to
trigger antitumor immune responses has been
validated by several studies. Its administration 3.3.4 Phagocytosis Receptors
with retinoic acid-inducible gene-I (RIG-I)
ligand triphosphate RNA triggers antitumor Phagocytes are specialized eating cells respon-
immune response by inducing the production sible for removing apoptotic cells in the body
of IFN-/IFN- and various immunogenic cyto- through a function of ligand-receptor interac-
kines, as well as activating antitumor immune tion. Dying tumor cells attacked by immune
response cells [74, 75]. cells or targeted by cytotoxic chemothera-
Taken together, RLHs ligands may be utilized peutic reagents are subject to recognition and
as adjuvants with other immunotherapies in order removal by phagocytic myeloid cells [85, 86].
to overcome immunosuppressive tumor microen- Molecules responsible for delivering eat
vironments. me signals, including milk-fat globule-EGF
factor 8 (MFG-E8), growth arrest-specific
6 (Gas-6), T-cell immunoglobulin-mucin
3.3.3 NOD-Like Receptors (NLRs) domain protein-4 (TIM-4), and calreticulin
(CRT), recognize the phosphatidylserine (PS)
NOD-like receptors (NLRs) are especially on apoptotic cells by integrin v3 on phago-
important for the recognition of sterile inam- cytes [8790]. On the other hand, the do not
mation such as uric acids and silica [76, 77]. eat me signal serves as negative regulators
for phagocytes. One example includes the (intercellular adhesion molecule 3) facilitates
interaction between CD47 and signal-regu- the cross talk between DCs and T lymphocytes,
latory protein- (SIRP-), which provides hence inuences immunogenic responses against
inhibitory signals that block phagocytosis [91] pathogens and tumors [94]. DEC-205 is highly
(Fig. 3.1a). expressed on DCs and promotes cross-presenta-
Manipulation of phagocytic systems has tion of tumor antigens to cytotoxic T lymphocytes
emerged as one of the tumor immune evasion [95]. Indeed, agonistic antibody targeting DEC-
machineries, and pharmacological targeting of 205 elicits potent antitumor immunity and durable
these pathways provides a feasible option to aug- tumor regression in various murine tumor models
ment host immune responses and eradicate [96]. In addition, C-type lectin domain family 9A
tumors. For example, blocking CD47 with a (CLEC9A) utilizes necrotic cells for uptake, anti-
monoclonal antibody triggers tumor destruction gen presentation, and immune response, hence
by inducing phagocytosis of malignant cells [90, raising the possibility that CLEC9A-mediated rec-
92], and the treatment with anti-MFG-E8 anti- ognition of immunogenic antigens may enhance
bodies elicits potent antitumor responses in com- antitumor immunity and clinical responses [97]
bination with conventional anticancer drugs [93]. (Fig. 3.1a). Therefore, CLRs serve as promising
candidates for improving therapeutic responses
to cancer immunotherapy. Moreover, deep under-
3.3.5 C-Type Lectin-Like Receptors standing of the mechanism through which CLRs
(CLRs) regulate innate immune response will lead to
improvement in cancer vaccines.
Carbohydrate-binding C-type lectin and lectin-
like receptors (CLRs) are a large family of
molecules expressed in innate immune cells 3.3.6 NK Cell Receptors
and play an important role in the regulation of
antitumor immunity. For example, the interac- NK cells possess various sets of pattern-
tion between DC-SIGN (dendritic cell-specic recognition receptors which activate or sup-
ICAM-3 grabbing non-integrin) and ICAM-3 press immune responses upon encountering
Fig. 3.1 Role of innate immune receptors in the regula- T-cell interactions. Phagocytosis receptors expressed on
tion of antitumor immunity. (a) The functions of the APCs interact with ligands on apoptotic cells and mediate
innate immune system are regulated by various receptors its removal by APCs. In some cases, ligand-phagocytosis
expressed in immune cells. C-type lectin-like receptors receptor interactions (such as CD47-SIRP-) provide an
(CLRs) regulate recognition and uptake of antigens (such inhibitory signal which blocks phagocytosis, a system uti-
as DEC-205), the interactions between immune cells lized by tumors to evade immune machineries. (b) The
(such as the interaction between DC-SIGN on APCs and balance between activating and inhibiting signals is criti-
ICAM-3 on T cells), and the recognition of dead cells, cal for NK cell activities. Upon interaction with corre-
such as CLEC-9A which recognizes necrotic cells and sponsive ligands, activating and inhibitory receptors
enhances cross-presentation of antigens derived from deliver a signal which is mediated by ITAM and ITIM in
necrotic cells to CD8+ T cells. Members of B7 family their cytoplasmic domain. Phosphorylated ITAM motifs
regulate the functions of APCs, such as B7-H1 and in activating receptors recruit adaptor proteins which acti-
B7-H4, which have immune suppressive effects, while vate downstream signaling pathways, while phosphory-
other members regulate the interaction with immune cells, lated ITIM motifs in inhibitor receptors recruit proteins
such as B7-H3, which interacts with NK cells and sup- such as SHP-1 which dephosphorylates downstream sig-
press its functions, and B7-1/B7-2 which regulates APCs- nal molecules and inhibit NK activities
b
their target cells. The balance between activation cytotoxicity receptor (NCR) family consists of
and inhibition signals is carefully mediated by three activating receptors: NKp30, NKp44, and
signals triggered by both activation and inhibi- NKp46, which are able to induce a strong cyto-
tion receptors in combination with cytokines. toxic reaction by NK cells. Expression levels
Signals delivered from NK receptors mainly of NCRs are correlated with cytotoxic ability
mediate through immunoreceptor tyrosine- of NK cells. MHC class I molecules counteract
based activation motif (ITAM) and immunore- with NCR-mediated activation signals; in addi-
ceptor tyrosine-based inhibition motif (ITIM). tion, the loss of MHC-I molecules, frequently
ITAM and ITIM bear conserved sequences of observed in transformed cells, activates NCRs
four amino acids repeated twice in the cytoplas- on NK cells [105107].
mic tails of NK cell receptors. Phosphorylation Killer cell immunoglobulin-like receptors
of tyrosine within ITAM motifs recruits adaptor (KIRs) are a family of cell surface molecules
proteins such as DNAX-activating protein-12 expressed on NK cells. KIRs have many mem-
(DAP12) and DNAX-activating protein-10 bers divided into two groups depending on the
(DAP10) involved in activating downstream sig- number of extracellular Ig domains (2D or 3D) or
naling pathways. On the other hand, phosphory- the length of their cytoplasmic tail, long vs. short
lation of tyrosine within ITIM motifs recruits (L or S). L-forms are shown to have inhibitory
proteins such as SHP which dephosphorylates functions, while S-forms enhance cytotoxic
downstream signal molecules to inhibit NK activities of NK cells in DAP12-mediated signal
stimulation [98] (Fig. 3.1b). pathways. KIRs regulate NK cells killing func-
Tumor cells evolve multiple strategies to tion through the interaction with MHC class I
evade NK cells by modulating ligand expression, molecules [100, 108].
ligand shedding, and upregulation of MHC mol- The interaction between inhibitory KIRs
ecules, in addition to the production of immu- and normal MHC-I molecules inhibits NK cell
nosuppressive cytokines. Thus, it is important stimulation. Correspondingly, NK cell stimu-
to understand the underlying mechanism of NK lation can occur due to an interaction between
cell activation and inhibition by their receptors, activating KIRs and polymorphic self-MHC
which eventually regulate immunosurveillance. class I molecules. Inhibitory KIRs were shown
NKG2D is a homodimeric C-type lectin- to be involved in the escape mechanism of acute
activating receptor expressed on NK, NKT, and myeloid leukemia (AML) from NK cell immune
activated CD8+ T cells [16, 99]. Ligands for surveillance, mechanism of which includes a
NKG2D include stress-induced proteins, such mismatch between donor KIRs and recipient
as MHC class I chain-related A and B (MICA human leukocyte antigen ligands [109]. Thus,
and MICB) as well as unique long 16 binding the understanding of KIR-mediated recognition
proteins (ULBPs) in human [99] and RAE1, of missing self is important in the treatment of
H60, and Mult1 in mice. NKG2D ligands are AML [110].
upregulated in stress conditions, such as viral Ly49 family is a large group of receptors
infection and transformation [99102]. Several expressed in mice but not in humans [111].
signaling pathways are involved in the induction Functionally Ly49 is similar to human KIRs,
of NKG2D ligands, including HSP70-mediated containing both activating and inhibitory recep-
cellular stress [101] and ATM/ATR-mediated tors. Inhibitory Ly94 receptors possess ITIM
DNA damage pathways [103]. Importantly, motifs which recruit SHP-1 to trigger an inhibi-
blocking of NKG2D pathways increases the tory signal, while activation receptors interact
susceptibility of mice to chemically induced car- with DAP12 to activate lytic machinery in NK
cinogenesis [104], indicating the importance of cells [112]. Ly49H is an activating NK receptor
NKG2D in tumor immunosurveillance. Natural which recognizes m157 glycoprotein encoded by
mouse cytomegalovirus (MCMV). Upon interac- tory signal which suppress cytotoxic activities of
tion with m157, Ly49H associates with DAP12 NK cell. In addition, blocking of B7-H3 could
and DAP10 to stimulate NK cell-mediated cyto- restore the antitumor effects of NK cells [116].
toxic activities against infected cells [113], sug- Finally, B7-H4 promotes protumorigenic and
gesting a role for Ly49H in the protection against immunosuppressive phenotypes of macrophages;
viral infection-associated tumors [114]. for example, the blockade of B7-H4 normalized
DNAM-1 (CD226) is an adhesion molecule immunogenicity of macrophages and augmented
expressed on the surface of NK cells, monocytes, antitumor immunity in ovarian tumor tissues
and a subset of T cells. DNAM-1 belongs to the [123] (Fig. 3.1a).
immunoglobulin superfamily containing 2 Ig-like
domains of the V-set. DNAM-1 is reported to
bind to two ligands: CD112 and CD155 [115].
CD112 and CD155 are highly expressed in some 3.4 The Role of Effectors
tumors like melanoma and neuroblastoma. Produced from Innate
Importantly, neuroblastoma cells that do not Immune Cells in Cancer
express CD112 and CD155 are resistant to NK and Antitumor Immunity
cells, indicating that NK lysis of this neuroblas-
toma cells requires DNAM-1 interaction with its 3.4.1 Interferons (IFNs)
ligands on tumor cells [116].
Type I IFNs are produced by many different cells
in response to viral or bacterial infections. Type I
3.3.7 B7 Family IFNs (IFN-/IFN-) enhance proliferation and
activation of innate immune cells such as DCs,
B7 family consists of co-stimulatory and co- macrophages, and NK cells [124]. In addition,
inhibitory receptors found on activated APC and they stimulate antigen processing and presenta-
T cells, which regulate the interaction between tion to antigen-specic lymphocytes, which
APCs and T cells. B7-1 and B7-2 are expressed greatly contribute to tumor immunosurveillance
on APCs and are involved in the stimulation of [125]. The importance of type I IFNs in tumor
T-cell response. B7-1 and B7-2 on APCs serve immunosurveillance also validated enhanced
as co-stimulatory molecules and play a critical susceptibility to tumorigenesis by treatment with
role in regulating antitumor immune responses anti-IFN-/IFN- neutralizing antibodies or in
through reciprocal interaction of their receptor mice with targeted mutations of type I IFN recep-
CD28 and cytotoxic T-lymphocyte antigen-4 tor [126, 127].
(CTLA-4) on T lymphocytes [117, 118]. B7-H1 Type II IFN (IFN-) is a cytokine involved in
(PD-L1) expression in DCs is induced by IL-10 the activation of adaptive immune cells. IFN- is
and VEGF at ovarian tumors [119]. B7-H1 on primarily produced by various innate immune
DCs suppresses IL-12 and promotes IL-10 secre- lymphocytes such as NK, NKT, and -T cells
tion, creating an immunosuppressive tumor and plays a critical role in the induction of Th1
environment. Moreover, the blockade of B7-H1 immune responses and the production of NO and
enhances antitumor immunity by DC-mediated ROS by macrophages, leading to enhanced cyto-
T-cell activation [119, 120]. In addition, treat- toxic activities against transformed cells [128].
ment with PD-1 neutralizing antibodies has been IFN- has an important role in the protection
found to decrease tumor growth and metastasis against transplanted tumors or chemically
in B16 melanoma and colon cancer models [121, induced tumors by increasing intrinsic immuno-
122]. B7-H3 on APCs bind to an unidentied genicity of tumor cells [129, 130]. IFNGR/
receptor on NK cells and transduce an inhibi- mice or mice decient in IFN--downstream
signaling molecule Stat-1 developed tumors immunogenic cytokines, such as IFN-, IL-2,
more rapidly and in greater frequencies com- TNF-, and GM-CSF, and also suppresses anti-
pared to wild-type mice [131, 132]. Thus, IFN-- gen presentation by APCs. On the other hand,
mediated regulation of tumor immunogenicity the carcinogen-mediated tumor incidence was
has a great impact on innate immunity and tumor increased in IL-10-knockout mice, whereas
immunosurveillance. IL-10 overexpression protects mice from aris-
ing tumors [138]. Thus, IL-10 has a complex
role in tumorigenesis, and the pro- and antitu-
3.4.2 Other Cytokines mor effects of IL-10 may depend on the differ-
ent experimental models. TGF- is a regulatory
Interleukins have an important role in regulat- cytokine which has important roles in the regu-
ing innate immune functions in tumor micro- lation of immune responses and immune toler-
environments. Several cytokines, such as IL-2, ance as well as carcinogenesis [139, 140]. TGF-
IL-12, IL-18, IL-15, and IL-21, serve as NK can inhibit the activities of NK cells through the
cell-stimulants, competent in targeting trans- suppression of IFN- production [141], as well
formed cells. Mice decient for IL-12p40 are as the downregulation of activating receptors
susceptible to carcinogen-induced tumorigen- such as NKp30 and NKG2D [142]. On the other
esis; in addition, IL-21/ mice showed reduced hand, TGF- negatively regulates recruitment
colitis-associated cancers [133], indicating the and differentiation of myeloid-derived suppres-
role of these cytokine in protecting hosts from sor cells (MDSCs) in tumor tissues derived from
arising tumors. With respect to the mechanisms mammary carcinomas, contributing to enhanced
of action, NKG2D systems are involved in the host immunity and tumor rejection [143].
enhancement of NK cell cytotoxic activities by Therefore, TGF- has different roles in antitu-
all cytokines suggested above, and perforin-gran- mor immunity and tumorigenicity, which are in
zyme pathways play an important role in exert- part dependent on the phase of tumor progres-
ing NK cell cytolysis by IL-18. Moreover, IL-21 sion and different cellular components in tumor
induces NK cell effector functions by increasing microenvironments [144]. Vascular endothelial
sensitivities to IFN-, and IL-15 regulates sur- growth factor-A (VEGF-A) also plays a critical
vival, activation, and proliferation of NK cells role in suppressing DC maturation and differ-
[134]. Cytokines produced from innate immune entiation, therefore impacting tumor immuno-
cells serve as feasible adjuvants in activating genicity and host immunosurveillance [145].
antitumor responses in patients with advanced Thus, various cytokines are responsible for
cancer. For example, the systemic administra- attenuating immunogenic potentials of innate
tion of high doses of recombinant IL-2 or the immune systems in tumors.
adaptive transfer of IL-2-stimulated NK cell can Several cytokines derived from innate lym-
trigger potent antitumor responses and medi- phocytes contribute to smoldering inammation
ate durable tumor regressions in patients with and tumor progression. IL-23-IL-17 pathway
advanced melanoma and renal cell carcinoma operated in endogenous tumor microenviron-
[135]. The clinical efcacy of IL-12 has been ments represents prototypical mediators which
evaluated as a monotherapy or in combination promote tumor-associate inammation. IL-23
with other immunotherapies in patients with can- promotes tumor cell growth and invasion through
cer; however, they did not induce durable clinical upregulation of proteins of the matrix metallo-
responses [136, 137]. proteinase-9 (MMP9), COX-2, and angiogen-
Several cytokines antagonize immuno- esis. In contrast, IL-23/ mice showed reduced
genic potential of tumors and innate lympho- inammation and thus attenuated tumor forma-
cytes. IL-10 downregulates the expression of tion [146]. IL-17 is elevated in various tumors,
where it plays an important role in tumor growth. cells which express appropriate chemokine
IL-17 can enhance tumor growth by direct effects receptors. Two types of chemokines have been
on tumor cells and tumor-associated stromal identied: CC chemokines that are chemotactic
cells by activating IL-6-Stat3 pathways [147]. for monocytes and CXC chemokines which
Furthermore, the altered composition of com- attract polymorphonuclear leukocytes (PMNs).
mensal microbes and disruption of epithelial Chemokines have a central role in tumor progres-
barrier functions facilitate differentiation of sion through the recruitment of innate immune
IL-17-producing T lymphocytes by IL-23 from cells into tumor site. Most studies have focused
myeloid cells in intestine, leading to increased on CCL2 and CCL5 as the major chemokines in
colon tumorigenesis [148, 149]. tumor microenvironment.
Granulocyte-macrophage colony-stimulating CCL2 (MCP-1) is produced by tumor cells
factor (GM-CSF) is produced in vivo by many and tumor-associated stromal cells and attracts
cells including mast cells, macrophages, T cells, CCR2+ inammatory monocytes to the tumor
broblasts, and endothelial cells in response to microenvironment, which differentiate into
immune activation and proinammatory cyto- tumor-associated macrophages and promote
kines. GM-CSF creates an immunosuppressive tumor aggressiveness, and the blockade of CCL2-
tumor microenvironment by differentiating CCR2 signaling by neutralizing antibodies sup-
immature myeloid-derived suppressor cells presses metastasis and prolongs overall survival
(MDSCs) into tumor tissues [150]. On the other of tumor-bearing mice [156]. The levels of CCL2
hand, the therapeutic administration of GM-CSF expression and macrophage inltration into
has been emerged as a potent immunogenic adju- tumors are correlated with poor prognosis and
vant to stimulate antitumor immunity by enhanc- metastases in human breast cancer, suggesting
ing APC functions [151]. signicance of CCL2-mediated immune regula-
Macrophage colony-stimulating factor M-CSF tion in cancer patients [157].
(also known as CSF-1) is a dimeric polypeptide CCL5, another important chemokine, plays an
growth factor which regulates the proliferation, important role in the recruitment of monocytes
differentiation, and survival of macrophages and into the tumor microenvironment [158]. CCL5
their bone marrow progenitors. CSF-1 expression induces expression of CCL2, CCL3 (MIP-),
is elevated in different tumors and is found to be CCL4 (MIP-), and CXCL8 (IL-8) by mono-
accompanied by high grade and poor prognosis cytes, which leads to the recruitment of myeloid
[152]. Targeting of CSF-1 has been evaluated in cells into tumor site [159]. CCL5 also induces
preclinical and clinical studies [153]. The admin- CCR1 expression on monocytes [160]. Hence,
istration of anti-CSF1R-neutralizing antibody chemokines lead to the recruitment of mono-
(AFS98) or a CSF-1R inhibitor (Ki20227) cytes, which produce more chemokines to further
resulted in reduced numbers of tumor-inltrated attract more monocytes as well as other leuko-
macrophages in an implanted osteosarcoma cytes into the tumor site. CCL5 enhances antitu-
model and reduced vascularization, angiogene- mor immune responses against tumors [161],
sis, and tumor growth [154, 155]. while it promotes tumorigenesis and metastases
in some conditions [162, 163]. These ndings
suggest dual function of CCL5 in cancer and
antitumor immunity.
3.4.3 Chemokines Taken together, the dynamic interactions
between tumor cells and innate immune cells
Chemokines are small cytokines secreted by governed by chemokine networks play a pivotal
many cell types in response to pathological con- role in the regulation of tumor immunosurveil-
ditions, in order to activate and attract effector lance and tumorigenicity (Fig. 3.2).
Fig. 3.2 Role of the innate immune system in cancer and metastasis. For example, IDO+ DC induces differentiation of
antitumor immunity. Innate immune system serves as the FoxP3+Treg cells which suppress antitumor immunity, and
rst defense line against cancers. Innate immune cells such molecules released by PMNs may have protumorigenic or
as DC, NK, NKT, -T cells are attracted into the tumor site, antitumor effects. Furthermore, tumors secrete chemokines
where they recognize the transformed cells and release mul- and cytokines that attract inammatory monocytes into the
tiple factors which initiate an antitumor immune response. tumor microenvironment and induce its differentiation into
On the other hand, other innate immune cells may also M2 macrophages, which play important roles in tumor pro-
involve in the promotion of tumor growth, angiogenesis, and gression, metastases, angiogenesis, and chemoresistance
3.5 Concluding Remarks Acknowledgments We apologize to the authors whose

work could not be cited due to space constraints.
This study is partially supported by a Grant-in-Aid for
Innate immune system serves as the rst line of Scientic Research and Scientic Research for Innovative
defense against pathogens and cancers. In tumors, Areas from the Ministry of Education, Culture, Sports,
innate immune cells are attracted into the tumor Science and Technology (MEXT) and the Ministry of
Health, Labour and Welfare, The Naito Foundation, and
site. Factors released from stressed cells at the the Astellas Foundation for Research on Metabolic
tumor microenvironment, such as PAMPs and Disorders (M.J.).
DAMPs, are recognized by another set of recep-
tors, including TLRs, RLRs, and NLRs, which
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B Cells in Cancer Immunology:
For or Against Cancer Growth? 4
Qiao Li, Qin Pan, Huimin Tao, Xiao-Lian Zhang,
Shiang Huang, and Alfred E. Chang
Contents H. Tao, MSc

Department of Surgery, University of Michigan,
4.1 Introduction ................................................... 48 3302 Cancer Center, 1500 E,
4.2 CD40-Activated B (CD40-B) Cells............... 48 Medical Center Drive, Ann Arbor,
MI 48109, USA
4.3 Tumor Killer B Cells ..................................... 50
Department of Hematology,
4.4 Tumor-Inltrating B Cells Wuhan Union Hospital, Wuhan, Hubei, China
(TIL-Bs) in Cancer ........................................ 52 e-mail: 747064551@qq.com
4.5 Resting B Cells and Regulatory X.-L. Zhang, PhD
B Cells in Cancer ........................................... 53 State Key Laboratory of Virology,
Department of Immunology,
4.6 Concluding Remarks .................................... 55 Hubei Province Key Laboratory of Allergy
References ............................................................... 57 and Immunology, Wuhan University School
of Medicine, Donghu Road 185#,
430071 Wuhan, Hubei, China
Department of Immunology,
Wuhan University School of Medicine,
Donghu Road 185#, Wuhan,
Hubei 430071, China
Q. Li, PhD (*)
e-mail: zhangxiaolian@whu.edu.cn
Department of Surgery, University of Michigan,
3302 Cancer Center, 1500 E, Medical Center Drive, S. Huang, MD
Ann Arbor, MI 48109, USA Hubei Province Stem Cell Research & Appling
e-mail: qiaoli@umich.edu Center, Wuhan Union Hospital, Wuhan, China
Q. Pan, PhD Department of Hematology,
Department of Surgery, University of Michigan, Wuhan Union Hospital, Wuhan, Hubei, China
3302 Cancer Center, 1500 E, Medical Center Drive, e-mail: sa2huang@hotmail.com
Ann Arbor, MI 48109, USA
A.E. Chang, MD
State Key Laboratory of Virology, Department of Surgery, University of Michigan,
Department of Immunology, Hubei Province Key 3302 Cancer Center, 1500 E,
Laboratory of Allergy and Immunology, Medical Center Drive, Ann Arbor,
Wuhan University School of Medicine, MI 48109, USA
Donghu Road 185#, 430071 Wuhan, Hubei, China
Division of Surgical Oncology,
Department of Immunology, Department of Surgery, University of Michigan
Wuhan University School of Medicine, Comprehensive Cancer Center,
Donghu Road 185#, Wuhan, Hubei 430071, China 3302 Cancer Center, Ann Arbor, MI, USA
e-mail: panqincn@whu.edu.cn e-mail: aechang@umich.edu

48 Q. Li et al.
4.1 Introduction tolerance regarding the induction of nave T

cells. Recent studies have shown that activa-
In the 1960s, B cells were rst dened in birds tion of mouse and human B cells using CD40L
when researchers found that removal of the bursa in vitro upregulates the expression of major his-
in newly hatched chicks severely impaired the tocompatibility complex (MHC) I, MHC II, and
ability of the adult birds to produce Abs [1, 2]. costimulatory molecules on B cells [69, 13, 14,
A decade later, it was found that mammalian B 16]. These B cells present exogenous antigens by
cells are derived from bone marrow and develop MHC class I or II molecules and stimulate anti-
into plasma cells that are the source of antibodies gen-specic T cells [7, 8]. CD40-B cells induce
(Abs). Over the years, most studies on B cell T cell proliferation, interferon- (IFN-) produc-
function in immune response have focused on tion, and specic cytotoxic T lymphocyte (CTL)
antigen presentation and antibody production. responses [69, 1115]. In mouse models, it has
However, recent advances in B cell biology have been shown that CD40-B cells directly present
capitalized on old ndings and demonstrated that antigen to nave CD8+ T cells, in order to induce
B cells can also act as effector cells or as regula- the generation of potent T effectors which are
tory cells [3, 4]. able to secrete cytokines and kill target cells [16].
B cells are often overlooked in tumor immu- Moreover, CD40-B cells express the full lymph
nology, likely because of the common notion that node homing triad CD62L, CCR7/CXCR4, and
humoral and cytolytic responses work in opposi- leukocyte function antigen-1 (LFA1), suggesting
tion. The eld of tumor immunology has focused that they could co-localize with T cells in the T
on CD8+ T cells due to their ability to directly kill cell-rich areas of secondary lymphoid organs [11,
tumor cells, as well as the close association 15]. This will facilitate CD40-B cell and T cell
between tumor-inltrating CD8+ T cells and can- contact for antigen presentation.
cer patients survival [5]. To date, the role of B Using a metastatic mouse model, Li et al. pro-
cells in tumor immunity has remained largely vided direct experimental evidence that the aug-
elusive. Results from different research groups mented antitumor activity by anti-CD40
are somewhat controversial. In this chapter, we monoclonal antibody (mAb)-stimulated tumor-
review the roles of B cells in tumor immunology, draining lymph node (TDLN) cells requires the
which may either positively or negatively affect presence of APCs, e.g., B cells as well as DCs.
tumor growth and patient outcomes. They found that anti-CD40 mAb augments anti-
tumor responses of TDLN cells via ligation to
CD40 on both B cells and DCs [17].
4.2 CD40-Activated B Typically, TDLN cells are composed of
(CD40-B) Cells approximately 60 % CD3+ T cells, 30 % CD40+ B
cells, and 5 % DCs. In a murine sarcoma model,
CD40-activated B (CD40-B) cells are thought to anti-CD3-/anti-CD40-activated MCA205 TDLN
be an excellent source of professional antigen- T cells secreted signicantly higher amount of
presenting cells (APCs) for antigen-specic tumor IFN- in an antigen-specic manner (in response
immunotherapy. They have demonstrated potent to MCA205 tumor, but not to MCA 207 tumor),
effects on cellular immunotherapy of cancers in comparison with solely anti-CD3-activated
[617]. CD40-B cells induce potent expansion of TDLN T cells (Fig. 4.1a). However, when B cells
antigen-specic CD4+ and CD8+ T cells, includ- were depleted from MCA205 TDLN cells, anti-
ing nave CD8+ T cells [69, 12, 16]. One reason CD3/anti-CD40 activation could not increase the
that dendritic cells (DCs) are considered as excel- IFN- anymore. This effect is very similar to DC
lent APCs in tumor immunotherapy is that they depletion (Fig. 4.1a). In vivo, adoptive transfer of
can powerfully prime nave T cells, while rest- anti-CD3-/anti-CD40-activated MCA205 TDLN
ing B cells cannot. Resting B cells poorly express T cells mediated signicantly higher MCA205
costimulatory molecules, resulting in immune tumor regression in a pulmonary metastasis
4 B Cells in Cancer Immunology: For or Against Cancer Growth? 49
Fig. 4.1 Anti-CD40 mAb a 3,000

augmented antitumor Anti-CD3: Unfrac.
responses of anti-CD3- Anti-CD3/anti-CD40: Unfrac.
activated TDLN cells via 2,500 * Anti-CD3/anti-CD40: CD19-
ligation to CD40 on both B Anti-CD3/anti-CD40: CD11c-
cells and DCs. (a) Activated Anti-CD3/anti-CD40: CD19-/CD11c-
2,000
total unfractionated (Unfrac)
TDLN cells were co-cultured
pg/ml
with MCA 205 vs. MCA207 1,500
tumor cells to determine
IFN- production. B cells
were removed by CD19 1,000
depletion (CD19), and DCs
were removed by CD11c 500
depletion (CD11c).
(b) Activated total TDLN
(Unfrac) cells or B cell, 0
T alone T cells + MCA205 T cells + MCA207
DC-depleted TDLN cells
(CD19 and/or CD11c) b 300
TDLN cells adoptively
>250 >250 >250
transferred into tumor- 250
bearing mice for therapy.
Pulmonary metastases
*p < 0.05 compare with

any other group in (a, b), 200
respectively (Adapted
by permission from 150
the American Association
of Immunologists,
Inc. Copyright 2005: 100 *
Li et al. [17])
50
0
No treatment IL-2only Unfrac. Unfrac. CD19- CD11c-CD19-/CD11c-
Anti-CD3 Anti-CD3/anti-CD40
setting, compared to anti-CD3-alone-activated They found that tumor growth inhibition was
TDLN T cells (Fig. 4.1b). However, B cell signicantly diminished in the B cell-decient
removal signicantly reduced the therapeutic mice after T cell + IL-2 + IL-21 combined ther-
efcacy conferred by CD40 engagement, and so apy (Fig. 4.2).
did DC removal. Together, these studies indicate In contrast to DCs, large numbers of B cells
that B cells, as well as DCs, are required in the can be obtained from the blood of patients after
generation of potent antitumor T effector cells ex vivo expansion (up to 1,000-fold) in the
from TDLN cells via simultaneous targeting of presence of CD40L [6]. For example, only
CD3 on T cells and CD40 on B and dendritic about 106 DCs can be generated from 10 ml of
cells. blood, while 1091010 B cells can be produced
In a separate study, Iuchi et al. reported that from the same volume of the blood sample.
host B cells were required for adoptive trans- Additionally, CD40-B cells can be continu-
ferred T cells to mediate optimal antitumor ously expanded in long-term culture (>65 days)
immunity [18]. Tumor-bearing mice were treated without the loss of APC functionality [6].
with adoptive transfer of T cells accompanied Therefore, CD40-B cells have the advantage
with IL-2 and IL-21 administration in wild-type over DCs in terms of isolation, generation, and
and B cell knockout (B/) animals, respectively. long-term expansion. These characteristics
50 Q. Li et al.
Fig. 4.2 Requirement for

host B cells in T cell transfer 60
+ IL-2 and IL-21 administra- WT: No treatment
tion-elicited antitumor 50
WT: T cells+IL-2+IL-21
immunity (Adapted by
B/: No treatment
Area (mm2)
permission from the 40
American Association B/: T cells+IL-2+IL-21
for Cancer Research: 30
Iuchi et al. [18])
20
10
0
0 5 10 15 20 25
Days after tumor injection
p<0.0001 WT No treatment vs. WT T cells + IL-2 + IL-21

p<0.0001 B/ No treatment vs. B/ T cells + IL-2 + IL-21
p=0.0071 B/T cells + IL-2 + IL-21 vs. WT T cells + IL-2 + IL-21
make CD40-B cells a promising alternative as ligand (TRAIL), programmed death ligands 1
cell-based vaccines. and 2 (PD-L1 and PD-L2), and granzyme B
In current B cell vaccine preparations, (GrB), which are potentially involved in B cell-
activated B cells can be loaded with antigens by mediated direct cytotoxicity against tumor cells
pulsing with peptides, proteins, tumor lysates, or [2129].
by transfection with DNA or RNA, or transduc- Due to the well-known fact that B cells can
tion with viral vectors [9, 10, 19]. Coughlin et al. produce Abs which lead to CDC and ADCC, as
loaded RNA on CD40-B cells from pediatric well as the recent ndings that B cells may
patients. Vaccination using these B cells resulted kill tumor cells directly through antibody-
in simultaneous targeting of multiple antigenic independent mechanisms, it is hypothesized that
epitopes and induced CTLs [9]. Chung et al. appropriately sensitized and activated B cells can
reported that B cells stimulated with iNKT function as effector cells to mediate antitumor
(CD1d-restricted invariant T cells) ligand immunity. Indeed, Li et al. [30] proved that
alpha-galactosylceramide (alphaGalCer) could in vivo sensitized and in vitro activated B cells
directly prime CTLs and generate long-lasting could mediate tumor regression in cancer adop-
cytotoxic antitumor immunity in vivo [10]. tive immunotherapy. In vivo sensitized TDLN
Furthermore, Garbe et al. reported that semi- cells were activated and expanded in vitro with
allogeneic fusions of microsatellite instability LPS/anti-CD40, resulting in B cell proliferation
(MSI) tumor cells with B cells primed B cells to and differentiation. These activated B cells were
induce MSI-specic T cell responses [19]. then adoptively transferred into tumor-bearing
recipients for therapy. These tumor-primed and
tumor-activated B cells signicantly reduced
4.3 Tumor Killer B Cells lung metastases in an adoptive immunotherapy
model (Fig. 4.3). Furthermore, total body irradia-
B cells can directly kill tumor cells through anti- tion (TBI) could enhance the antitumor activity
body (Ab)-independent mechanisms [20]. Recent of the adoptively transferred B cells. This study
studies have shown that B cells express death- represents one of the early studies demonstrating
inducing ligands and can therefore mediate cell that effector B cells could confer antitumor
death under many circumstances. Evidence has immunity after adoptive transfer into tumor-
emerged that B cells express Fas ligand (FasL), bearing mice [30].
tumor necrosis factor-related apoptosis-inducing
1,000 a 150 p < 0.05

No treatment
TBI p < 0.05
Tumor size (mm2)
750
Number of pulmonary
B cell transfer
metastatic nodules
100
TBI+ B cell transfer
500
50
250
*
0
0 5 10 15 20 25 30 0
T cell B cell IL-2 No
Days after tumor inoculation Treatment Treatment only treatment
*p < 0.03 vs. any other group.
p < 0.05
60
Fig. 4.3 TBI (total body irradiation) signicantly b
augmented the therapeutic efcacy of adoptively trans-
ferred B cells in the s.c. D5 tumor model (Adapted by
Number of pulmonary
metastatic nodules
permission from the American Association of 40
Immunologists, Inc. Copyright 2009: Li et al. [31])
Using a murine 4T1 pulmonary metastatic 20
model, it was found that adoptive transfer of

4T1-primed and LPS-/anti-CD40-activated
TDLN B cells signicantly inhibited 4T1 pulmo- 0
B cell T+B cell T+B cell No
nary metastasis in tumor-bearing mice [31] Treatment Treatment Treatment treatment
(Fig. 4.4). The efcacy mediated by B cells was (10 106) (10 106) (20 106)
comparable to that mediated by an equal number Fig. 4.4 (a) Adoptively transferred 4T1 TDLN B cells
of T cells, which served as a positive control in mediated effective inhibition of the spontaneous pulmo-
the experiment (Fig. 4.4a). Of note, adoptively nary metastasis of 4T1 breast cancer cells similarly to
transferred 4T1 TDLN T + B cells mediated inhi- equal numbers of T cells. (b) adoptively transferred 4T1
TDLN T + B cells mediated inhibition of the spontaneous
bition of the spontaneous pulmonary metastasis pulmonary metastasis of 4T1 better then B cells alone,
of 4T1 in a dose-dependent manner (Fig. 4.4b). and the efcacy was dose dependent (Adapted by permis-
This study also showed that activated 4T1 sion from the American Association for Cancer Research:
TDLN B cells caused tumor cell lysis directly Li et al. [31])
in vitro in the absence of Ab and other effector
cells and this direct cytotoxicity was tumor spe- In line with these ndings, Kemp et al. dem-
cic (Fig. 4.5). In these experiments, 4T1 mam- onstrated that CpG-A oligodeoxynucleotide
mary carcinoma murine tumor-primed TDLN B (CpG-A ODN) stimulation of human PBMCs
cells were activated with LPS and anti-CD40 leads to high levels of functional TRAIL/Apo-2L
mAb, washed thoroughly, and then co-cultured expression on B cells, and these B cells mediate
with 4T1 tumor cells or irrelevant tumor controls, TRAIL-/Apo-2L-dependent tumor cell lysis [25].
Renca (renal cell carcinoma) and TSA (sarcoma). Additional studies support the observation
The effector B cells killed 4T1 cells directly in a that B cell can function as effector cells in antitu-
dose-dependent way and were signicantly more mor responses. For example, Penafuerte et al.
effective than their killing of the control tumors. reported that B effector cells activated with a
These data support the conclusion that tumor chimeric protein consisting of IL-2 and the
antigen-primed and in vitro activated B cells are ectodomain of TGF- receptor II (also known as
able to kill tumor cells independent of Ab or FIST) induce potent antitumor immunity [32].
complement. However, the mechanism(s) by In this study, the B effector cells were
which the killer B cells lyse tumor cells directly characterized by the production of TNF and
in such a setting remains to be identied. IFN- and potent antigen presentation properties
52 Q. Li et al.
Expt 1 Expt 2
25 4T1 20 4T1
Renca **
TSA
20 *
15
Cytotoxicity (%)
Cytotoxicity (%)
15
10
10
*
5
5
0 0
1:1 3:1 10:1 25:1 3:1 25:1
E:T E:T
*p < 0.01, 4T1 vs. Renca at the **p < 0.05, 4T1 vs. TSA at the
same E:T ratio same E:T ratio
Fig. 4.5 Activated 4T1 TDLN B cells mediate direct and tumor-specic cytotoxicity of 4T1 cells (Adapted by
permission from the American Association for Cancer Research: Li et al. [31])
[32]. In addition, Forte et al. found that patients [36, 37, 42, 44, 45], lower relapse rate
administration of a specic CD73 inhibitor, ade- [41], or low metastasis [42]. In a study on immune
nosine 5-(, -methylene) diphosphate (APCP), inltrates in high-grade serous ovarian cancer, it
to melanoma-bearing mice induced signicant was revealed that intraepithelial CD20+ TIL-Bs
tumor regression [33]. They observed that after are associated with increased disease-specic
APCP administration, the presence of B cells in survival [37]. Importantly, the association
the melanoma tissue was more than that observed between the immune inltrates and survival was
in control mice. This was associated with the pro- dependent on histological subtype, because
duction of IgG2b within the melanoma, implying immune inltrates were less prevalent in the
a critical role for B cells in the antitumor activity other histological subtypes compared to the high-
of APCP [33]. Together, these studies suggest grade serous cases [37]. In breast cancer, TIL-Bs
that the mechanisms underlining B cell-mediated are present in about 24 % of tumors and comprise
antitumor immunity may involve multiple cellu- up to 40 % of the lymphocytic inltrates [34].
lar and molecular events, as well as direct killing TIL-Bs have been shown to undergo antigen-
of the tumor cells. driven clonal proliferation and afnity matura-
tion in situ [35]. Very recently, in a large patient
cohort of different histological and biological
4.4 Tumor-Inltrating B Cells subtypes, Mahmoud and colleagues provided
(TIL-Bs) in Cancer evidence for a favorable outcome when high
numbers of CD20+ TIL-Bs were present [36].
Tumor-inltrating B cells (TIL-Bs) have Additionally, TIL-Bs may be involved in humoral
revealed controversial roles in antitumor immu- immune response in situ. Using recombinant Ab
nity. They have been found in breast cancer cloning techniques, Hansen et al. reported an
[3436], ovarian cancer [37], lung cancer [38], antigen-driven humoral immune response
colorectal cancer [39, 40], cervical cancer [41], directed against -actin exposed on apoptotic
cutaneous melanoma [42], and prostate cancer mammary carcinoma cells [46]. Yasuda and
(CaP) [43]. A few studies have indicated that coworkers identied TIL-Bs which produce
TIL-Bs are correlated with favorable survival of tumor-specic Abs against mutated p53 [47].
Maletzki et al. also reported that TIL-Bs from enhanced T cell antitumor activity and signicant
colorectal carcinoma show an activated improvement in survival rate [4852]. It has been
immunophenotype (CD23+, CD80+) and produce reported that there are increased effector T cells
IgGs that specically bind to allogeneic target [48], increased T cell inltration of tumors [52],
tumor cells [40]. higher Th1 cytokine and antitumor CTL response
On the other hand, TIL-Bs may produce cyto- [49, 51, 52], and even reduced T regulatory cell
kines contributing to tumor development. It has (Treg) frequencies [53] in these B cell-decient
been reported that TIL-Bs in castration-resistant mice. Some studies explored the possible mecha-
CaP produce lymphotoxin by an inammation- nisms involved. B cells present in the priming
responsive IB kinase (IKK)--dependent path- phase result in disabled CD4+ T cell help for
way, which then in turn activates IKK- and CTL-mediated tumor immunity [51]. B cells
STAT3 in tumor cells to enhance hormone-free produce IL-10 which can repress antitumor
tumor survival [43]. In this study, B cell inltra- immunity [49, 54]. Similarly, Abs were shown to
tion was detected in 100 % of human CaP sam- promote primary tumor formation in a trans-
ples, while B cells were undetectable in normal genic mouse model of inammation-associated
prostate or benign prostatic hyperplasia [43]. carcinogenesis [55]. Autoantibody responses to
Castration-resistant CaP growth was delayed in self-proteins triggered by cancer vaccines may
mice reconstituted with bone marrow from JH/ inuence the efcacy of vaccination [56].
mice, which lack mature B cells [43]. It was fur- Additionally, B cells have been shown to have
ther found that these CaP allografts exhibited other pro-tumorigenic roles. For example,
IKK- nuclear translocation, which was depen- enhanced NK cell antitumor activity has been
dent on IKK- in B cells. IKK- deletion abol- reported in B cell-decient mice [48, 50, 52];
ished lymphotoxin expression by B cells. When however, the mechanisms are poorly understood.
lymphotoxin- was ablated in B cells, growth of We hypothesize that the effects of B cells on
castration-resistant CaP was delayed. Similarly, antitumor immunity depend on the presence of
another study showed that tumor-inltrating T B cell subsets mainly involved under certain
and B cells were not associated with long-term tumor conditions. In the past two decades,
survival of patients with non-small-cell lung can- investigators have identied B cell subsets
cer [38]. which are capable of suppressing the immune
The roles of TIL-Bs may be complicated, response. Suppression of an immune response
since the tumor environment is dynamic and was rst reported in 1974 where spleen B cells
changes during tumor onset and progression. were found to impair delayed-type hypersensi-
TIL-Bs need to contact other immune cells or tivity (DTH) responses in guinea pigs [57, 58].
tumor cells to be activated or regulated, so their This nding led to the conclusion that DTH
contributions to immune responses are likely to responses and T cell function can be regulated
vary in different cancers and during the course of by suppressor B cells. Subsequently, convincing
cancer. data have demonstrated that IL-10-producing B
cells, termed regulatory B cells (Bregs) by
Mizoguchi et al. [59], can suppress inamma-
4.5 Resting B Cells tory responses in experimental autoimmune
and Regulatory encephalomyelitis (EAE), collagen-induced
B Cells in Cancer arthritis (CIA), and colitis [5961]. Recently,
Bregs and their potential immunomodulatory
In contrast to activated B cells, there is abundant activities have been examined in several
evidence indicating that resting B cells can immune-related diseases. In the majority of
promote the development or progression of these studies, the function of Bregs is dependent
cancer. Resting B cells are small B cells in the G0 on IL-10 production, whereas the mechanisms
stage of cell cycle, prior to activation. Studies are still undened partly because of conicting
have shown that B cell-decient mice exhibit results regarding the phenotypic characterization
54 Q. Li et al.
of IL-10-producing cells. For example, the alongside an increase in IFN--producing CD8+

following B cells have been reported as putative T cells in the spleen [54]. In this study, Tnf/
mouse Bregs: CD1dhigh subset of B cells in mice were resistant to chemical carcinogenesis
chronic colitis in TCR-decient mice [59], of the skin. LPS-stimulated CD19+ B cells iso-
CD21highCD23low B cells in contact hypersensi- lated from Tnf/ mice produced less IL-10.
tivity (CHS) mouse model [62], CD21highCD23high These mice had a reduced absolute number of
T2-MZ precursor B cells in CIA model [63], IL-10+CD19+ B cells in their spleens, and Tnf/
CD1dhighCD5+ B cells (termed B10 cells by mice were decient for CD19+CD21high B cells.
Yanaba et al.) in CHS [64] and EAE models The authors speculated that resistance to carci-
[65], CD138+CD19+ plasmablasts in Salmonella nogenesis in Tnf/ mice may result from
typhimurium infection [66], and T cell Ig increased CD8+ IFN--producing T cells and
domain and mucin domain protein (TIM)-1+ B decreased IL-10-producing B cells. In another
cells [67]. For human, CD19+CD24hiCD38hi B study, Horikawa et al. reported that production
cells have been found as putative Bregs [68, 69]. of IL-10 by Breg inhibits lymphoma depletion
Triggering Toll-like receptors (TLRs) [7072], during CD20 immunotherapy in mice [76].
the BCR [64], CD40 [73], or combinations They found that adoptive transfer of
thereof have been shown to promote IL-10 CD1dhighCD5+ B cells (that are enriched for B10
production by B cells. BCR-mediated Ca2+ ux cells) eliminates the therapeutic benet of CD20
appears to be required for IL-10 production, since mAbs in mouse lymphoma model. The trans-
B cells decient in the calcium sensors stromal ferred B10 cells in this model downregulated
interaction molecule (STIM) 1 and STIM2 have a the expression of MHC II molecules and CD86
profound defect in IL-10 secretion and abrogated on macrophages and reduced LPS-induced
suppression abilities in vivo [74]. Nuclear factor nitric oxide and TNF- production by macro-
of activated T cells (NFAT) 1, a transcription fac- phages, indicating that B10 cells suppress the
tor, is involved in Ca2+-dependent IL-10 produc- antitumor response at least partly by downregu-
tion [74]. Therefore, their proposed model for lation of macrophage activity. Our unpublished
IL-10 production by B cells is that, after BCR data support that Bregs play a negative role in
stimulation, STIM and Orai-dependent Ca2+ antitumor immunity. In melanoma and breast
increase by store-operated Ca2+ entry (SOCE) carcinoma models, depletion of IL-10-producing
activates calmodulin/calcineurin and then B cells from TDLN cells resulted in the genera-
NFAT1, leading to IL-10 expression. In addition, tion of potent effector B cells which dramati-
the TLR signaling pathway is also required cally inhibit tumor metastasis after adoptive
for IL-10 secretion [7072]. Given that TLR transfer in two genetically distinct immune
stimulation does not induce Ca2+ mobilization in competent hosts, B6 and Balb/c mice,
B cells, crosstalk between Ca2+ and Ca2+- respectively.
independent TLR cascades may be involved in Although little is known about the mecha-
IL-10 production. nisms by which Bregs undermine effective
IL-10 is an immunomodulatory cytokine antitumor immunity, several possibilities are
and inhibits Th1 polarization, prevents Th2 suggested by studies on inammation and auto-
responses, and suppresses pro-inammatory immunity. Bregs impair Th1 immune responses.
cytokine production by monocytes and macro- The initial nding about Th1 response regulated
phages [75]. So far, the potential role of Bregs by Bregs was reported by Skok et al. [77]; they
in tumor immunology is not clear, but several found that IL-10 produced by B cells is involved
studies suggest that Bregs can negatively in the feedback regulation of Th1 development.
regulate antitumor immunity. Using a mouse It has been reported that Bregs suppress the Th1
chemical carcinogenesis model, Schioppa et al. cell-mediated immune reactions in a number of
found that resistance to papilloma development mouse models, including EAE, CIA, CHS, and
in Tnf/ mice was associated with a signicant diabetes mellitus [60, 61, 64, 65, 72, 78, 79].
reduction in IL-10-producing B regulatory cells Fillatreau et al. reported that B cell IL-10
deciency correlates with enhanced type I transitional 2 immature (T2) B cells stimulated
autoreactivity; in addition, transfer of IL-10+ B with agonistic anti-CD40 (T2-like Bregs) to
cells was found to result in resolution of EAE, convert autologous effector T cells into Tr1 cells
characterized by enhanced encephalitogenic [86]. Sayi et al. also showed that B cells activated
Th1 response [60]. Later, Lampropoulou et al. by Helicobacter TLR-2 ligands produce IL-10
showed that TLR signaling in B cells suppresses and induce IL-10-producing CD4+CD25+ Tr1
inammatory T cell responses (both Th1 and cells depending on TCR signaling and a direct
Th17) and stimulates recovery from EAE [72]. T-B cell interaction through CD40/CD40L and
Similarly, using mouse model of CIA, Mauri CD80/CD28 pathways [85].
et al. showed that transfer of IL-10-producing B
cells inhibits T helper type 1 differentiation and
prevents arthritis development [61]. Yanaba 4.6 Concluding Remarks
et al. also revealed that CD1dhighCD5+ B cell
transfer normalized inammation in CHS model B cells are phenotypically and functionally het-
[64]. Using NOD mouse model of type 1 diabe- erogeneous. Characterization of B cell subpopu-
tes (T1D), Hussain et al. found that BCR- lations is shown in Table 4.1. B cells play multiple
stimulated B cells produce IL-10 and attenuate roles in tumor immunity (Fig. 4.6). On one hand,
islet inammation by polarizing CD4+ T cell accumulating literature indicate that B cells are
response toward a Th2 phenotype [79]. signicantly involved in antitumor responses. In
Bregs induce the differentiation of Tregs. this regard, B cells present tumor antigens to T
Given that MT/ B cell-decient mice display cells to generate antitumor CTLs. Upon tumor
reduced Treg frequencies in comparison with antigen stimulation, B cells can differentiate into
wild-type mice [53] and that these mice develop plasma cells to produce antibodies to target tumor
exacerbated EAE and Ag-induced arthritis (AIA) cells via ADCC and/or CDC. In addition, B cells
[60, 80], a role for Bregs in modulating Tregs was may act as killer cells to directly cause tumor cell
proposed. Several disease models have demon- lysis in the absence of antibodies. B cells migrate
strated that IL-10 produced by Bregs is important to tumor tissue and become TIL-Bs which may
for the generation and/or maintenance of Tregs. induce humoral immune response or act as killer
Sun et al. reported that after oral tolerance cells in situ. On the other hand, regulatory B cells
induction, Treg cells increase much more in WT have been described which downregulate antitu-
than in MT/ mice. However, adoptive transfer mor responses by producing immunomodulatory
of B cells before treatment normalized Treg cytokine IL-10, suppressing Th1 immune
cell development in MT/ mice [81]. In this responses, and enhancing Treg and Tr1 responses.
study, they found that sublingual tolerization Further characterization of B cell subsets respon-
with OVA/CTB (Ag conjugated to cholera toxin sible for these conicting functions demonstrated
B subunit) enhances the tolerogenic activity of B in tumor immunity and understanding of the
cells and their production of IL-10, which was molecular mechanisms involved would help
associated with the generation of Ag-specic develop novel clinical strategies for cancer
Foxp3+CD25+CD4+ Tregs [81]. This relationship immunotherapy.
between Bregs and Tregs is further supported
by the results from mouse models of airway Acknowledgments This work was supported in part by
sensitization. These results showed that Bregs the Gillson Longenbaugh Foundation, the National
Outstanding Youth Foundation of China (81025008,
prevent and reverse allergic airway inammation
Xiao-Lian Zhang), and the National Natural Science
via FoxP3+ T regulatory cells [82, 83]. Foundation of China (31270176, Qin Pan).
Additionally, Bregs can induce the differentiation
of T regulatory 1 (Tr1) cells [8486]. Gray et al.
reported that autoimmune inammation could be Competing Interests
protected by the induction of Bregs which induce The authors have declared that no competing
T cell-derived IL-10 [84]. Blair et al. used the interest exists.
56 Q. Li et al.
Table 4.1 Phenotypic characterization of B cell subpopulations

Marker Source References
Resting B cells Human CD19+CD38IgD+CD27 Tonsils [87, 88]
CD38IgM+IgD+CD27 Blood [88]
Mouse IgMlowIgDhighHSAlowCD21intCD23brightMel14 Lymph node [89]
(CD62L)brightCD44intCD69
IgMhighIgDhighCD23bright Spleen [90]
CD40 B cell Human CD19+CD38+CD80+CD86+CD71+ Tonsils [87]
CD95+CPM(carboxypeptidase-M)+
CD19+CD23+CD54+CD58+CD80+ Blood [6]
CD86+MHCIhighMHCIIbright
Mouse B7.1highB7.2highICAM+MHCIhigh Spleen [90, 91]
MHCIIbright
Putative Breg Human CD19+CD24highCD38high Blood [68, 69]
Mouse B220+CD1dhighCD21intermediate(int) Lymph nodesa [59]
CD62lowIgMintCD23high
B220+CD21highCD23low Spleen in CHS model [62]
B220+CD21highCD23high IgMbrightCD1dhigh Spleen in CIA model [63]
CD1dhighCD5+ CD19+ B220+ Spleen in CHS model [64]
CD1dhighCD5+ CD19+ Spleen in EAE model [65]
CD138+CD19+ Spleen of mice infected [66]
with Salmonella
TIM-1(T cell Ig domain and mucin domain Spleen [67]
protein)+CD19+
TIL-Bs Mostly unknown. Related to cancer types and progression
Human CD19+CD20+ CD23+CD80+ From colorectal [40]
carcinomas
Killer B Unknown
a
From TCR-decient mice
Fig. 4.6 Potential roles Tumor cell

played by B cells in
tumor immunity. ADCC
antibody-dependent Tumor
cellular cytotoxicity, CDC antigen
complement-dependent
cytotoxicity, TIL-Bs IL-10
Treg
tumor-inltrating B cells B cell Breg
Tr1
Antigen
presentation
Plasma
Th1
cell
Effector
T cell Macrophage
Effector
B cell Th17 IFN-
Tumor ADCC TNF-
Lysis CDC
Tumor
Lysis Tumor cell IL-17
TIL-Bs
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The Role of Exhaustion in Tumor-
Induced T Cell Dysfunction 5
in Cancer
Heriberto Prado-Garcia, Susana Romero-Garcia,

and Jose Sullivan Lopez-Gonzalez

5.1 Introduction ................................................ 61
T cells are divided into two major functional
5.2 T Cell Activation......................................... 62
types: helper and cytotoxic T cells. Helper T cells
5.3 T Cell Anergy.............................................. 63 (CD4+) release an array of cytokines and orches-
5.3.1 T Cell Anergy in Cancer............................... 64
trate diverse immune responses, which integrate
5.4 T Cell Exhaustion ....................................... 65 both adaptive and innate effector mechanisms.
5.4.1 Mechanisms for Inducing Cytotoxic T cells (CD8+ effector T cells) are pri-
T Cell Exhaustion ......................................... 65
5.4.2 Identication of Exhausted T Cells .............. 66 marily involved in the recognition and elimina-
tion of body cells compromised by intracellular
5.5 T Cell Exhaustion in Cancer ..................... 67
pathogens or oncogenic transformation.
5.5.1 A Particular Case: T Cell Exhaustion
in Lung Cancer Patients ............................... 69 Thus, T cells are essential components of the
immune system, which have been the major
5.6 Concluding Remarks ................................. 72
focus of immunotherapeutic strategies to boost
References ............................................................... 72 endogenous antitumor immunity. However,
despite homing to tumor sites, inltrating T cells
seldom control tumor growth, as a consequence
of the tumor microenvironment, which contains a
wide array of suppressive mechanisms that allow
tumors to escape T cell effector functions.
Even when T cell anergy has been considered
responsible for T cell hyporesponsiveness in can-
cer patients, cancer is also regarded as a chronic
disease, similar to chronic viral infections, where
H. Prado-Garcia, PhD (*) S. Romero-Garcia, PhD T cells are continuously stimulated. Thus, with
J.S. Lopez-Gonzalez, PhD
Departamento de Enfermedades Cronico-Degenerativas, chronic stimulation, tumor-specic T cells grad-
Instituto Nacional de Enfermedades Respiratorias ually become less functional until they undergo
Ismael Coso Villegas, Calzada de Tlalpan 4502, cell death, a phenomenon known as T cell
Col. Seccion XVI, Mexico City, Distrito Federal exhaustion. This chapter will focus on the latter
14080, Mexico
e-mail: hpradog@yahoo.com; sugar_cia@yahoo.com; mechanism and its participation in cancer-
slopezgonzalez@yahoo.com induced T cell dysfunction.

62 H. Prado-Garcia et al.
5.2 T Cell Activation which interact with CD28 expressed on the

T cell membrane [1] (Fig. 5.1). CD28 is the
T cell activation requires two signals delivered primary costimulatory molecule for nave T
by antigen-presenting cells (APCs). The rst cells; this molecule is essential for initiating
signal involves the presentation of antigen by T cell responses. The interaction of CD80 and
APCs, in the form of peptides bound to MHC CD86 with CD28, together with TCR signal-
class I or class II molecules, to the T cell recep- ing, promotes the expansion and differentiation
tor (TCR), expressed on the surface of the T cell. of antigen-stimulated T cells into effector and
The second signal, or costimulatory signal, stim- memory cells. The interaction of CD28 with its
ulates T cells in conjunction with the antigen. ligands (1) enhances the production of interleu-
The molecules expressed on APCs engage their kin-2 (IL-2) and other cytokines, (2) promotes
corresponding costimulatory receptors on the energetic metabolism, (3) induces the cell cycle
surface of T cells generating costimulatory sig- progression, (4) promotes T cell survival, and
nals. CD80 (B7-1) and CD86 (B7-2) are well- (5) maintains T cell responsiveness upon subse-
characterized costimulatory signal molecules, quent restimulation [1].
Fig. 5.1 T cell activation

requires recognition of the
antigen and costimulatory
signals. Inammation
generated by tissue damage
or infections activates
antigen-presenting cells
(APCs) and stimulates the
expression of costimulatory
molecules, such as CD80/
CD86. Presentation of the
antigen to the T cell receptor
(TCR), in the context of major
histocompatibility complex
(MHC) molecules and CD80/
CD86 that interact with
CD28, stimulates the
expansion and differentiation
of nave T cells (top panel).
Resting APCs express few or
no costimulatory molecules
and fail to activate T cells,
this leads to anergy (middle
panel). CTLA-4 is a
coregulatory molecule that
binds CD80 and CD86 and is
upregulated on activated T
cells. CD80/CD86-CTLA-4
interactions inhibit T cell
responses and mediate
tolerance
5 The Role of Exhaustion in Tumor-Induced T Cell Dysfunction in Cancer 63
Although costimulatory molecules were 5.3 T Cell Anergy

initially identied as stimulators of T cell
responses, some costimulatory (coregula- T cell anergy induces peripheral tolerance; this
tory) receptors inhibit T cells [1]. Cytotoxic mechanism protects the host from autoimmune dis-
T-lymphocyte-associated antigen-4 (CTLA-4) eases. In addition, anergy has been suggested to
is a CD28 homolog that also binds CD80 and play an important role in the induction of T cell dys-
CD86. Nevertheless, CTLA-4 expression is function in cancer patients. T cell anergy is a toler-
inducible after T cell activation, and is involved ance mechanism in which, after antigen encounter,
in the induction and maintenance of tolerance, as the T cell is intrinsically and functionally inacti-
its ligation inhibits IL-2 production and blocks vated [5]. The cell remains alive in this hyporespon-
cell cycle progression [1]. sive state for an extended period of time. Anergic T
After the discovery of homologs of CD28/ cells neither produce nor respond to proliferative
CTLA-4 and their ligands, many other coregu- signals and are unable to exert effector functions,
latory molecules have been identied, some of such as cytolysis or cytokine secretion. A character-
which include the inducible T cell costimula- istic of anergy is that it must be cell autonomous,
tor (ICOS or CD278) with its ligand CD275 which distinguishes this process from immunoregu-
(ICOS-L, B7h, B7-RP), the inhibitory pro- lation mediated through other regulatory cells, such
grammed death-1 (PD-1, CD279) with its as regulatory T cells (Tregs) [5, 6].
ligands PD-L1 (B7-H1, CD274) and PD-L2 There are at least ve distinct sets of circum-
(B7-DC, CD273), and the B- and T-lymphocyte stances that lead to T cell anergy [5, 7]: (1) TCR
attenuator (BTLA, CD272) which binds the ligation in the absence of full costimulation; (2)
herpesvirus entry mediator (HVEM). BTLA exposure to partial agonists, peptides with minor
is an additional receptor of the immunoglobu- sequence differences from native agonist antigenic
lin superfamily that negatively regulates T cell peptides that exhibit reduced avidity for TCR liga-
activation. In addition, HVEM interacts with tion; (3) full signaling without IL-2 receptor-
another negative regulator of T cells, CD160. driven cell division; (4) TCR ligation in the
Recent studies of the lymphocyte activation presence of IL-10 or transforming growth factor-
gene-3 (LAG-3, CD223) suggest that this mol- (TGF-); and (5) anergy induced through CTLA-4
ecule also plays an important role in the regula- or other coregulatory molecules (Fig. 5.1).
tion of T cell responses. Moreover, the T cell Thus, anergy is the consequence of factors
immunoglobulin domain and mucin domain-3 that negatively regulate proximal TCR-coupled
(TIM-3), with its ligand galectin-9, are involved signal transduction, together with active tran-
in terminating Th1 cell responses and establish- scriptional silencing, which is reinforced through
ing tolerance [2, 3]. epigenetic modications [8]. This state of nonre-
T cells that recognize antigen in the absence sponsiveness is molecularly distinct from T cell
of costimulation either fail to respond and exhaustion. T cell anergy is induced upon the rst
undergo cell death or enter a state of unrespon- encounter with the antigen and is quickly initi-
siveness, a phenomenon known as anergy. Thus, ated, in contrast with T cell exhaustion, which
costimulation is a key factor in the outcome of T is progressive. Gene expression proles show
cell interactions with the antigen. Signicant that anergy and exhaustion are partially distinct.
efforts have been undertaken to characterize Genes, such as Rnf128 (Grail), Egr2, and Egr3,
costimulatory molecules in order to augment are upregulated in anergic but not in exhausted
antitumor responses; recent evidence has demon- T cells, whereas NFAT is upregulated under
strated the importance of coregulatory molecules both conditions [9]. The detailed characteriza-
in the inhibition of immune responses. Thus, tion of the differences between anergy and T cell
interfering with these regulatory pathways has exhaustion will have important implications for
gained interest as a potential strategy for cancer therapeutic interventions in immune-mediated
therapy [4]. diseases and chronic infections.
Fig. 5.2 T cell exhaustion during chronic inammation. stages of dysfunction, losing effector functions (cytolysis
In an acute inammatory process, nave T cells are primed and secretion of cytokines) and proliferative potential in a
by an antigen, costimulatory molecules, and cytokines that hierarchical manner. Finally, deletion of T cells by apop-
promote differentiation into effector T cells. After clear- tosis occurs. As antigen load increases or CD4+ T helper
ance of the antigen and once inammation is resolved, a subpopulation decreases, T cells become more exhausted.
subset of effector T cells differentiates to become mem- Expression of coregulatory receptors is correlated with the
ory cells. During chronic processes, such as viral infec- level of exhaustion. The scale of each activity is presented
tions, the antigen persists, and T cells go through several from high (+++) to low ()
5.3.1 T Cell Anergy in Cancer coregulatory molecules, such as PD-L1, PD-L2,

ICOS-L (B7-H2, CD275), and B7-H3 (CD276),
Anergy has been proposed to play a role in the indicating a poor costimulatory and a high inhibi-
impairment of T cell function in human cancers. tory anergy-promoting environment. Evidence
Tumor cells are poor APCs, as these cells express that cancer induces T cell anergy comes from
antigens on MHC class I molecules but do not studies where the transfection of CD80 in tumor
express costimulatory molecules to provide a cells or the blockage of the B7 family coregula-
second signal for full T cell activation; thus, tory molecules results in reduced tumor growth
tumor-inltrating lymphocytes (TILs) are ren- or tumor rejection in mice models [2, 1114].
dered anergic [10]. In addition, immature Analysis of the functional state of TILs has
myeloid-derived dendritic cells (mDCs), demonstrated that these cells are characterized by
plasmocytoid dendritic cells (pDCs), myeloid- impairment of cytolytic activity, decreased cyto-
derived suppressor cells (MDSCs), and tumor- kine secretion, reduced expression of IL-2R
associated macrophages (TAMs) potentially (CD25), and diminished activation of extracellu-
induce anergy in TILs [8, 11, 12]. Several studies lar signal-regulated kinase (ERK) after TCR acti-
have shown that human tumor cells, mDCs, vation. Thus, T cell anergy occurs in the tumor
pDCs, MDSCs, and TAMs express high levels of microenvironment of some human tumors [1416].
Nevertheless, direct evidence that T cell anergy late stages of exhaustion. Finally, during the most
occurs in cancer has been difcult to obtain due extreme stages of exhaustion, deletion of T cells
to the lack of surface markers to identify anergic occurs through apoptosis [19, 24] (Fig. 5.2). Like
T cells [8]. CD8+ T cells, CD4+ T cells also lose function
Based on mouse tumor models, the induction during chronic infections; however, there is little
of antigen-specic T cell anergy has been sug- information about the mechanisms of exhaustion
gested to be an early event in the progression of in this T cell subpopulation [19].
tumors, which occurs in the equilibrium phase of Exhausted T cells possess a molecular prole
immunoediting, before immunosuppression that is distinct from those of memory, effector,
takes place in advanced tumors (escape phase) and anergic T cells [9]. First, many membrane
[10, 17]. However, Klein et al. showed that highly inhibitory receptors are upregulated, for instance,
immunogenic tumors evade immunosurveillance PD-1, LAG-3, and TIM-3. Second, transcription
due to antigen overload and an insufcient num- of genes encoding molecules involved in TCR
ber of tumor-specic T cells, resulting in the signaling (such as Lck and NFATc) and cytokine
exhaustion of the immune cells [18]. Thus, from receptors (IL7 and IL-15 receptors) are down-
a temporal perspective, T cell anergy predomi- regulated. Third, the pattern of genes involved in
nantly occurs during the early stages of tumor chemotaxis, migration, and adhesion is changed.
progression, whereas T cell exhaustion might Fourth, there is an altered pattern of differentia-
play a crucial role in T cell dysfunction during tion compared with memory or effector T cells.
the late stages of cancer [10]. Finally, exhausted T cells present deciencies in
translational, metabolic, and bioenergetic pro-
cesses, such as the Krebs cycle [9].
5.4 T Cell Exhaustion
T cell exhaustion has been dened as a stage of T 5.4.1 Mechanisms for Inducing
cell differentiation where T cells have poor effec- T Cell Exhaustion
tor functions, sustained coregulatory receptor
expression, and a transcriptional state distinct Immunoregulation is critical in T cell exhaustion.
from that of functional effector or memory T Coregulatory receptors play a key role in many
cells [19]. Originally, this phenomenon was iden- aspects of adaptive immunity, including self-
tied in chronic viral infections in mice and later tolerance, prevention of autoimmunity, and can-
in chronic viral infections in humans, e.g., human cer. The mechanisms of regulation through
immunodeciency virus (HIV), hepatitis B virus coregulatory receptors have not been character-
(HBV), and hepatitis C virus (HCV) [1922]. ized in detail; nevertheless, several studies sug-
Chronic bacterial and parasitic infections have gest that these receptors attenuate T cell responses
been demonstrated to induce T cell exhaustion; in many ways. Accumulating evidence highlights
also, cancer has been suggested to induce a simi- the pivotal role of the PD-1/PD-L1 pathway in
lar phenomenon [20, 23]. maintaining an immunosuppressive tumor micro-
During chronic infections, antigen-specic environment. This pathway has been proposed to
CD8+ T cells initially acquire effector functions, be the most important coregulatory pathway
but gradually become less functional as the infec- involved in T cell exhaustion [25, 26].
tion progresses. The dysfunction of exhausted T A transmembrane receptor of the Ig superfam-
cells is hierarchical, showing the initial loss of ily, PD-1 (CD279), is upregulated in mice chroni-
properties, such as cytotoxic activity, prolifera- cally infected with lymphocytic choriomeningitis
tive potential, and interleukin 2 (IL-2) synthesis; virus (LCMV) [25]. PD-1 interacts with its
followed by diminished tumor necrosis factor- ligands PD-L1 (B7-H1, CD274) or PD-L2 (B7-
alpha (TNF-) secretion and subsequent loss of DC, CD273), which are members of the B7 fam-
interferon-gamma (IFN-) production during the ily [26]. PD-1 is rapidly upregulated on activated
T cells; then, after antigen clearance, the expres- secretion of effector cytokines, such as IFN-,
sion of this receptor is reduced on effector T TNF-, and IL-2 [19]. Interestingly, CD8+ T cells
cells. Upon subsequent antigen stimulation, expressing both coregulatory receptors also pro-
effector T cells show upregulated PD-1 expres- duce the suppressive cytokine IL-10 [37].
sion. Thus, the continuous stimulation of T cells Remarkably, functional effector T cells express
(nave or effector) during chronic infections coregulatory receptors during activation; however,
induces the accumulation of PD-1+ T cells [19]. as indicated above, the prolonged and increased
High levels of PD-L1 expression on APCs (or expression of multiple coregulatory receptors is
tumor cells) might sustain PD-1 expression on T a key feature of CD4+ and CD8+ T cell exhaus-
cells, impair T cell effector maturation, and allow tion. However, exhausted T cells do not necessar-
the progression of chronic infection [2729]. ily coexpress all of the coregulatory molecules.
Studies in mouse tumor models demon- The pattern as well as the level of expression of
strated that the inhibition of PD-L1 or PD-1 coregulatory receptors simultaneously expressed
using blocking monoclonal antibodies (mAbs) in the same CD8+ T cell might considerably inu-
increases the cytolytic activity of CD8+ T ence the severity of dysfunction [38].
cells and reverses T cell dysfunction [30, 31]. Several factors, such as duration of the infec-
Subsequently, Barber et al. showed that the inhi- tion, level of antigen exposure, availability of
bition of PD-1 using anti-PD-1 mAbs in chroni- CD4+ T cell help, and the type of APCs that
cally infected mice enhances the proliferation present the antigen, have been implicated in the
and effector functions of exhausted T cells [25]. severity of T cell exhaustion. Ligand availability
Since the publication of these seminal reports, for coregulatory receptors could also inuence
many other studies have shown that PD-1 with the degree of exhaustion, as well as environmen-
its ligand (PD-L1) is crucially involved in T cell tal factors such as the presence of immunoregu-
exhaustion in chronic human pathogen infec- latory cytokines [19]. In chronic viral infections,
tions and cancer [2124, 3234]. IL-10 expression is associated with T cell dys-
In addition to PD-1, many other cell surface function [38, 39]. In addition, TGF- has also
inhibitory receptors also participate in T cell been linked to exhaustion in chronic infections
exhaustion. These coregulatory receptors regu- in humans [40, 41]; nevertheless, the mecha-
late distinct cellular functions. For instance, PD-1 nisms underlying IL-10 and TGF--mediated T
pathway affects T cell survival and proliferation, cell exhaustion are unclear. Interestingly, both
whereas LAG-3 affects cell cycle progression, cytokines are secreted by several human tumors
but has less inuence on apoptosis [19]. Several [42, 43].
receptors belonging to the tumor necrosis recep-
tor family are upregulated in exhausted T cells,
such as Fas, TNF-R, and tumor necrosis factor- 5.4.2 Identication of Exhausted
related apoptosis-inducing ligand (TRAIL) T Cells
receptors; hence, these death receptors have been
implicated in the induction of exhaustion, as T Exhausted T cells show a poorly differentiated
cells might become prone to activation-induced phenotype (CD27hiCD28loCD57loCD127loCCR7-
cell death (AICD) [19, 35, 36]. CD45RA+ or CD27+CD45RO+) correlated with T
TIM-3 is an inhibitory molecule that down- cell dysfunction. Although PD-1 upregulation in
regulates effector Th1 responses; upregulation of T cells was initially considered as a hallmark of T
this molecule has been found in HIV-specic and cell exhaustion, this molecule is upregulated
HCV-specic CD8+ T cells in patients with pro- along with activation markers, such as CD38 or
gressive HIV and HCV infections, respectively. HLA-DR [44]. In healthy adults, the percentage
Importantly, the coexpression of TIM-3 and PD-1 of PD-1+ cells varies from 40 to 80 % of
has been associated with severe CD8+ T cell (CCR7+/CD45RA) memory T cells; remark-
exhaustion in terms of the proliferation as well as ably, these cells do not exhibit characteristics of
exhaustion [45]. Thus, PD-1 is also associated tumor-specic T cells, virus-specic T cells are
with T cell activation and differentiation. more frequent and easily detectable, facilitating
Many cell surface coregulatory receptors are the ease in identication, phenotypic character-
expressed in exhausted T cells. LAG-3, TIM-3, ization, and isolation of T cells [10]. However, in
CD244 (2B4), CD160, CTLA-4, and the recently the tumor microenvironment, inltrating T cells
described B- and T-lymphocyte attenuator become dysfunctional and show reduced effector
(BTLA) are coexpressed in antigen-specic CD8+ functions. Several reports suggest that PD-L1
T cells during chronic infection. The pattern and expression on tumor cells plays an important role
level of coregulatory receptors simultaneously in tumor-induced T cell dysfunction. PD-L1
expressed in the same CD8+ T cell consider- membrane expression has been observed using
ably inuence the severity of dysfunction [38]. immunohistochemistry on many human tumors,
However, depending on the chronic infections or such as melanoma, lung, larynx, colon, breast,
cancer, exhausted T cells might express a differ- cervix, and stomach [26]. In breast, esophageal,
ent pattern of coregulatory molecules. gastric, and renal carcinomas, the increased
Genomic strategies have provided a molecular expression of PD-L1 on the surface of tumor
prole for exhausted T cells. These genomic cells is strongly associated with poor prognosis
studies support the notion that T cell exhaustion [26, 47]. Thus, T cell exhaustion has been pro-
represents a particular state of differentiation, posed as a mechanism for inducing T cell dys-
different from that of effector or memory T cells function through the PDL-1/PD-1 pathway.
[9, 19]. However, as previously indicated, PD-1 expres-
Several transcriptional pathways have been sion cannot be considered as the sole marker of T
associated with T cell exhaustion. The increased cell exhaustion in chronic diseases and cancer;
expression of transcriptional repressor Blimp-1 is hence, other markers must be considered.
associated with the upregulation of many coregu- In human metastatic-melanoma lesions, TILs
latory receptors (PD-1, LAG-3, CD160, and show upregulation of PD-1 expression, accompa-
CD244). In addition, the transcription factor nied with reduced production of IFN- TNF-, and
NFATc1 (NFAT2) is also upregulated but shows a IL-2. Both tumor-inltrating CD8+ T cells, par-
dysregulated function [9]. The transcription fac- ticularly MART-1-specic, and tumor-inltrating
tor T-bet also plays a role in protection against T CD4+ T cells show signicantly higher levels of
cell exhaustion, as T-bet promotes terminal dif- PD-1 expression than CD8+ and CD4+ T cells
ferentiation after acute infection, and the from peripheral blood and normal tissues from
increased expression of this transcription factor cancer patients. In addition, a large proportion
inhibits the expression of coregulatory receptors of CD8+ T cells from TILs were PD-1+CTLA-4+
during chronic viral infection. T-bet expression is cells compared with normal tissues and blood.
downregulated through persistent antigenic stim- Furthermore, PD-1+CD8+ cells from TILs lacked
ulation, resulting in T cell exhaustion [46]. CD25 as well as IL-7RA expression, suggesting
that these cells were unable to proliferate, pro-
duce effector cytokines, and differentiate into
5.5 T Cell Exhaustion in Cancer memory cells [48]. PD-1+NY-ESO-1-specic
CD8+ T cells, from patients with advanced mela-
Cancer and chronic viral infections have been noma, upregulate TIM3 expression and are more
thought to share similar mechanisms in establish- dysfunctional than TIM3-PD-1+ and TIM3-PD-
ing high antigen load and an immunosuppressive 1NY-ESO-1-specic CD8+ T cells, producing
environment. However, there is a fundamental less IFN-, TNF-, and IL-2 [49].
difference between both diseases: viral antigens Derr et al. showed that tumor antigen
are exogenous and extremely immunogenic, (Melan-A/Mart-1)-specic CD8+ T cells express
whereas tumor antigens are self-molecules that high levels of BTLA and are susceptible to func-
are weakly immunogenic. Thus, compared with tional inhibition through its ligand HVEM [50].
In addition, Baitsch et al. recently showed that in defects in proliferation and cytotoxicity, but with
melanoma, tumor antigen-specic CD8+ T cells increased production of IFN- and TNF-, nor-
with effector phenotypes simultaneously express mal production of IL-2, and increased expression
four or more of the inhibitory receptors BTLA, of T-bet. Thus, although CD8+ T cells show fea-
TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1, or tures of T cell exhaustion, these cells retain the
CTLA-4 [51]. Moreover, tumor antigen-specic ability to produce cytokines [57]. However, head
CD8+ T cells present a large variety of genes with and neck cancers that are positive for human pap-
a similar genetic prole as that of exhausted T illomavirus (HPV) present a high inltration of
cells from chronic viral infections [52]. Taken PD-1+ T cells, and the numbers of PD-1+ cells are
together, these reports show that tumor antigen- positively associated with a favorable clinical
specic CD8+ T cells are exhausted in melanoma outcome. Nevertheless, these PD-1+ T cells
patients. express activation markers, 50 % of this popula-
Additional evidence for T cell exhaustion in tion lack TIM-3 expression, and are functional
other cancers comes from studies in patients with after the blockade of the PD-1/PD-L1 pathway,
ovarian cancer. Matsusaki et al. reported that suggesting that PD-1+ T cells are activated rather
NY-ESO-1-specic CD8+ T cells, from the than exhausted [58].
peripheral blood of patients with ovarian cancer, Interestingly, Haymaker et al. proposed that
show impaired effector functions along with PD-1highCD8+ T cells in cancer patients are not
coexpression of the inhibitory molecules LAG-3 exhausted [59]. This hypothesis is based on the
and PD-1. The expression of LAG-3 and PD-1 on observation that CD8+ T cells from the TILs of
the surface of CD8+ T cells is upregulated through melanoma patients recover their proliferative
IL-10, IL-6, and tumor-derived APCs. In addi- potential ex vivo, despite expressing high levels
tion, LAG-3+PD-1+CD8+ T cells are decient in of PD-1. These TILs mediate antitumor responses
IFN-/TNF- secretion compared with LAG- upon adoptive transfer into patients [60, 61].
3+PD-1 or LAG-3PD-1 subsets [53]. Under this hypothesis, inltrating and peripheral
In hepatocellular carcinoma, PD-1+CD8+ T blood CD8+ T cells, expressing PD-1, BTLA,
cells are higher in tumor tissues than in non- along with other coregulatory receptors, are not
tumor tissues, presenting decreased proliferative exhausted. Instead, these cells are highly acti-
abilities as well as effector functions, as demon- vated effector-memory cells T cells that can be
strated by reduced granule and cytokine expres- stimulated through immunotherapy [59].
sion compared with PD-1CD8+ T cells [54]. Nevertheless, these observations have been pri-
Nevertheless, no other marker of T cell exhaus- marily achieved in melanomas. In other cancers,
tion was analyzed. the reduced proliferative and effector capacities
PD-L1 expression is upregulated in Hodgkin persist, even after stimulation, and immunothera-
lymphoma (HL) and several T cell lymphomas, peutic strategies have failed to induce potent anti-
but not in B cell lymphomas. In addition, PD-1 is tumoral responses [53, 57, 62, 63].
upregulated in TILs as well as peripheral blood Some of the phenotypic, functional, and
T cells from HL patients and the blockade of the molecular changes that occur in T cells during
PD-1 pathway restores IFN- production in chronic infections are exhibited in TILs as well
T cells [55]. Moreover, LAG-3 is expressed on as peripheral blood T cells from several cancer
TILs from patients with this malignancy [56]. types. The initial aim of tumor immunotherapy
Taken together, these reports suggest that TILs was to prevent anergy and tolerance towards
from patients with HL are exhausted. tumor antigens. However, the efcacy of this
In patients with chronic lymphocytic leuke- strategy is potentially limited by T cell exhaus-
mia (CLL), CD8+ and CD4+ effector T cells show tion [10]. Interestingly, Hailemichael et al.
the increased expression of CD244, CD160, and showed that in mice vaccinated with gp100 mela-
PD-1, with the expansion of the PD-1+ BlimpH1 noma peptide, the persisting tumor antigen at
subset. CD8+ T cells from CCL patients show vaccination sites induces the sequestration of
CD8+ T cells, resulting in the dysfunction and from chronic viral infections [24], which sug-
death of these cells [63]. gests that CD8+ T cells are exhausted.
PD-1 blockage results in the recovery of T cell Recently, pleural effusion CD8+ T cells,
effector functions in vitro and in animal models derived from lung cancer patients, have been
in several tumors, thus signicantly enhancing shown to be susceptible to AICD. This phenome-
antitumor immunity [30, 31, 49, 64]. This knowl- non is preferentially observed in memory as well
edge has been translated into several clinical tri- as terminally differentiated CD8+ T cells. AICD
als [34, 65]. Recently, Brahmer et al. showed that is associated with upregulation of FasL and
the antibody-mediated blockade of PD-L1 TRAIL molecules. Interestingly, CD4+ T cells
induced durable tumor regression along with pro- from malignant pleural effusions are not prone
longed disease stabilization in patients with to AICD [75]. Thus, chronic stimulation by the
selected advanced cancers, including non-small lung tumor mass may sensitize CD8+ T cells to
cell lung cancer [65]. Thus, understanding T cell AICD, as it has been shown in TILs from vari-
exhaustion in cancer will contribute to the ous types of human cancers [75]. Nevertheless,
advancement of tumor immunotherapy. evaluation of exhaustion in lung tumor-specic
CD8+ T cells has not been possible, since lung
tumor-associated antigens are not shared among
5.5.1 A Particular Case: T Cell all lung cancer patients [62].
Exhaustion in Lung Cancer Here, it is shown PD-1 expression on CD8+
Patients and CD4+ T cells from pleural effusions and
peripheral blood of lung cancer patients who
Lung cancer is the leading cause of cancer-related were admitted to the National Institute of
mortality in developed countries and the second Respiratory Diseases Ismael Coso Villegas.
leading cause of death in countries with emerging Pleural uid was obtained by thoracocentesis
economies. Lung cancer is one of the most com- used for routine diagnostic procedures. Diagnosis
monly diagnosed cancers worldwide, represent- was established by histological examination of
ing 13 % of all cancer cases and approximately pleural biopsy or cytological observation of
18 % of all cancer deaths [66]. Some reports malignant cells in pleural effusion. None of the
show that the presence of TILs with memory patients received any type of anticancer therapy
phenotype in lung cancer is predictive of a favor- before the study or had clinical signs of acute or
able clinical outcome [6769]. Also, it has been chronic infection, which might interfere with the
shown that CD8+ T cell subpopulation is PD-1 analysis. For comparison, two groups of
decreased in the pleural compartment with patients with acute (pneumonias) and chronic
respect to peripheral blood from lung cancer (tuberculosis) infections that presented pleural
patients, whereas the CD4+ T cell subpopulation effusion were included. In lung cancer patients,
is increased [70, 71]. PD-1 was expressed on average at about 40 % of
Both in TIL and in the pleural compartment, pleural effusion CD8+ T cells, which was signi-
CD8+ T cells are functionally impaired and are cantly higher compared to percentages of
poorly responsive or unresponsive to several T PD-1+CD8+ T cells from pleural effusions sec-
cell-activating stimuli, even though memory cells ondary to acute or chronic tuberculosis infec-
inltrate lung tumors. CD8+ T cells present low tions. Also, PD-1 was expressed in more than
proliferation rate, diminished production of some 30 % of peripheral blood CD8+ T cells from lung
Th1 cytokines, and reduced cytotoxic potential cancer patients; in contrast, approximately 23 %
[70, 7274]. Pleural effusion CD8+ T cells from of peripheral blood CD8+ T cells from healthy
lung cancer patients express cell markers associ- subjects expressed PD-1 (Fig. 5.3). With respect
ated with a memory phenotype (CD45RA- to CD4+ T cells, the percentages of PD-1+ cells
CD45RO+CD27+Granzyme AlowPerforin), were signicantly higher in malignant effusions
similar to those markers found in CD8+ T cells compared to tuberculosis and acute effusions
Fig. 5.3 Determination of PD-1 on CD4+ and CD8+ T FITC-conjugated anti-PD-1 or isotype control mAb. Cells
cells. Pleural effusion (PE) and peripheral blood (PB) of were washed, xed with 1 % paraformaldehyde, and ana-
lung cancer patients (n = 23), patients with acute diseases lyzed using ow cytometry. FSC vs. SSC dot-plot graphs
(non-chronic n = 8), patients with tuberculosis (TB, n = 9), were done for cellular debris and necrotic cell exclusion.
and PB from healthy donors (HD, n = 9) were evaluated. From a lymphocyte gate containing 50,000 lymphocytes,
For PD-1 membrane staining, peripheral blood mononu- CD4+ or CD8+ cells were gated from a CD4+ or CD8+ vs.
clear cells (PBMCs) or pleural effusion mononuclear cells SSC dot-plot graph. For the analysis of PD-1 expressions,
(PEMCs) were incubated with anti-CD4 PE or anti-CD8- and to rule out nonspecic antibody binding and autouo-
PECy5 monoclonal antibodies (mAbs), in addition to rescence, quadrants were set according to isotype control
(Fig. 5.3). Similar percentages of PD-1+CD4+ T peripheral blood. However, TIM-3 expression on
cells were found in peripheral blood, between CD8+ T cells was not associated with any clinical
lung cancer patients and healthy donors. Thus, a pathological parameter in lung cancer patients
greater percentage of CD8+ and CD4+ T cells (e.g., tumor size, lymph node metastasis, and
from the pleural compartment are PD-1+, which tumor stage) [77].
is a consequence of the underlying pathology, In this chapter, TIM-3 expression on
rather than the anatomical compartment. CD4+ and CD8+ T cells derived from pleu-
Recently, Zhang et al. reported that tumor- ral effusion of lung cancer patients is shown
inltrating CD8+ T cells derived from patients (Fig. 5.4). Percentages of TIM-3+ cells were
with non-small cell lung carcinoma express signicantly higher in pleural effusion CD8+
increased levels of PD-1 [76]. These CD8+ T T cells in comparison with CD4+ T cells from
cells are impaired in cytokine production as well the same cancer patients (Figs. 5.4 and 5.5).
as proliferative potential. Blockade of the PD-1/ Interestingly, pleural effusion CD8+ T cells
PD-L1 pathway by anti-PD-L1 antibody partially from cancer patients showed higher percent-
restores cytokine production and cell prolifera- ages of TIM-3+ cells compared to those from
tion. However, PD-1 expression cannot be con- the nonmalignant group (tuberculosis). Hence,
sidered as the sole marker of T cell exhaustion; TIM-3 is likely to be upregulated in response
additionally, TIM-3 has been shown to mark to tumor-derived environmental factors absent
exhausted CD8+ T cells [38]. In a study by Gao in pleural effusions from patients with tuber-
et al., TIM-3 was found to be highly upregulated culosis. Nevertheless, in contrast with results
on both CD4+ and CD8+ T cells from lung tumor reported by Gao et al., who found that in lung
tissues, but almost undetectable on T cells from tumor tissues the majority of TIM-3+TILs are
250K
Cancer Tuberculosis
4 7.2 4.5 4 0.22 0.29
200K 10 10
150K
91 Gated on
100K 10
3
10
3 CD8+ cells
CD3+ cells
50K
5
0 10 3 32.2 3 51.4
19 10 10
0 50K 100K 150K 200K 250K 4
10
SSC 3 4 3 4
0 10 10 0 10 10
3
10
250K 73 10
4
0.34 0.43 10
4
0.18 0.30
65
CD8
200K 3
10
3 3 4 5
150K 10 0 10 10 10 3 3
10 10 CD4+ cells
100K CD4
FSC
50K 3 35.1 103 56.9

10
TIM-3
0
3 3 4 5 3 4 3 4
10 0 10 10 10 0 10 10 0 10 10
CD3 PD-1
Fig. 5.4 Gating strategy for the analysis of PD-1 and were plotted on PD-1 (FITC) vs. TIM-3 (APC) 5 % con-
TIM-3 expression on T cells. PEMCs were plotted rst on tour outlier plots; quadrants were set according to isotype
SSC vs. FSC with selection of the lymphocyte population. controls. Immunostaining was done as indicated in
Gated cells were then plotted on CD3 (PE-Texas Red) vs. Fig. 5.3. Representative data from patients with lung can-
FSC and further gated on CD4 (Alexa 700) vs. CD8 cells cer or tuberculosis are shown
(APC-Cy7). CD4+ (lower row) or CD8+ (upper row) cells
Fig. 5.5 (a) Percentage of

TIM-3+ cells on CD4+ and
CD8+ T cells from lung
cancer (n = 9) and tuberculo-
sis patients (n = 5). (b)
Percentage of cells express-
ing PD-1 and TIM-3 in
pleural effusion CD8+ T
cells. Determination of
TIM-3 and PD-1 was done
as indicated in Fig. 5.4.
* p = 0.015, ** p = 0.023.
Bars depict mean standard
error
PD-1+ [77], in the pleural compartment, most tion of exhausted CD8+ T cells. Nevertheless,
PD-1+CD8+ T cells did not coexpress TIM-3 TIM-3 expression is likely responsible for
(Figs. 5.4 and 5.5). Thus, PD-1+CD8+ T cells the absence of CD8+ T cell responses in lung
might be activated in a microenvironment that cancer patients.
does not provide the sufcient signals to fully Interestingly, the administration of PD-1 anti-
differentiate into effector T cells. Because body as a blocking agent against PD-1 pathway
PD-1 was not coexpressed in TIM-3+ CD8+ T has shown durable partial tumor regression in
cells (Fig. 5.5), further studies are required to patients with lung cancer, which was long thought
dene whether this subset belongs to a popula- to be a non-immunogenic tumor [65]. Thus,
reactivation of immune responses in lung cancer [52]. Nevertheless, it is not clear whether
patients, via blocking PD-1, TIM-3, or other reg- exhausted T cells share similar molecular and
ulatory pathways, in combination with other ther- genetic patterns in patients with chronic infec-
apeutic modalities, such as radiotherapy or tions and other types of cancer.
chemotherapy, will provide major clinical bene- Understanding the mechanisms of tumor-
ts to lung cancer patients. induced T cell exhaustion will conduce to the
development of vaccine-induced T cells aimed at
promoting tumor rejection. Preliminary clinical
5.6 Concluding Remarks ndings with blockers of immune-regulatory
pathways, such as the PD-1/PD-L1 pathway, sug-
T cell exhaustion is a stage of T cell differentia- gest that this strategy is promising for enhancing
tion where T cells show poor effector functions, antitumor immunity with the potential to produce
sustained coregulatory receptor expression, as long-lasting clinical responses.
well as a transcriptional state distinct from mem-
ory, effector, and even anergic T cells. From a
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Regulatory T Cells and Th17 Cells
in Cancer Microenvironment 6
Chang H. Kim

6.1 Introduction ................................................... 77
Organs and tissues in the body are highly
6.2 Diversity of Tumor Microenvironments
and Tumor Tissue Factors ............................ 79
heterogeneous in producing tissue factors that
affect the development and maintenance of
6.3 Generation of Tregs and Th17 Cells ............ 80
immune cells. In general, organs and tissues in
6.4 Impact of Tumor-Derived Factors the body maintain highly tolerogenic conditions.
on Regulation of T-Cell Differentiation....... 81 This is important to prevent unwanted autoim-
6.5 Migration of Tregs and Th17 Cells mune or inammatory responses to harmless
into Tumors .................................................... 82 antigens and immune stimulants. Tumors, formed
6.6 Impact of Tregs and Th17 Cells in tolerogenic tissue environments, are naturally
on Antitumor Immune Responses ............... 84 hypoimmunogenic and utilize a number of mech-
6.7 Concluding Remarks .................................... 85 anisms to actively suppress the generation of
effector T cells [1, 2]. Tumors maintain tolero-
References ............................................................... 86
genic environments to avoid antitumor immune
responses. Tumors harbor high numbers of
FoxP3+ T cells (commonly called Tregs). Despite
the tolerogenic nature of the tumor microenviron-
ment, tumors variably produce many factors that
affect T-cell differentiation and maintenance. The
numbers of effector T-cell populations in tumors
are relatively more variable. Certain cancers are
linked to chronic inammation [3]. Cancers
formed in certain tissues, such as the intestine
and in patients with chronic infection, are
exposed to microbes, which can form inamma-
tory conditions in tumors. Cancers formed in
C.H. Kim, PhD (*) these tissues would be inuenced by inamma-
Laboratory of Immunology and Hematopoiesis, tory conditions. Necrotic tumor cells also induce
Department of Comparative Pathobiology, inammation through damage-associated molec-
College of Veterinary Medicine, Weldon School of
Biomedical Engineering, Center for Cancer Research,
ular pattern (DAMP) receptors such as TLR2,
Purdue University, West Lafayette, IN, USA TLR4, and the receptor for advanced glycation
e-mail: chkim@purdue.edu end products (RAGE) [4]. Inammatory tumors

78 C.H. Kim
a b Non-inflammatory c Inflammatory
Th1
FoxP3 Th17 Th17 FoxP3

Naive Th17
FoxP3
Naive Th17 Th1/CD8/

FoxP3 FoxP3 FoxP3 Th17/Th1/CD8/
Fig. 6.1 Potential roles of FoxP3+ T cells and Th17 cells priming conditions. (b) FoxP3+ T cells can promote tumor
in tumors. (a) FoxP3+ T cells are made in the thymus as growth by suppressing antitumor immune responses at
nave-type FoxP3+ T cells, which migrate to lymphoid tis- early and late stages. On the other hand, Th17 cells can
sues. These FoxP3+ T cells can become the memory type induce immune responses that lead to eradication of
after activation in secondary lymphoid tissues. Induced tumor cells in a manner similar to other effector, CD8+,
FoxP3+ T cells with memory-type FoxP3+ T cells and and T cells. (c) In inammatory conditions, FoxP3+ T
Th17 cells are made from nave CD4+ T cells. FoxP3+ T cells and Th17 cells have the potential to play different
cells suppress effector T cells and other immune cells and roles. Th17 cells cause inammation in tissues; hence,
decrease tissue inammation. Th17 cells produce IL-17 to inammatory tumors are formed and stimulated to grow.
induce inammatory responses. FoxP3+ T cells and Th17 FoxP3+ T cells suppress the function of Th17 cells and
cells can trans-differentiate into other T-cell subsets such other inammatory T cells, leading to suppression of the
as Th1 and T-FH cells in appropriate cytokine and antigen tumorigenic process in inamed tissues
harbor FoxP3+ T cells and effector T cells, includ- cells [79]. In certain cancers, FoxP3+ T cells
ing Th17 cells and Th1 cells [5, 6]. FoxP3+ T increase, whereas Th17 cells decrease in number
cells can suppress the function of antitumor as cancers advance to more aggressive stages [9].
effector T cells and other immune cells to pro- The presence of FoxP3+ T cells and Th17 cells in
mote tumorigenesis (Fig. 6.1). On the other hand, tumors and associated tissues not only reects
FoxP3+ T cells can suppress tissue inammation the nature of tumor microenvironments but also
to prevent the emergence of tumor cells follow- indicates the types of active T-cell-mediated
ing chronic tissue inammation. Effector T cells immune responses in tumors. In this chapter, we
produce inammatory cytokines that promote will discuss tumor factors that regulate T-cell dif-
tumorigenesis by increasing tissue inammation ferentiation into Tregs and Th17 cells, migration
and angiogenesis, but they can also promote anti- of the T-cell subsets into tumors and associated
tumor immunity. An inverse correlation was lymphoid tissues, and the functions of Tregs and
observed between frequencies of FoxP3+ T cells Th17 cells in regulating antitumor immune
and effector T cells such as Th17 cells and Th1 responses.
6 Regulatory T Cells and Th17 Cells in Cancer Microenvironment 79
6.2 Diversity of Tumor cytotoxicity and cytokine secretion by CD8+ T

Microenvironments cells are impaired at the low pH range [19].
and Tumor Tissue Factors Cells in the tumor microenvironment produce
various cytokines and growth factors [20]. Some
Tumor microenvironment is highly heterogeneous, of these factors are drained into lymphatic ves-
depending on tumor types and sites of formation. sels and form tumor-associated microenviron-
Together with tumor cells, broblasts, myobro- mental milieu in lymph nodes. If tumors have
blasts, endothelial cells, mast cells, and other tissue tumor-specic or tumor-associated antigens,
cells make up tumors. Moreover, immune cells are these antigens are drained or transported into
an important component of tumors and are mainly lymph nodes and presented to T cells via antigen-
composed of T cells, B cells, innate lymphoid cells, presenting cells (APCs). Effector and regulatory
and myeloid cells. Tumor-associated myeloid cells T cells can be made during this antigen priming
are heterogeneous as well and contain immature process. The cytokine milieu is critical in deter-
and mature myeloid cells. mining the fate of differentiating T cells in tumor-
Myeloid-derived suppressor cells (MDSC) are draining lymph nodes. Again, the type and
immature myeloid cells and highly enriched in amount of cytokines and other factors produced
tumors [10]. MDSC are composed of heteroge- in tumors are highly diverse among tumor types.
neous myeloid cells at various different stages. Expression of IL-1, IL-1, IL-6, IL-11, and
Compared to mature myelocytes such as dendritic TNF- was observed in colon carcinoma, colon
cells (DCs) and macrophages, MDSC do not adenoma, ovarian cancer, and gastric cancer [21
highly express cytokines, co-stimulatory mole- 27]. IL-2 and IL-15 are expressed in melanoma.
cules, and MHC class molecules. Therefore, they IL-10 and TGF- are expressed in myeloma,
poorly support antitumor effector T-cell responses. colon cancer, lung cancer, and mammary carci-
Moreover, MDSC express various molecules that noma [28, 29]. Expression of IL-17, IFN-, and
dampen immune responses. MDSC produce per- IL-4 has been observed in certain tumor types
oxynitrite for nitration and nitrosylation of many [3032]. Expression of M-CSF, GM-CSF, and
proteins in the tumor environment [11, 12]. A IL-3 has been observed as well [3335]. These
major target protein for nitration and nitrosylation tumor-derived hematopoietic cytokines regulate
is TCR, which becomes ineffective at activating myeloid cell-mediated inammation and affect
T cells after the modications [13]. They also T-cell activity in tumors. Chemokines such as
express Arg1, inducible nitric oxide synthase CXCL chemokines (CXCL1, 3, 6, 8, 10, and 12)
(iNOS), and TGF-1, among others [14]. Tumors and CCL chemokines (CCL1, 2, 5, 17, 25, and
also harbor many macrophages, which can be 28) are expressed in various tumor types [3639].
made from MDSC or myeloid progenitor cells Growth and angiogenic factors such as VEGF,
[15]. Dendritic cells express indoleamine EGF, and HGF are broadly expressed in a num-
2,3-dioxygenase (IDO) to regulate available tryp- ber of cancer types [40, 41]. The cell types pro-
tophan [16]. Other immune cells such as mast ducing these factors are not limited to tumor cells
cells, NK cells, CD8+ T cells, and B cells are fre- but can be from various cell types in tumors. For
quently found in many tumor types. example, tumor-associated macrophages produce
The tumor environment is low in both oxygen both inammatory and immunosuppressive cyto-
and pH. Tumor cells rapidly divide and therefore kines such as IL-1, IL-6, IL-10, and TGF- [42].
vigorously consume oxygen supplied via blood T-cell receptor (TCR) activation signals are
vessels. Tumor cells mainly utilize the aerobic gly- modied by the signals from co-stimulatory and
colysis pathway to generate energy [17]. This can co-inhibitory molecules, which are expressed by
accumulate lactic acid and protons, leading to low tumor cells and tumor-associated APC [43]. These
extracellular pH [18]. The most common pH range molecules include B7-1, B7-2, programmed
in tumors is 66.5. The low acidic tumor environ- cell death-1 ligand (PD-L1), PD-L2, ICOS-L,
ment leads to immune cell anergy. For example, B7-H2, B7-H3, B7-H4, and B7-H6. Among these,
80 C.H. Kim
PD-L1-PD and B7-1/2-CTLA-4 play important RALDH2 expression is induced during immune
roles in the formation of Tregs in tumor micro- responses to increase the concentration of RA
environments [4446]. Moreover, TNF receptor available in local tissue environments. Inamed
family members such as OX40, GITR, 4-1BB, and tissues or tumors are low in expression of
CD40 are expressed in tumors and regulate antitu- RA-producing RALDH but are high in expres-
mor immune responses [47, 48]. sion of RA-catabolizing CYP26 [58, 59]. In sum,
Inammatory mediators are produced in the tumor microenvironment is made of highly
tumors. Cyclooxygenase-2 (COX-2) is highly diverse factors. Some are from tumor cells, while
expressed in malignant tumors [49, 50]. COX-2 others are from tissue cells and immune cells.
expression is induced in hypoxic conditions or by These factors have profound effects on T cells in
cytokines and growth factors [51]. COX-2 gener- tumors and associated lymphoid tissues as dis-
ates prostaglandin H2 from arachidonic acid, cussed in detail later in this chapter.
which is processed to generate major inamma-
tory mediators such as PGD2, PGE2, PGI2, and
TXA2. These mediators regulate angiogenesis 6.3 Generation of Tregs
and various aspects of inammatory responses in and Th17 Cells
tumors [49].
Some tumor types are under the inuence of FoxP3+ Tregs are made in the thymus as natural
microbe-associated molecular pattern (MAMP) FoxP3+ T cells. They are also induced in the periph-
receptor ligands if tumors are formed in barrier tis- ery from nave CD4+ T cells. In addition, IL-10-
sues such as the intestine or in patients infected producing Tregs (Tr1 cells) are made from nave
with pathogens. In mucosal tissues, decreased bar- CD4+ T cells. Tregs produce suppressive cytokines
rier functions due to tumorigenesis or preexisting such as IL-10, IL-35, and TGF- [6062]. These
inammation can lead to bacterial invasion and Tregs play critical roles in preventing autoimmune
induction of inammatory responses. Furthermore, diseases. Tregs are generally made whenever effec-
tumors that are associated with infection by papil- tor T cells are formed during immune responses.
lomavirus (uterine cervical carcinoma), hepatitis This is important to limit the potentially inamma-
B virus (hepatocellular carcinoma), Epstein-Barr tory activities of effector T cells.
virus (Burkitts lymphoma), human T-cell leuke- Induction of effector T cells and Tregs occurs
mia virus (adult T-cell leukemia), or herpes virus mainly in secondary lymphoid tissues. One reason
(Kaposis sarcoma) would be inuenced by viral for this is that naive CD4+ T cells that become effec-
MAMPs. MAMPs and DAMPs activate Toll-like tor T cells and Tregs migrate mainly to secondary
receptors (TLRs) [52]. TLR activation can induce lymphoid tissues. However, memory/effector T
tissue inammation that promotes cancer [53]. cells can trans-differentiate into each other at any
MYD88 signaling is also required for activation of tissue sites upon antigen priming (Fig. 6.1a). Th1
dendritic cells for proper formation of effector T cells are the most readily made effector T cells from
cells. Without proper MYD88 signaling, Th2 cells nave CD4+ T cells. IL-12, a cytokine produced
ineffective in antitumor immunity can be made from DCs, promotes the generation of Th1 cells.
[54]. TLR signaling can work together with STAT3 Th2 cells are made when IL-4 is abundant. Th17
and Notch pathways to activate MAPK and NFkB, cells are generated when IL-6, TGF-, and other
which promote the survival and proliferation of inammatory cytokines are present during T-cell
tumor cells [55]. priming. MAMPs and TLR activation in tissues
Retinoic acid is an anticancer agent. Retinoic promote the generation of Th17 cells. Th1 cells are
acids such as all-trans retinoic acid (ATRA) and efcient in the promotion of cell-mediated immu-
9-cis RA are produced from retinol (vitamin A) nity through production of IFN- Th17 cells that
by retinol metabolizing enzymes such as ADH are effective at inducing inammatory conditions
and RALDH [56]. Epithelial cells and APCs in through producing IL-17. A number of inamma-
the intestine highly express these enzymes [57]. tory cytokines, neutrophil-attracting chemokines, and
inammatory mediators are induced by IL-17 [63]. molecules such as B7-1 and B7-2 at high levels.
IL-2 is required for the induction of T-cell prolifera- In tumors, the signals to maturate DCs are diverse
tion. IL-7 and IL-15 drive proliferation of T cells and not as apparent as those in infection. Thus,
in an antigen-independent manner in lymphopenic APCs maturated in tumor microenvironment do
conditions [64, 65]. IL-2 suppresses the formation not highly express the co-stimulatory molecules
of Th17 cells [66]. IL-4, while inducing Th2 cells, [75]. Moreover, tumor-associated APCs express
suppresses the formation of induced FoxP3+ T cells co-inhibitory receptor ligands such as PD-L1 and
and Th1 cells [67, 68]. IL-27 promotes the genera- PD-L2 [76, 77]. This affects T-cell activation and
tion of Tr1 cells [69, 70]. Expression or activation differentiation. Therefore, DCs in or from tumors
of specic transcription factors is required for the have low activation potentials for T cells. This con-
generation of specialized effector T cells and Tregs. dition typically generates induced FoxP3+ T cells
For example, RORt, STAT3, and AHR are impor- but not effector T cells. Other APCs in tumors,
tant for Th17 cells. FoxP3 and STAT5 are important such as macrophages and MDSC, are also ineffec-
for the formation of induced Tregs. c-Maf and aryl tive in generating effector T cells but are prone to
hydrocarbon receptor (AHR) are important for for- induce Tregs [78].
mation of Tr1 cells [61, 60, 71]. Beyond cytokines, As mentioned, the hypoxic condition in the
many other factors can modulate the generation of tumors is another regulatory factor for T cells [79].
Tregs and Th17 cells. This subject will not be dis- It is expected that draining lymph nodes or tertiary
cussed in detail, as the generation of Tregs and Th17 lymphoid tissues within tumors have low oxygen
cells during basic immune responses is exhaustively levels. T cells become FoxP3+ T cells when they are
discussed elsewhere. activated in hypoxia [80]. This is in part mediated
by a transcription factor called HIF-1. The high
glycolytic activity in tumors leads to accumulation
6.4 Impact of Tumor-Derived of lactic acid [8183]. This promotes the generation
Factors on Regulation of FoxP3+ T cells. TGF-1 is a characteristic cyto-
of T-Cell Differentiation kine produced in the tumor environment [8486].
TGF-1 is the most efcient cytokine that induces
Most T cells in tumors are memory T cells [72]. FoxP3+ T cells in the periphery. Along with TGF-
Both antigen-specic and nonspecic bystander T 1, IL-10 acts to suppress antitumor immune
cells would be present in tumors. In general, the responses and the promotion of Tregs [87, 88].
presence of memory T cells and CD8+ T cells is IL-10 is produced by various cell types, including T
linked to positive prognosis in cancer patients. This cells, myeloid cells, B cells, and tumor cells.
indicates that it is benecial to have these T cells in PGE2 is highly produced in the tumor envi-
tumors. About 3050 % of CD4+ T cells in various ronment. PGE2 induces FoxP3+ T cells. This
tumors formed in animals are FoxP3+ T cells [72]. induction is mediated by EP4 and EP2 receptors
Th17 cells are also found in tumors, particularly [89, 90]. In this regard, inhibition of cyclooxy-
tumors formed in mucosal tissues [73, 7, 74]. In genase-2 (COX-2) decreased FoxP3 expression
contrast, Th17 cells are hard to nd in transplanted in tumors and reduced tumor burden [91].
tumors in animal models at ectopic sites [72]. Interestingly, FoxP3+ Tregs express COX-2 and
Many factors of the tumor microenvironment can produce PGE2 [92]. The PGE2 produced by
promote the generation of FoxP3+ T cells. First, Tregs suppresses effector T cells. In addition,
APCs in tumor environments are prone to generate prostaglandin D2 (PGD2) acts on DCs to induce
FoxP3+ T cells. During infection, DCs uptake anti- FoxP3+ T cells [93]. This effect is mediated
gens and undergo maturation in response to TLR through the D prostanoid receptor and cyclic
activation. Activated DCs emigrate tissue sites of AMP-dependent protein kinase A. In this regard,
infection and migrate into secondary lymphoid enforced expression of COX-2 in head and neck
tissues through lymphatic vessels. Only mature squamous cell carcinoma led to expansion of
DCs express MHC molecules and co-stimulatory IL-10+ FoxP3+ T cells [94].
82 C.H. Kim
Commensal bacterial products that activate and integrins mediate rolling and rm adhesion
TLR2 are implicated in selectively promoting of leukocytes on endothelial cell vessels [106,
FoxP3+ T cells and Th17 cells. Segmented la- 107]. Chemokines induce integrin activation
mentous bacteria (SFB) promote Th17 cells in the between rolling and rm adhesion of immune
small intestine [95]. Certain bacterial groups such cells on endothelial cells. Chemokines also
as Clostridium and Bacteroides fragilis promote induce chemotaxis for migration of immune cells
the generation of FoxP3+ T cells in the intestine within tissues. Organs and tissues express distinct
[96, 97]. Tumors, formed in the intestine, female and overlapping chemokines and adhesion mole-
reproductive tract, and skin, are expected to be cules for regulation of immune cell migration
heavily inuenced by commensal bacteria. In [108]. Since tumors are formed within special-
these tumors, bacterial MAMPs would activate ized organs and tissues, there are similarities in
APC and T cells to regulate the generation of expression of trafcking signals between normal
FoxP3+ T cells and Th17 cells. Thus, depending tissues and tumors formed within the tissues.
on the bacterial group that is dominant in the Compared to normal tissues, however, tumors
tumor environment, FoxP3+ T cells and Th17 have altered expression of chemokines and adhe-
cells can be differentially generated. sion molecules [109]. The trafcking signals and
As mentioned, retinoic acid is an important receptors required for T-cell migration into the
tumor factor. Retinoic acid affects T cells and intestine are well established. In the intestine,
tumor cells. Retinoic acid promotes the generation CCL20 and CCL25 are, respectively, expressed
of FoxP3+ T cells but suppresses that of Th17 cells in the subepithelial cell dome (SED) of Peyers
[98, 99]. Retinoic acid affects the development of patches and by small intestinal epithelial cells
DCs and generates tolerogenic DCs expressing [110113]. Endothelial cells in the intestine,
Arg1 [100]. These DCs promote the generation of Peyers patches, and mesenteric lymph nodes
FoxP3+ T cells but suppress the formation of Th17 express mucosal addressin cell adhesion mole-
cells. This function seems to be mediated through cule-1 (MAdCAM-1) [114]. T cells migrating to
RAR-. It is also reported that retinoic acid at low the small intestine express CCR9 and 47 [115
concentrations (i.e., 0.55 nM) is required for nor- 117]. Memory T cells migrating to the Peyers
mal function of effector T cells [101, 102]. Low patches express CCR6 [118, 119]. Nave T cells
concentrations of RA are found in bodily uids in migrating to Peyers patches, MLN, and PLN
most tissues. In vitamin A deciency, the migration express CCR7, 47, and CD62L [120]. Memory
and function of effector T cells are severely T cells migrating to other tissues or inamed tis-
impaired. As mentioned, tumor cells express sues variably express CCR1-6, CCR8, CCR9,
CYP26 and can decrease retinoic acid concentra- CCR10, CXCR3, CXCR5, and CXCR6 [108].
tion in tumors and associated tissues [58]. This Effector T cells frequently express P-selectin gly-
hyporetinoic acid condition would signicantly coprotein ligand-1 (PSGL-1), E-selectin ligand-1
affect the T-cell prole in tumors and associated (ESL-1), CXCR3, CCR5, and CCR4 [105, 120].
lymphoid tissues. Moreover, retinoic acid can pro- The trafcking receptors of Tregs and Th17
mote differentiation of tumor-associated MDSC cells have been determined. FoxP3+ T cells that
into dendritic cells and macrophages [103]. are freshly made in the thymus express CCR7,
CXCR4, and CD62L [121, 122]. FoxP3+ T cells
activated or induced in the periphery express
6.5 Migration of Tregs and Th17 memory-type trafcking receptors that are fre-
Cells into Tumors quently expressed by Th1 or Th2 cells. Th17 cells
express most memory-type chemokine receptors
Migration of T cells, including Tregs and Th17 [123, 124]. CCR6 is a characteristic chemokine
cells, is regulated by trafcking receptors such as receptor expressed by most Th17 cells. In general,
chemokine receptors and adhesion molecules FoxP3+ Tregs and Th17 cells follow the trafck-
[104, 105]. Adhesion molecules such as selectins ing pattern of conventional nave and memory/
effector T cells. Conventional nave CD4+ T cells nd universal trafcking signals which govern
expressing CCR7 and CD62L lose these receptors T-cell trafcking in most tumors.
upon T-cell activation in the secondary lymphoid Our group investigated the trafcking recep-
tissues and migrate into nonlymphoid or inamed tors expressed by tumor-inltrating FoxP3+ T
tissues. Various tissue factors inuence the cells [72]. FoxP3+ T cells account for 2550 %
expression of trafcking receptors on FoxP3+ T of CD4+ T cells inltrating A20, CT26, 4T1, and
cells and Th17 cells [125, 126]. For example, reti- B16 tumors. Most of these FoxP3+ T cells are
noic acid acts on T cells undergoing activation to memory CD44+ CD62- T cells, which are down-
induce gut-homing receptors such as CCR9 and regulated for CD62L and CCR7. Downregulation
47. FoxP3+ T cells and Th17 cells express these of CCR7 was critical for the migration of FoxP3+
gut-homing receptors and migrate to the intestine T cells into tumors, as CCR7high FoxP3+ T cells
[98, 127]. In vitamin A deciency, the number of were not efcient at migrating into tumors [72].
FoxP3+ T cells and Th17 cells in the gut is signi- Downregulation of CCR7 and CD62L occurs
cantly decreased in part because most T cells do in tumor-draining lymph nodes during antigen
not migrate to the small intestine [128]. In addi- priming. Therefore, migration of T cells into sec-
tion, TGF-1 is a major cytokine that induces the ondary lymphoid tissues is required to acquire a
expression of CCR6 on FoxP3+ T cells and Th17 proper trafcking receptor phenotype for migra-
cells [123]. Moreover, IL-2 is a cytokine that tion into tumors. While downregulated for CCR7
effectively downregulates CCR6 expression and CD62L, tumor-inltrating FoxP3+ T cells
induced by TGF- 1. Thus, cytokines and tissue express CCR8 and CXCR4 at high levels [72].
factors can co-regulate the expression of trafck- This trafcking receptor phenotype reects the
ing receptors on T cells. differentiation status of the tumor-inltrating T
Researchers have been searching for che- cells and/or the trafcking receptor requirement
mokines that regulate immune cell trafcking for FoxP3+ T-cell migration into the tumors.
and antitumor immune responses [129133]. Induction of FoxP3+ T cells from FoxP3- T cells
Chemokines such as CCL3-5, CCL20, and in tumors was assessed, and the results indicate
CXCL10, often expressed in inamed tissues, are that this induction is inefcient [72]. Thus, the
also expressed in tumors [134139]. Chemokines tumor-inltrating FoxP3+ T cell in these tumors
induce chemotaxis of immune cells and tumor is largely from the FoxP3+ T cells made in the
cells. They can co-stimulate T cells and pro- thymus or secondary lymphoid tissues rather
mote angiogenesis [140, 141]. CCR2-10 and than FoxP3+ T cells induced directly in tumors.
CXCR3-5 regulate T-cell trafcking in various However, this can be quite different in other types
tumors [132]. Most of these receptors are highly of tumors where the tumor microenvironment is
expressed by FoxP3+ T cells and Th17 cells more conducive in priming T cells for differ-
in mice and humans [105, 123, 124, 121, 122, entiation into Tregs. In tumors, FoxP3+ T cells
142]. CCL17 and CCL22 are highly expressed in appear highly stable in maintaining their FoxP3
gastric cancer with CCR4-expressing FoxP3+ T expression. While detailed information on Th17
cells [131]. CCR7 is expressed by some T cells cell migration into tumors is not available, Th17
in colorectal cancers and is predictive of positive cells would probably utilize the same tissue- or
prognosis [143]. CXCR4+ T cells are increased inammation-associated trafcking signals uti-
in lung adenocarcinoma [144]. Chemokines lized by Th17 cells for regulation of general
expressed in tumors also attract hematopoietic immune responses. Th17 cells are prevalent in
progenitors, myeloid cells, NK cells, and CD8+ the gastrointestinal (GI) tract and other muco-
T cells [136, 145, 10]. An important point is sal tissues. High numbers of Th17 cells were
that chemokine signals in cancer patients are observed in aggressive forms of GI cancers [73,
highly diverse among different tumors. They are 7, 74]. Thus, these tumors would have trafcking
also affected by tissue sites and inammatory and cytokine signals appropriate for recruitment
responses in tumors. Therefore, it is difcult to and maintenance of Th17 cells or their progenitors.
84 C.H. Kim
Th17
HPC
Blood FoxP3 Tumor
vessels
DC
FoxP3
Back to circulation
Th17 Th1
MDSC
Naive Humoral Mac

tumor
factors
DC
Th17
FoxP3 LN
Lymphatic
vessel
FoxP3
Memory type
Naive
trafficking receptors DC
CCR7
Th17
CD62L
Fig. 6.2 Migration of FoxP3+ T cells and Th17 cells into CCR5, CCR8, CCR10, and/or CXCR4. Dendritic cells
tumors. Natural FoxP3+ T cells made in the thymus can (DCs) transport and present tumor tissue antigens and
migrate into lymph nodes, but cannot migrate directly into play important roles in the generation of FoxP3+ T cells
tumors unless tumors are formed in lymphoid tissues. and Th17 cells in lymph nodes. Soluble tumor tissue fac-
FoxP3+ T cells can migrate into tumors after they are anti- tors are collected in tumor-draining lymph nodes, and
gen primed in secondary lymphoid tissues and gain the some affect T-cell priming and differentiation. In tumors,
memory/effector-type trafcking receptors. Loss of CCR7 macrophages (Mac), DCs, and MDSC suboptimally acti-
and CD62L occurs during antigen priming and is required vate T cells in tumors. These APCs play potentially
for migration of antigen-primed FoxP3+ T cells into important roles in maintaining the phenotype of FoxP3+ T
tumors. Induced FoxP3+ T cells in the tumor-draining cells and Th17 cells in tumors. There is no such thing as
lymph nodes can migrate into tumors, as they are down- tumor-specic trafcking receptors. Instead, T cells vari-
regulated for CCR7 and CD62L but upregulated for mem- ably use conventional trafcking receptors to migrate into
ory/effector-type trafcking receptors such as CCR4, different tumors
Migration of FoxP3+ T cells and Th17 cells into frequencies of memory CD4+ T cells and CD8+ T
tumors and draining lymph nodes is summarized cells in many cancer types. Tumorigenesis is
in Fig. 6.2. increased in pan-T-cell- or -T-cell-decient
animals or humans [148]. Strikingly, T cells
have a small negative effect on tumor numbers,
6.6 Impact of Tregs and Th17 but a greater positive effect on tumor size. This
Cells on Antitumor Immune implies that T cells are composed of heteroge-
Responses neous subsets with different functions, and some
of these T cells may even promote tumor growth.
The presence of T cells in tumors is a highly reli- FoxP3+ T cells and other regulatory T cells are
able prognostic factor for survival of cancer likely the T cells that suppress antitumor immune
patients [146, 147]. There is a strong posi- responses. FoxP3+ T cells can inhibit antitumor
tive correlation between patient survival and immune responses and promote tumor growth
[149]. Many FoxP3+ T cells are self-reactive and inammatory tumors with high expression of
effective in preventing autoimmune diseases. The inammatory cytokines would harbor high
same function can be used to promote tumor numbers of Th17 cells. Tumors are heteroge-
growth. This is because tumor cells basically neous in the tumor microenvironment even
express self-antigens, and FoxP3+ T cells can within the same group of cancers, and not all
effectively suppress immune responses to self- tumors t into the inammatory vs. noninam-
antigens [150]. In the same line, the frequencies matory tumor model. While there is an inverse
of FoxP3+ T cells in many tumor types are correlation between FoxP3+ T cells and Th17
inversely correlated with patient survival rates cells, both T-cell subsets can be increased or
[151, 147]. However, lack of correlation or posi- decreased depending on the balance of cytokines
tive correlation has been noticed as well [152, and other tissue factors. An example for this situ-
153]. A good example is colorectal carcinoma, in ation is invasive ductal breast carcinoma [157].
which high frequencies of FoxP3+ T cells are
associated with a favorable prognosis [5]. It is
expected that FoxP3+ T cells can even prevent the 6.7 Concluding Remarks
formation of some tumors by suppressing tissue
inammation at early stages of tumorigenesis. As discussed throughout this chapter, FoxP3+ T
Therefore, FoxP3+ T cells have the potential to cells and Th17 cells play both positive and nega-
either promote or suppress tumorigenesis depend- tive roles in regulating antitumor immune
ing on tumor type, tissue site, and immune responses (Fig. 6.1). Despite the presence of
response. The potentially complex functions of these T cells, some tumors still develop and grow.
Tregs in tumorigenesis are depicted in Fig. 6.1. Thus, these T cells by themselves are not suf-
It has been observed that Th17 cells can procient to effectively mount antitumor immune
mote CD8+ T-cell-mediated antitumor immune responses. More detailed studies on FoxP3+ T
responses in a mouse model [154]. Moreover, cells and Th17 cells in various tumors can pro-
polarization of CD8+ T cells into Tc17 cells vide systematic information regarding the tumor
increased their antitumor immunity [155]. Th17 microenvironment and therapeutic interventions.
cells may become Th1 cells or activate CD8+ T It is important to develop novel strategies to boost
cells to increase antitumor immunity. the benecial effects of the T-cell subsets and to
Paradoxically, Th17 cells can cause inammation suppress their tumor-promoting effects. The key
to initiate development of inammatory tumors is to alter tumor microenvironment to regulate
at early stages of tumorigenesis. In colorectal these T-cell subsets. This is expected to be
cancer, Th17 cells are linked to poor prognosis, achieved through control of antigen-presenting
whereas Th1 cells are positively linked to patient cells, metabolism, cytokines, chemokines, co-
survival [156]. The major cytokine product of stimulatory/inhibitory receptors, inammatory
Th17 cells, IL-17, can induce tissue inamma- mediators, and nuclear hormone receptor ligands
tion and the expression of certain angiogenic fac- such as retinoic acid. Regulation of multiple fac-
tors, including CXCL8, MMP-2, MMP-9, and tors at the same time would provide more effec-
VEGF [157]. The function of Th17 cells in can- tive strategies in tipping the T-cell balance toward
cer can be complex and appears to be determined tumor-eradicating immune responses. A one-
again by cancer type, stage, and site. The poten- size-ts-all approach is not likely to be effective
tially complex functions of Th17 cells in tumori- in changing the microenvironment and T-cell
genesis are depicted in Fig. 6.1. activity in all tumors. In this regard, another point
Apart from their effector functions, the fre- is that antitumor therapy strategies should be
quencies of FoxP3+ T cells and Th17 cells reect tailor-made based on cancer type, tissue site, and
the context of the tumor microenvironment. tumor microenvironment. It is expected that
Noninammatory tumors with low expression of application of wrong immunotherapy strategies
IL-6 and other inammatory cytokines would to regulate the T-cell subsets could even worsen
have high numbers of FoxP3+ T cells, whereas the prognosis of cancer patients. More research
86 C.H. Kim
into classication of cancer types based on tumor 12. Bentz BG, Haines 3rd GK, Radosevich JA. Increased
protein nitrosylation in head and neck squamous cell
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S, Kang L, et al. Altered recognition of antigen is a
Acknowledgments The author thanks Kim Lab mem- mechanism of CD8+ T cell tolerance in cancer. Nat
bers and F. Chu (Purdue University) for their inputs and Med. 2007;13(7):82835.
assistance in preparation of this chapter. This study was 14. Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello
supported, in part, by grants from the NIH (R01AI074745, P, Mandruzzato S, et al. Myeloid-derived suppressor
R01DK076616, 1R01AI080769, and 1S10RR028293), cell heterogeneity and subset denition. Curr Opin
the Crohns and Colitis Foundation of America, and the Immunol. 2010;22(2):23844.
National Multiple Sclerosis Society to CHK. 15. Kusmartsev S, Gabrilovich DI. STAT1 signaling regu-
lates tumor-associated macrophage-mediated T cell
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Role of Cytokines in Tumor
Immunity and Immune Tolerance 7
to Cancer
Murugaiyan Gopal

7.1 Introduction ................................................. 93
Strong evidence has been accumulated
7.2 Cytokine Regulation
of the Antitumor Immune Response .......... 94
demonstrating that cancer cells in humans and
7.2.1 IL-12 .............................................................. 95 animals are recognized in general as nonself
7.2.2 IL-27 .............................................................. 100 by the immune system [1, 2]. Both innate and
7.3 Cytokines in Immune Tolerance adaptive immune reactions to cancer have been
to Cancer ...................................................... 101 described. Many cases of spontaneous tumor
7.3.1 TGF- ............................................................ 101 regression in patients with cancer have been
7.3.2 IL-17 .............................................................. 105
reported. In addition, such spontaneous regres-
7.3.3 IL-23 .............................................................. 108
7.3.4 IL-35 .............................................................. 108 sions normally occur following an infection.
7.3.5 IL-10 .............................................................. 109 Moreover, immunosuppressed patients are at
7.4 Concluding Remarks .................................. 111 increased risk for virally induced tumors [3]. In
fact, the presence of highly adaptive immune cell
References ................................................................ 111
inltrates within the tumor can be a positive prog-
nostic indicator of patient survival [4]. Murine
models of spontaneously arising or chemically
induced tumors have been useful in demonstrat-
ing that the immune system naturally surveys for
aberrant cells and has an important role in pre-
venting tumor formation [2].
An antitumor immune response is initiated
when the cells of the innate immune system
become alerted to the presence of a growing
tumor, at least in part owing to the local tissue
damage that occurs as a result of stromal remod-
eling process integral to the basic physiology of
M. Gopal, PhD (*) solid tumor development [2, 5]. Once solid tumors
Department of Neurology, Center for Neurologic reach a certain size, they begin to grow invasively
Diseases, Brigham and Womens Hospital, and require an enhanced blood supply that arises
Harvard Medical School, Harvard Institutes
as a consequence of the production of angiogenic
of Medicine, 77 Avenue Louis Pasteur, NRB-641,
Boston, MA, 02115, USA proteins [6]. Invasive growth causes minor disrup-
e-mail: mgopal@rics.bwh.harvard.edu tions within the surrounding tissue that induces

94 M. Gopal
inammatory cytokines and chemokines leading Tumors use several mechanisms that facilitate
to recruitment of cells of the innate immune system immune escape and prevent tumor elimination
[7]. The innate response includes several cellular including impairment of antigen presentation,
factors, such as natural killer (NK) cells, natural activation of negative co-stimulatory signals, and
killer T (NKT) cells, T cells, macrophages, elaboration of immunosuppressive factors [12].
dendritic cells (DCs), and neutrophils [8]. These In addition, tumor cells may promote the expan-
cells can reject tumors either by direct killing of sion and/or recruitment of regulatory immune
the tumor cells or by inhibition of angiogenesis. cell populations which can contribute to the
The components of innate immunity use pattern immunosuppressive network; these populations
recognition receptors and other cell surface mol- include regulatory T cells (Tregs), myeloid-
ecules to detect tumor cells. Cancer cells express derived suppressor cells (MDSCs), and distinct
families of stress-related genes such as MHC subsets of immature and mature regulatory DCs
class I-related stress-inducible surface glycopro- [12]. All these host-derived immune cell popula-
tein A and B (MICA and MICB), which function tions can impair antitumor effector cell responses,
as ligands for NKG2D receptors expressed on NK both locally at the tumor microenvironment and
cells [9]. In addition, NK cells can be triggered for systemically in the lymphoid organs. In fact, both
cytolytic activity by DCs depending on direct cell tumor-promoting and tumor-inhibitory immune
contact through their expression of cell surface cell populations have been found in patients with
molecules such as CD48 and CD70 which are various cancers. Several recent studies have
ligands for NK cell-activating receptors 2B4 and found correlations between particular immune
CD27, respectively [9]. The DCs that have been cell inltrates in tumors and patient prognosis.
recruited to the tumor site become activated either Inltration of CD8+ T cells and mature DC is
by exposure to the cytokine milieu created during associated with a favorable prognosis in patients
the ongoing attack by the innate immune system with cancer. However, an extensive macrophage
or by interacting with NK cells. The activated inltration correlates with poor patient prognosis
DC can acquire tumor antigens directly by inges- in most of the cancers analyzed. Thus, the com-
tion of tumor cell debris or potentially through plexity of the immune cell populations inltrat-
indirect mechanisms involving transfer of tumor ing tumors with their synergistic or opposing
cell-derived heat shock protein/tumor antigen effects may inuence tumor growth differently,
complexes [10]. The activated antigen-bearing depending on their cytokine secretion. A number
DCs then migrate to the draining lymph nodes, of immune-enhancing cytokines have been
where they trigger the activation of tumor antigen- shown to promote or inhibit antitumor immunity
specic CD4+ Th1 cells. In addition, DCs activate in multiple experimental models and in patients
CD8+ cytotoxic T lymphocytes (CTL) via cross- with cancer. This chapter reviews the role of anti-
presentation of tumor antigenic peptides on MHC tumor cytokines IL-12 and IL-27 in tumor immu-
class I molecules [11]. Activated tumor-specic nity and immunotherapy while discussing the
CD4+ and CD8+ T cells home to the tumor site role of pro-tumor cytokines (TGF-, IL-17,
where they kill tumor cells. Mice lacking adaptive IL-23, IL-35, and IL-10) that have pathogenic
immunity (RAG-2 gene-decient mice lacking signicance in cancer progression.
T cells) were more susceptible to carcinogen-
induced and spontaneous primary tumor forma-
tion. Thus, the development of adaptive immunity 7.2 Cytokine Regulation
may provide the host with the capacity to com- of the Antitumor Immune
pletely eliminate the developing tumor. However, Response
the development of clinically evident cancers
indicates that these innate and adaptive immune Cytokines comprise a large family of intracellu-
responses are not always enough to prevent dis- lar communicating molecules that play important
ease progression as cancer cells manage to escape roles in immunity, inammation, and repair, as
host-tumor immunity. well as general tissue homeostasis. In addition,
7 Role of Cytokines in Tumor Immunity and Immune Tolerance to Cancer 95
cytokines functions extend to many other aspects boosts antitumor immunity by contributing to
of biology, including cancer [13]. In the tumor the development of NK cells and CTLs without
microenvironment, cytokines are produced by any toxic side effects.
host stromal and immune cells, in response to
molecules secreted by the cancer cells. In addi- 7.2.1.2 IL-12: Linking Innate
tion, cancer cells also produce cytokines in the and Adaptive Antitumor
same environment. Increased levels of circulating Immunity
cytokines and their receptors have been found in IL-12 plays an essential role in the interaction
patients with various types of cancer, both at between the innate and adaptive arms of antitu-
diagnosis of the primary disease and in those mor immunity [17] (Fig. 7.1). It induces IFN-
with metastases, compared with healthy people production by NK cells and T cells. In fact, NK
[14, 15]. The cytokine repertoire present at the cells and T cells were rst shown to express high-
tumor site determines the type of host response afnity receptors for IL-12 [18]. Tumor eradica-
directed against the tumor. Immunosuppressive tion after vaccinations supported by IL-12 is
cytokines secreted by tumor cells or tumor- dependent on NK cells in several animal models
inltrating immune cells can impair the host anti- [1921]. IL-12 enhances in vitro lysis of both NK
tumor response, whereas cytokines promoting cell-sensitive and NK cell-resistant tumor cells.
the development of T-cell-mediated immunity Consistent with animal studies, in patients with
can induce or enhance antitumor immunity. cancer, IL-12 enhances the cytolytic activity of
Studies of cytokine-decient mice have revealed NK cells and increases the expression of CD2,
dual role for the immune system in suppressing LFA-1, and CD56 molecules which mediate NK
and promoting tumor growth. cell migration [22]. Moreover, IL-12 was shown
to enhance the cytotoxicity mediated by NK cells
from healthy donors against cancer cells derived
7.2.1 IL-12 from patients with cancer.
In addition to its effect on NK cell cytotoxic-
7.2.1.1 Overview ity, IL-12 enhances T-cell-mediated cytotoxicity
IL-12 is a heterodimeric cytokine containing a and has an enhancing effect on CD8+ T cells [23].
35 kD and a 40 kD subunit that signals through DCs play a crucial role in facilitating the interac-
a receptor of the type I family of cytokine recep- tion between CD4+ T cells and antigen-specic
tors. The principal sources of IL-12 are APCs CD8+ T cells. Priming of CTL is enabled by the
such as DCs and macrophages. Secretion of ligation of CD40 on DC and its ligand CD154 on
IL-12 is generally activated via the physiologi- activated CD4+ T cells [24, 25]. The induction of
cal stimuli of CD40 and toll-like receptors IL-12 synthesis that occurs as a result of CD40
which recognize structurally conserved mole- ligation suggests an important role for IL-12 in
cules derived from microbes [16]. IL-12 plays a the molecular mechanisms responsible for CTL
major role in the development of antitumor priming [26]. It was then shown that IL-12, in the
immune responses [17]. Numerous studies presence of antigen, acts directly on naive CD8+
report that IL-12 promotes an effective destruc- T cells to promote clonal expansion and differen-
tion of cancer cells through the induction of the tiation [27]. In addition, priming of CD8+ T cells
innate and adaptive arms of antitumor immu- in the absence of IL-12 rendered them unrespon-
nity. In addition, IL-12 has potent antiangio- sive to the same antigen [28]. Agonistic CD40
genic activity. Due to these features, IL-12 has antibodies (Abs) were shown to substitute the
been used as a systemic cancer therapeutic function of CD4+ T cells in murine models of
agent, but the clinical development of IL-12 has T-cell-mediated immunity, resulting in rapid
been hindered by its signicant toxicity and dis- expansion of CTLs that cleared established lym-
appointing antitumor effects seen in cancer phomas and provided long-term protection
patients. However, emerging studies suggest against tumor rechallenge [29, 30]. These obser-
that IL-12 in combination with other cytokines vations provided an explanation for the impaired
96 M. Gopal
Innate immunity
IFN-
Tumor
Tumor Macrophage
Cytolysis
IL-12
Tumor Ag
on
biti
hi
in NK
is
ne
s IL-12 IL-12
ge
gio
IL-12
An
DC NKT
Endothelial cells
Perforin CTL
Granymes&
IFN- IL-12
CD8+T
B cell IL-2,IFN-
Th1 CD4+T
IL-12
Tumor
IL-12
Adaptive immunity Antibodies Plasma cell
Fig. 7.1 IL-12 links innate and adaptive antitumor immu- addition, IL-12 has a direct toxic effect on the some tumor
nity. IL-12 utilizes several mechanisms to induce antitu- cells. Furthermore, IL-12 secretion by DCs can induce
mor effects. IL-27 activates innate effectors, such as NK adaptive arms of antitumor immunity. IL-12 can augment
cells, NKT cells, and T cells and promotes their cyto- Th1 response necessary for cellular immune response.
lytic activity and cytokine production. IL-12 induces Il-12 stimulates the differentiation and lytic capacity of
IFN- production in macrophages that can have a cyto- CTL and promotes immune memory. Finally, IL-12 can
toxic effect on tumor cells. IL-12 induces the production mediate antibody-mediated tumor clearance via B-cell
of antiangiogenic molecules from endothelial cells. In activation
tumor antigen-specic CTL activation in CD40- ducted by different groups, indicated that the
decient mice and conrmed the key role of the injection of IL-12 directly into subcutaneous
CD40-IL-12 pathway in the regulation of antitu- tumors results in CTL response against the tumor
mor immunity. A series of experiments, con- in mice [3133].
The rejection of tumors requires CD8+ T cells Favorable antitumor responses were related to the
whose activation and maintenance depends on synthesis of Abs against tumor antigens inducing
CD4+ T cells. Upon stimulation, nave CD4+ T tumor cell lysis in a complement-dependent cyto-
cells differentiate into different lineages of T toxicity assay [42].
helper subsets including Th1, Th2, Th17, and The ability of IL-12 to facilitate cell-mediated
Tregs. These distinct CD4+ T-cell subsets have immune responses, including enhancement of
varied impact on tumor growth. While Th1 cells NK cytotoxicity, generation of CTL, and DC
promote CD8+ T-cell-mediated immunity to activation, suggests its role in both the innate and
tumors, the other CD4+ T-cell subsets Th2 and adaptive immunity resistance mechanisms
Tregs negatively regulate CD8+ T-cell function. against tumors. Experimental studies of systemic
In the presence of IL-12, nave CD4+ T cells dif- administration of the cytokine have indicated that
ferentiate into IFN--secreting Th1 cells [34]. IL-12 exerts potent antitumor activity against a
Th1 cytokines, IL-2, and IFN-, stimulate the variety of metastatic tumors and can even prevent
cytolytic activity of NK cells. High production spontaneous tumor development in HER-2/neu
of IFN- by CD8+ T cells and a Th2 to Th1 shift transgenic mice. In addition, models based on
in the cytokine secretion prole of CD4+ T cells intra-tumor cytokine delivery or in vivo transfer
were also seen in the IL-12-treated mice [35]. By of cytokine-secreting tumors have indicated that
altering the balance between Th1 and Th2 cyto- IL-12 has signicant dose-dependent antitumor
kines, IL-12 plays a critically important role in activity against a wide spectrum of murine tumors
antitumor immune responses. A shift from Th1 including melanoma and breast, ovarian, and
to Th2 cytokine production has been reported bladder tumors [17, 43, 44]. All these studies
in progressive cancer patients, and a vaccine have demonstrated that IL-12 can inhibit tumor
inducing Th2 to Th1 shift in a murine model of growth and improve the survival of tumor-bearing
tumor was shown to induce tumor rejection [36]. animals that are dependent on not only its ability
In addition, Th2 cytokines have been shown to to activate the innate and adaptive arms of antitu-
accelerate tumor growth in multiple experimental mor immunity but also through its antiangiogenic
models [37]. In fact, CD4+ T cells can directly activity.
interact with CD8+ T cells via CD40-CD154
interactions [38], which directly contrast with 7.2.1.3 IL-12 and Angiogenesis
the early notion that CD4+ and CD8+ T cells are Inhibition
brought together on the same antigen-presenting Accumulating evidence indicates that the antitu-
cell for the effective delivery of IL-2 to neighbor- mor effects of IL-12 are mediated, at least in part,
ing CD8+ T cells. Moreover, a full CD8+ T-cell through mechanisms involving angiogenesis and
response is elicited by a temporal release of IL-2 its direct effects on tumors. Angiogenesis is an
from CD4+ T cells, which is consistent with the essential process for tumor growth and metasta-
ndings that neutralization of IL-2 signicantly ses. In addition, it is the result of a complex bal-
limits CD8+ T-cell growth [39]. IL-12 also plays ance between angiogenic and antiangiogenic
an important role in the establishment of mem- factors. The balance between angiogenic and
ory CD8+ T cells [40]. A strong specic CTL angiostatic molecules in the tumor microenviron-
response was observed in patients with advanced ment can determine tumor growth and survival.
melanoma after administration of IL-12. The The antiangiogenic properties of IL-12 were rst
number of tumor-specic CTL increased in the observed by Voest et al. who demonstrated that
circulation, and inux of specic memory CD8+ IL-12 treatment almost completely inhibited neo-
T cells into metastasized lesions was docu- vascularization in immunocompetent mice,
mented [41]. Additionally, IL-12 was shown to severe combined immunodecient mice, and
stimulate humoral immunity. In a model of colon T-cell-decient nude mice [45]. However, sup-
carcinoma, vaccination with IL-12-transduced pression of angiogenesis by IL-12 was dependent
tumor cells cured 40 % of tumor-bearing mice. on its ability to induce IFN- expression.
98 M. Gopal
Accordingly, administration of IFN- reproduced age, and NK cell inltration surrounding small
the antiangiogenic effects promoted by IL-12. vessels [54]. These results documented that
Moreover, it was shown that inhibition of tumor NK cell cytotoxicity of endothelial cells is a
growth by IL-12 or IFN- required an intact sig- potential mechanism by which IL-12 can sup-
naling from IFN- receptors expressed in neo- press neovascularization. The antiangiogenic
plastic cells. This indicated that IL-12 could program activated in lymphocytes by IL-12 can
inhibit tumor growth by inducing neoplastic cells also directly affect gene expression in neoplastic
to produce antiangiogenic factors. Two of the cells. In fact, upregulation of signal transducers
most relevant factors were identied as the IFN-- and activators of transcription-1 (STAT-1) and
inducible genes, IFN-inducible protein 10 (IP- angiopoietin 2 together with down-modulation
10) and monokine induced by interferon- (MIG) of vascular endothelial growth factor (VEGF)
[46, 47]. Local and systemic treatment with IL-12 has been observed in neoplastic cells exposed
was associated with the expression of IFN-, to soluble factors released by IL-12-stimulated
IP-10, and MIG in the tumor; in addition, intra- lymphocytes [55]. In addition, IL-12 treat-
tumor delivery of MIG into subcutaneously ment reduced the production of metallopro-
growing tumor in nude mice led to tumor necro- teases, playing a role in matrix remodeling, a
sis associated with vascular damage. process required during neoangiogenesis [56].
Administration of neutralizing Abs to IP-10 and Moreover, the activation of integrin V3 on
MIG substantially reduced the antitumor effects endothelial cells is reduced by the IL-2-induced
of IL-12 [48]. IP-10 and MIG interact with their IFN-, which leads to decreased endothelial cell
receptor CXCR3 to mediate their angiostatic adhesion and survival [57]. IL-12-induced secre-
activity. These results support the notion that tion of IFN- leads to an increase in p53 activity,
these chemokines, both ligands of the receptor which subsequently results in tumor suppression
CXCR3, contribute to the antitumor effects of due to the induction of apoptosis in cancer cells
IL-12 through their inhibitory effect on tumor [58]. Furthermore, IL-12 dramatically decreased
vasculature. In addition to IFN- stimulation, tumor-supportive activities of tumor-associated
IL-12 promotes the expression of interferon regu- macrophages (TAMs), which are involved in
latory factors 1 (IRF-1) and 4 (IRF-4), which are tumor angiogenesis and metastasis. The anti-
necessary for Th1 cell differentiation [49]. IRF-1 angiogenic mechanisms mediated by IL-12 are
has tumor suppressor activities in cancer cells complex and dependent not only on the direct
in vitro and decreases the tumorigenicity of cells effect on endothelial cells of the proinamma-
inoculated into athymic nude mice [50, 51]. tory cytokine/chemokines induced by IL-12 but
Similarly, IRF-4 suppresses c-Myc-induced leu- also on the recruitment of immune effector cells
kemia in animal models and inhibits BCR/ABL- such as NK and T cells.
induced B-cell acute lymphoblastic leukemia
[52, 53]. 7.2.1.4 Regulation of IL-12 in Tumor
Emerging evidence indicates the involve- Microenvironment
ment of lymphocyte-endothelial cell crosstalk Although controlled Th1 and CTL responses can
at the beginning of the process of angiogen- exert a signicant antitumor immunity, the same
esis inhibition by IL-12. It has been shown that responses, if exaggerated, may result in host-
neutralization of NK cell function reversed tissue destruction and autoimmunity. Therefore,
IL-12 inhibition of angiogenesis in athymic as a part of immune homeostasis, the inamma-
nude mice. Immunohistochemistry analysis tory responses need to be counter-regulated.
revealed that neovascularization inhibited by Tregs play a major role in controlling unwanted
IL-12 displayed accumulation of NK cells and immune response to self-antigens [59]. Studies
IP-10-positive cells. In addition, experimental have revealed a signicant role for Tregs in defec-
Burkitt lymphomas treated locally with IL-12 tive immune responses to tumor antigens. Treg
displayed tumor tissue necrosis, vascular dam- functions are mediated in part through secre-
tion of immunosuppressive cytokines IL-10 and only one partial response (renal cell carcinoma)
TGF-. Both TGF- and IL-10 can inhibit DC and one transient complete response (mela-
antigen presentation, IL-12 secretion, and effec- noma), among the 40 enrolled patients. However,
tor functions of both CD4+ and CD8+ T cells common signs and symptoms of toxicity such as
[12]. Thus, it is possible that as an immunosup- fever/chills, nausea, vomiting, fatigue, and head-
pressive environment develops in the growing ache were observed [64]. Administration of IL-12
tumor, DCs secreting IL-12 become scarce. This resulted in stabilization of the disease in several
might be due to an absence of DC activation sig- renal cancer patients and partial regression of a
nals, CD40, or inhibition of activated CD4+ T metastatic lesion, but has not proceeded further
cells which could themselves activate in clinical development due to signs and symp-
DC. Moreover, the CD40-CD40L interaction toms of toxicity, including fever, vomiting, and
between DCs and T cells leads to the induction elevation of hepatic enzymes [65]. Clinical trials
of not only IL-12 but also IL-10, a pro-tumor of IL-12 treatment in combination with rituximab
cytokine that may act in an autocrine or a para- in patients with B-cell non-Hodgkin lymphoma
crine manner to downregulate IL-12 secretion (NHL) did not result in clinical response [66].
from DCs [60]. Indeed, reduced CD40 expres- However, several clinical studies revealed posi-
sion on DC or CD40-L on T cells from tumor- tive results with IL-12 administration. During
bearing hosts may explain the reason for reduced IL-12 treatment in patients with NHL, 21 % of
levels of IL-12 observed in patients with cancer the patients had a partial or complete response
[61]. In accordance with this, reduced expres- without major side effects [67]. Similarly, sub-
sion of IL-12 was observed in patients with cutaneous IL-12 treatment resulted in complete
advanced cancer types including glioblastoma, response in 56 % of the treated patients with T-cell
renal cell carcinoma, head and neck squamous lymphoma with minor toxicity [68]. Furthermore,
cell carcinoma, gastric cancer, melanoma, clinical trials on metastatic melanoma revealed
colorectal cancer, hepatocellular carcinoma, and that IL-12 administration induces tumor shrink-
gastric cancer [15]. Moreover, IL-12 production age in patients accompanied with increased fre-
by stimulated peripheral blood mononuclear quency of circulating antitumor CTLs [41]. The
cells decreased signicantly in patients with gas- low efcacy of IL-12 in the abovementioned
tric and colorectal cancer with advanced disease. clinical trials may be due to an immunosuppres-
In addition to the immunosuppressive cytokines sive microenvironment in advanced tumors. In
TGF- and IL-10, other factors present in the addition, IL-12 may self-limit its own therapeutic
tumor microenvironment can downregulate efcacy by inducing IL-10 and other suppressive
IL-12 production, for example, prostaglandin E2 factors. For example, IFN- induced by IL-12
(PGE2) produced by tumor cells or tumor- can activate immunoregulatory molecules such as
associated host cells (macrophages, endothelial programmed death ligand-1 (PD-L1) and indole-
cells, and stromal cells) known to inhibit IL-12 amine 2,3 dioxygenase (IDO) on a variety of cells
production [62]. (DC, T cells, and endothelial cells) [69]. Both
PD-L1 and IDO can abrogate antitumor immu-
7.2.1.5 Clinical Studies with IL-12 nity through various mechanisms. Furthermore,
Based on the provocative preclinical studies, other factors such as environment and diet may
IL-12 was evaluated in patients with differ- alter the effectiveness of IL-12-mediated antican-
ent malignancies. However, clinical experience cer immunity. Although systemic administration
with IL-12 in humans is limited. Several phase of IL-12 in patients is limited by its signicant
I clinical trials of IL-12 in patients with solid toxicity, emerging studies in animal models indi-
tumors and hematological malignancies have cate that IL-12 in combination with other cyto-
been reported [63]. IL-12 administration in kines boosts antitumor immunity without any
patients with advanced colorectal cancer (CRC), toxic side effects [44]. Thus, selective targeted
melanoma, and renal cell carcinoma resulted in delivery of IL-12 to tumors and/or reducing the
100 M. Gopal
dose of IL-12 while combining it with other ther- with these observations, IL-27R/ mice failed
apeutics may yield better outcome. to regulate tumor growth in vivo, reiterating the
importance of IL-27 signaling in the genera-
tion of antitumor immunity [82]. Most recently,
7.2.2 IL-27 DC-derived IL-27 has been shown to induce
NK and NKT cell-dependent antitumor immu-
7.2.2.1 Overview nity against methylcholanthrene-induced bro-
IL-27 is a member of the IL-12 family cytokine sarcoma and transplanted B16 melanoma [83].
that plays potent antitumor effects against vari- Moreover, IL-27 in combination with other cyto-
ous tumor models via different mechanisms, kines such as IL-2 and IL-12 boosts antitumor
depending on the characteristics of each tumor immunity by contributing to the development of
[70]. Unlike IL-12, IL-27-mediated antitumor CTLs and NK cells [84].
functions are independent of IFN-, and IL-27- In addition to the direct effect of IL-27 on
treated mice do not manifest any toxic side CD8+ T-cell activation, the inuence of IL-27 on
effects. IL-27 is mainly produced by activated CD4+ T-cell responses might provide further
APCs including DCs and macrophages. DCs therapeutic opportunities. Initial studies have
secrete IL-27 on exposure to physiological indicated that IL-27 leads to the differentiation of
stimuli such as type I and type II interferons Th1 cells [85]. IL-27 synergizes with IL-12 to
(INF) and CD40 [7173]. In addition, IL-27 enhance IFN- production [86]. Moreover, it has
expression is induced in APCs upon stimula- been shown that IL-27 inhibits Th2 polarization
tion by various TLR ligands such as poly(I:C), of nave CD4+ T cells and suppresses Th2 cyto-
lipopolysaccharide (LPS), and CpG-DNA, kine production from in vitro polarized Th2 cells
which are agonists of toll-like receptors [8789]. By altering the balance between Th1
(TLR)3, TLR4, and TLR9, respectively and Th2 cytokines, IL-27 plays a critically impor-
[7476]. tant role in antitumor immune responses. In line
with this, a recent study conrmed IL-27s capa-
7.2.2.2 IL-27 in Antitumor Immunity bility in the reversion of the Th2 polarization of
IL-27 has a wide array of functions necessary in vivo primed lymphocytes from pancreatic can-
for the induction of antitumor immune response. cer patients [90]. IL-27-dependent enhancement
IL-27 has been shown to act on NK cells to of preexisting antigen-specic Th1 responses has
enhance their cytotoxic activity both in vitro also been demonstrated [36]. IL-27 may promote
and in vivo; in addition, therapeutic administra- tumor regression through the inhibition of Tregs.
tion of IL-27 increased NK cell susceptibility of IL-27 inhibits the generation of Foxp3+ Tregs
tumors [77]. By activating NK cells, IL-27 might both in vitro and in vivo. IL-27 blocks Treg dif-
enhance adaptive immunity to tumors in part; the ferentiation through a mechanism that is at least
killing of tumor target by NK cells could provide partially dependent on STAT-3 [91, 92]. In addi-
DCs with increased access to tumor antigens; tion, IL-27 can limit Treg cell expansion by
thus, IL-27 serves as a link between innate and inhibiting IL-2, a cytokine necessary for Treg
adaptive antitumor immunity. In addition to NK development [93]. In a murine model of neuro-
cell activation, IL-27 acts on CD8+ T cells and blastoma, IL-27 has been shown to inhibit IL-2-
induces the generation of CTL through enhanc- induced Treg expansion in the tumor, promoting
ing the expression of effector molecules such as antitumor immune responses [84]. IL-27 also
granzyme B and perforin [78]. Similar to mice, induces tumor-specic Ab response which coop-
IL-27 promotes IFN- and granzyme B produc- eratively elicits ADCC activity [94].
tion from human CD8+ T cells [79]. The over-
expression of IL-27 in highly immunogenic 7.2.2.3 Direct Effect of IL-27 on Tumors
murine tumor cells facilitated CTL development IL-27 possesses multiple antitumor effects medi-
with enhanced IFN- production [80, 81]. In line ated by mechanisms involving angiogenesis
and its direct effects on tumors. IL-27 has been ne balance with other myeloid cell populations
shown to have antiproliferative activities which especially tumor-inltrating macrophages and
inhibit tumor growth and metastasis in murine DCs expressing OPN and IL-27.
melanoma [95]. The major antitumor role of
IL-27 relies on its antiangiogenic property of sur- 7.2.2.4 Advantages of IL-27 Over IL-12
rounding endothelial cells and broblasts. IL-27 in Tumor Immunity
signicantly inhibited tumor growth in SCID IL-27-mediated antitumor mechanisms are com-
mice through the induction of antiangiogenic plex. Similar to IL-12, IL-27 utilizes effector
factors such as IP-10 and MIG from endothelial mechanisms of innate and adaptive immunity
cells [96]. Consistent with these results, IL-27 to mediate antitumor immunity. IL-27 promotes
has been shown to directly act on human umbili- tumor immunity through the induction of Th1
cal cord endothelial cells and induce production and CTL responses while inhibiting immu-
of the antiangiogenic chemokines such as IP-10 nosuppressive Th2 and Tregs. Unlike IL-12,
and MIG [97]. IL-27 strongly inhibited tumor IL-27-mediated antiangiogenic functions are
growth of primary multiple myeloma (MM) independent of IFN-. Thus, IL-27-treated mice
cells through inhibition of angiogenesis [98]. In are not observed with any toxic side effects [104].
addition, IL-27 downregulated a wide panel of The central role of IL-27 in orchestrating both the
proangiogenic genes, including matrix metallo- innate and adaptive arms of immunity together
proteinase-9 (MMP-9), TGF-, and VEGF with a with multiple antiangiogenic functions explains
concomitant upregulation of the angiostatic che- the essential contribution of this molecule to
mokines IP-10 and MIG. the development of antitumor immunity against
IL-27 may further promote tumor regression both high and poor immunogenic tumors. These
through the inhibition of a proangiogenic cyto- observations together with the lack of toxicity
kine IL-17. IL-27 suppresses the Th17 key tran- observed in vivo in preclinical trials with IL-27
scription factor RORt and thus inhibits treatment highlight the enormous therapeutic
expression of IL-17 by T cells both in humans potential of this approach.
and mice [99, 100]. Accordingly, mice decient
in either the IL-27 EBI3 subunit or IL-27R have
increased levels of IL-17 [101]. Among the Th17 7.3 Cytokines in Immune
suppressive molecules found in the tumor micro- Tolerance to Cancer
environment, IL-27 is one of the most potent
inhibitors of Th17 differentiation. IL-27 can be Although certain cytokines produced in the tumor
induced in tumor-inltrating DCs by galactin-1, microenvironment can function to inhibit tumor
IFN-, and apoptotic tumor cells in the tumor growth, others can promote tumor growth and
microenvironment [71, 102, 103]. However, the progression. Several cytokines were found to
proangiogenic molecules which dominate the serve as growth and survival factors that act on
microenvironment in advanced tumors can limit premalignant cells, stimulate angiogenesis and
the availability of IL-27. Osteopontin (OPN), a metastasis, and maintain tumor-promoting
proinammatory cytokine, inhibits the expres- immunosuppression and inammation.
sion of IL-27 in DCs while inducing Th17 dif-
ferentiation [72]. OPN promotes tumor growth
through mechanisms involving angiogenesis, 7.3.1 TGF-
tumor migration, and metastasis, suggesting that
OPN may release the brake on Th17 cell 7.3.1.1 Overview
responses by suppressing IL-27 in DCs. Both Transforming growth factor- (TGF-) is a pleio-
OPN and IL-27 are expressed in DCs and macro- tropic cytokine with broad tissue distribution that
phages; thus, the outcome of Th17 accumulation plays critical roles during embryonic develop-
in tumor microenvironment may depend on the ment, normal tissue homeostasis, and cancer [105].
102 M. Gopal
Fig. 7.2 TGF--mediated Innate immunity

immunosuppression. TGF-
affects components of both
innate and adaptive immune
DC maturation
systems. TGF- inhibits NK
cell activation and its effector
function. In addition, TGF- Th1
Ma ti on
inhibits DC maturation and cro iva
pha ct
antigen-presenting function ge e ll a
while promoting polarization pol
ariz Tc Th2
atio 4+
of M2 macrophages. TGF- n CD
inhibits CD8+ T-cell-
mediated antitumor immune Treg
response. TGF- also has a
signicant impact on CD4+
T-cell differentiation and CD Th17
on 8+
function. TGF- induces vati Tc
cti ell
ll a
B cell activation
Treg and Th17 differentiation ac
ce tiv
ati
while inhibiting Th1 and Th2 NK on
differentiation. Furthermore,
TGF- inhibits B-cell
proliferation and antibody
secretion
Adaptive immunity
Elevated TGF- serum concentrations were TGF- growth inhibitory signals which is an
observed in patients with different malignancies important reason for the shift from being a tumor
and were associated with poor prognosis. TGF- suppressor to a tumor promoter. Subsequently,
is released not only by a variety of cells in human cancer cells start secreting nonphysiological lev-
and murine tumors including T cells, macro- els of TGF- in an autocrine and paracrine man-
phages, and DCs but also by tumor cells them- ner which may affect the differentiation of the
selves [106]. Almost all human cell types are tumor cells and the surrounding cellular environ-
responsive to TGF-, which signals through type ment, respectively, leading to development of the
I and type II TGF- receptors (TRI and TRII, tumor and metastasis [108]. Notably, TGF-
respectively). Upon binding of TGF- to TRII, induces epithelial-mesenchymal transition
TRI is recruited and activated to phosphorylate (EMT), whereby epithelial tumor cells acquire an
the downstream mediators, SMAD2 and SMAD3. invasive, mesenchymal-like phenotype accompa-
Phosphorylated SMAD2 and SMAD3 combine nied by changes in the expression of cell-cell
with SMAD4 to enter the nucleus to modulate adhesion molecules and secretion of metallopro-
gene transcription [107]. teinases, leading to metastasis [109, 110]. The
The function of TGF- in cancer is complex. potent regulatory activity of TGF- on immune
TGF- can act as a tumor suppressor or a tumor cell functions represents an important mechanism
promoter depending on the stages of tumor devel- of immune tolerance to tumors. The presence of
opment. Initially, it acts as a tumor suppressor TGF- in the microenvironment of the develop-
since it induces apoptosis and inhibits the growth ing tumor is predicted to disable effective immu-
of normal and premalignant tumor cells [108]. At nosurveillance by multiple mechanisms, most of
later stages of tumor progression, TGF- acts as which converge on the impairment of tumor cell
a tumor promoter. At this stage, cancer cells pro- killing by innate and adaptive immune cells
tect themselves and tend to acquire resistance to (Fig. 7.2).
7.3.1.2 Effect of TGF- on Innate Macrophages, the predominant leukocyte,

Immunity to Tumors play a key role in tumor growth. The role of
TGF- is an important regulator of NK cell tumor-associated macrophages (TAMs) in
function, being a potent antagonist of IL-12- tumors is controversial [123]. TAMs origi-
induced production of IFN- in NK cells [111]. nate from recruited myeloid cells, such as
In addition, TGF- inhibits the activity of NK blood monocytes or MDSCs by a number of
cells by limiting expression of the activating chemoattractants produced by tumor cells
receptor NKG2D, NKp30, and DNAM-1 [112]. and stromal cells. Tumor-derived chemokine
In fact, reduced expression of NKG2D is associ- CCL2 is critical for the recruitment of macro-
ated with elevated levels of TGF- in patients phages to the tumor site [124]. Macrophages
with cancer. It has been shown that surface- can exert different properties when polarized
bound TGF- on MDSCs can inhibit NK cell with distinct inducers. Differential cytokine
cytolytic activity against mammary adenocarci- production is a key feature of polarized mac-
noma [113, 114]. Moreover, TGF- has been rophages. When stimulated with IFN-, M1
shown to suppress MHC class I and MHC class macrophages secrete high levels of IL-12, but
II expression in a number of cell populations low levels of IL-10. In contrast, M2 macro-
[115117]. Importantly, the TGF--dependent phages express high levels of IL-10 but low
decrease of MHC class I expression in tumor levels of IL-12 [125]. Due to their different
cells has been shown to result in reduced tumor cytokine proles, these polarized macrophages
cell lysis by NK cells [117]. Although NK cells have distinct functions. For example, the IL-12
are the major innate effectors, they require acti- produced by M1 macrophages can promote the
vation by DCs. TGF- has been demonstrated to differentiation of Th1 cells, which can improve
impair DC function both in vitro and in vivo. antigen phagocytosis and contribute to antitu-
TGF- inhibits upregulation of critical co-stim- mor immunity. Whereas, the IL-10 expressed
ulatory molecules on the surface of DCs and by M2 macrophages can promote the produc-
reduces cytokine production and their antigen- tion of IL-4 and IL-13 by Th2 cells. Both IL-4
presenting capacity [118, 119]. TGF- can and IL-13 have been shown to impair antitu-
immobilize DCs, thereby interfering with their mor T-cell responses. TGF- promotes tumor-
migration and the transport of antigen to drain- associated macrophage polarization to an M2
ing lymph nodes for presentation to T cells. vs. M1 phenotype, which further promotes
Moreover, TGF- can also induce apoptosis of TGF- production and deepens immunosup-
DCs [120]. In recent years, more correlative pression [126]. In most tumors, the inltrated
clinical data has supported the inhibitory role of macrophages are considered to be of the M2
TGF- in the observed defects in cancer. phenotype. TAMs orchestrate various aspects
Increased serum TGF- in human colorectal of cancer, such as tumor progression, angio-
cancer correlates with reduced circulating DCs genesis, metastasis, and immunosuppression
[121]. Moreover, tumor-inltrating DCs both [127]. It has been shown that NKT cells can
secrete and respond to TGF-, in either an auto- suppress CTL responses through mechanisms
crine or paracrine manner. These TGF-- involving TGF-. Therefore, blockade of
secreting DCs promote the formation of Tregs TGF- signaling not only enhances the fre-
[122] that potently inhibit the function of other quency of antitumor CTLs but also restores the
T cells, and that Treg production of TGF- can activities of the cytolytic machinery and pre-
inhibit NKG2D-mediated NK cell cytotoxicity, vents NKT cell-mediated immunosuppression
thereby enhancing tumor growth and metastasis. [128]. Furthermore, TGF- also inhibits effec-
In addition to DCs, TGF- can suppress or alter tor functions of other innate immune cells such
the activation and function of other innate as -T cells [106]. Thus a dampened innate
immune cells such as NKT cells, macrophages, immune response leads to poor adaptive immu-
and neutrophils [106]. nity, resulting in persistence of the tumor.
104 M. Gopal
7.3.1.3 Effect of TGF- on Adaptive limit antitumor resistance. In contrast, depletion

Immunity to Tumors of Tregs with anti-CD25 Ab in animal models
The presence of TGF- in the tumor microenvi- enhances antitumor immunity and tumor regres-
ronment can have a profound impact upon antitu- sion, further suggesting the involvement of Tregs
mor activity of T cells. It has been shown that in tumor growth. Furthermore, when tumor-spe-
TGF- can suppress CTL differentiation and cic CD8+ T cells were adoptively transferred
CTL-mediated lysis of tumor cells [129, 130]. with either Tregs or CD4+CD25 T cells into
TGF- acts on CTLs to specically repress the hosts with tumor, CD8+ T-cell-mediated immu-
expression of different cytolytic effector mole- nity was abolished in those receiving Tregs but
cules such as perforin, granzyme A, granzyme B, not CD4+CD25 T cells [137, 138]. Collectively,
Fas ligand (FasL), and IFN-, which are collec- these studies provide strong evidence that Tregs
tively responsible for CTL-mediated tumor kill- can attenuate the antitumor immunity by down-
ing [131]. Blockade of TGF- in tumor models regulating the antitumor immune responses and
has been shown to reduce tumor burden by ultimately facilitating the development of cancer.
improving CD8+ T-cell-mediated tumor immu-
nity [131]. Furthermore, TGF- could suppress 7.3.1.4 TGF-, Treg, and Tumor
IL-2 production and IL-2-induced T-cell differ- Angiogenesis
entiation [132]. Tumor cells transfected with Angiogenesis and tumor-associated immuno-
TGF- were shown to attenuate the efcacy of suppression are hallmarks of tumorigenesis. The
DC-based tumor vaccines [118]. In addition, association between angiogenesis and immunosup-
TGF- functionally regulates the differentiation pression is related to hypoxia which induces both
of T helper cell subpopulations both in vitro and angiogenesis and immunosuppression via activa-
in vivo. TGF- inhibits Th1 and Th2 cells, tion of hypoxia-induced factor 1 (HIF-1). The
whereas it promotes Treg and Th17 cell differen- induction of VEGF during hypoxia is primarily
tiation [133]. Most recently, TGF- has also been mediated by HIF-1. HIF-1-induced VEGF pro-
shown to play an important role in the develop- motes angiogenesis by inducing the recruitment
ment of IL-9-secreting Th9 cells [134]. of various proangiogenic bone marrow-derived
Although there are many sources of TGF- in cells including endothelial progenitors and
the tumor microenvironment, it has been shown myeloid cells [139]. Although hypoxia-VEGF
that Tregs can provide a signicant source of axis has been thought to be solely involved in
TGF- responsible for attenuation of tumor anti- vascular growth and permeability, recent stud-
gen expanded CTLs. Tregs hamper the functions ies suggest its role in immunosuppression in
of Th1, CD8+ T cells, NK cells, DCs, and other the tumor microenvironment. Within the tumor
key effector cells of antitumor immunity [106]. microenvironment multiple cell types with
Accordingly, Treg-mediated immunosuppression established roles in immunosuppression have
has been proposed to be one of the important been shown to promote angiogenesis. Among
mechanisms involved in tumor immune evasion. the immunosuppressive cell types found in the
An accumulation of Tregs in tumors can dampen tumor microenvironment, Tregs are consid-
T-cell immunity to tumors and is thus the main ered pivotal regulators of immunosuppression.
obstacle to successful immunotherapy [59]. The Tregs can trafc to tumors from the periphery
frequency of Tregs present in the peripheral blood under the inuence of chemokines in the tumor
of patients with various cancers is higher than microenvironment. It has been shown that tumor
that of normal population [135]. Notably, Tregs hypoxia leads to the recruitment of Tregs via
isolated from peripheral blood and solid tumors chemokine CCL28 [140]. Forced expression of
remain suppressive to T-cell activation in vitro CCL28 in mouse tumor cells resulted in accel-
[136]. Likewise, Tregs from tumor-bearing mice erated tumor growth and Treg accumulation
inhibited tumor rejection, indicating that Treg associated with increased VEGF levels and
cells suppress tumor-specic immunity and angiogenesis. In addition, Tregs were shown
to express CCR4, the receptor for CCL22, and improve tumor immunity. There are many TGF-
can therefore migrate to CCL22 present in the signaling antagonist agents under development at
tumor microenvironment [141, 142]. Beyond both the preclinical and clinical stages. Mice with
recruitment of Tregs through chemokines, the fully or partially disrupted TGF- function have
tumor microenvironment promotes the continued phenotypes with severe self-reactive immune
expansion of Tregs and the generation of Tregs responses [146, 147]. However, clinical trials of
due to a tumor microenvironment rich in TGF-. TGF- antagonists, such as a monoclonal Ab or
The recruited Tregs in turn dampen the antitumor small molecules that interfere with TGF- recep-
immune response and promote angiogenesis. The tor signaling, in cancer patients have been tested
accumulation of Tregs at tumors has been cor- and are ongoing. In phase I/II clinical trials, intra-
related with VEGF overexpression and increased tumoral administration of AP-12009, an anti-
angiogenesis in cancers, providing evidence for sense oligonucleotide to TGF-, resulted in a
an association between Tregs and angiogenesis signicant increase of survival time [148]. Some
[143, 144]. Tregs can contribute to tumor angio- clinical benet without apparent induction of
genesis through different mechanisms. They pro- autoimmune disease was found in a clinical trial
mote angiogenesis indirectly by suppressing Th1 of a human monoclonal anti-TGF- in melanoma
cells that release angiostatic cytokine IFN-, as patients [149, 150]. In addition, a vaccine that
well as interferon-induced chemokines such as contains allogeneic tumor cells that are modied
CXCL9 and CXCL10. Indeed, Tregs have been to express antisense TGF- has been tested in a
shown to promote tumor angiogenesis by spe- phase I/II clinical trial. Using this approach, a
cically inhibiting tumor-reactive T cells. Tregs response rate of 30 % has been reported in non-
can signicantly contribute to the direct promo- small cell lung carcinoma (NSCLC), with no
tion of tumor angiogenesis through the induc- serious toxicities observed [151]. LY2157299 is a
tion of VEGF and endothelial cell proliferation small molecule inhibitor which is selective for
[144]. Additional therapeutic opportunities may the kinase domain of the type 1 TGF- receptor.
be provided by Tregs capability in suppress- LY2157299 is currently being evaluated in
ing tumor-specic immunity while promoting patients with metastatic malignancies.
tumor angiogenesis by well-planned manipu-
lations of Tregs, including depletion, block-
ing trafcking into tumors, and reducing their 7.3.2 IL-17
differentiation and suppressive mechanisms. It
will be benecial to tumor eradication by com- 7.3.2.1 Overview
bining this strategy with various current thera- IL-17 is a proinammatory cytokine produced by
peutic approaches. In an early phase I clinical Th17 cells [152]. In addition to Th17 cells, IL-17
trial in patients with metastatic breast cancer, can also be produced by cells other than T helper
the anti-CD25 Ab daclizumab signicantly cells, such as iNKT, CD8+ T, and -T cells [153
depleted Tregs and enhanced the immunogenic- 155]. Since Th17 cells produce large quantities of
ity of a cancer vaccine [145]. In addition, block- IL-17A, most Th17-mediated effects are attrib-
ing Treg function using Abs targeted against uted to this cytokine. Many factors are required
glucocorticoid-induced tumor necrosis factor for the induction and stabilization of Th17 cells.
receptor-related protein (GITR) and cytotoxic Of these, TGF- and IL-6 are the most crucial
T-lymphocyte antigen 4 (CTLA-4) is under cytokines for its differentiation. IL-6 induces
clinical evaluation in patients with cancer. production of IL-21, which subsequently favors
Th17 differentiation in an autocrine manner [156,
7.3.1.5 TGF- in Clinical Trials 152]. These cells require CD40-induced IL-23 to
As a result of the wide variety of effects of TGF- maintain their Th17 phenotype in vivo. The dif-
on tumorigenesis, blockade of TGF- and its sig- ferentiation of Th17 cells into IL-17-secreting
naling pathway can be a potent approach to cells requires the expression of the transcription
106 M. Gopal
factor ROR-t [157]. It has been shown that Th17 T cells inhibits angiogenesis and induces major
cells are gradually increased in the tumor micro- histocompatibility complex I in tumor cells, thus
environment during tumor development. In addi- favoring immune recognition and subsequent
tion, Th17 cells have been found in patients with arrest of tumor growth [166]. In contrast, IL-17
different tumors. The frequent association of favors angiogenesis and tumor growth; therefore,
raised IL-17 concentrations with negative prog- replacing IFN- with IL-17 in the tumor micro-
nosis links the increased systemic IL-17 con- environment may have severe consequences for
centrations with the later stages of cancer. Many immune recognition and surveillance.
factors released by the tumor cells and molecules
secreted by tumor-inltrating immune cells such 7.3.2.3 Tumor-Promoting Functions
as TGF-, IL-6, prostaglandin E2 (PGE2), IL-21, of IL-17
IL-23, osteopontin, IL-1, and TNF- can play Many functions of IL-17 in the tumor micro-
major roles in the induction of Th17 differentia- environment contribute to tumor progression,
tion [158161]. besides their minor direct effect on the prolifera-
tion and survival of tumor cells [167]. The major
7.3.2.2 Th17 Differentiation in the Tumor pro-tumor role of IL-17 in cancer relies on its
Microenvironment proangiogenic property of the surrounding endo-
There are many sources for Th17 cells in the thelial cells and broblasts. For example, IL-17-
tumor microenvironment. Th17 cells found in the overexpressing human cervical cancer cells and
tumor microenvironment can either be migrated NSCLC cells show greater ability in developing
from the periphery or differentiated from nave tumors in immunocompromised mice compared
T cells under the inuence of tumor microen- with control cells with no IL-17 expression [168,
vironmental factors. Th17 cells can trafc to 169]. In addition, IL-17 overexpression in bro-
tumors from the periphery under the inuence of sarcoma cells enhances their tumorigenic growth
tumor microenvironmental chemokines such as in syngenic mice, primarily owing to the proan-
RANTES and monocyte chemotactic protein-1 giogenic activity of IL-17. Moreover, the levels
(MCP-1) [162]. In addition, high levels of che- of Th17 cells were positively correlated with
mokines CXCL12 and CCL20 have been found microvessel density in tumors [170]. By acting
in tumor microenvironments, which could facili- on stromal cells and broblasts, IL-17 induces a
tate Th17 cell trafcking and migration into the wide range of angiogenic mediators [171, 172]
tumor sites. Moreover, Th17 cells in the tumor including VEGF. In addition, they markedly
microenvironment might clonally expand follow- promote inammatory and tumor angiogenesis
ing stimulation by tumor-associated macrophages [173]. IL-17 is able to upregulate VEGF pro-
[163]. In addition, Th17 cells can be induced duction by broblasts and therefore promotes
and differentiate in the tumor microenvironment broblast-induced new vessel formation in the
[164]. It has become clear that IL-17 producing inammatory microenvironment and tumors. The
Th17 cells and Tregs share a common pathway. IL-17-VEGF loop that modulates angiogenesis
Although TGF- favors differentiation of nave T includes another angiogenic factor, TGF-. IL-17
cells into Tregs, the simultaneous presence of both induces VEGF, which in turn induces TGF- fol-
TGF- and IL-6 promotes the differentiation of lowed by VEGF-mediated angiogenesis [174].
Th17 cells. Given the tight association of TGF- TGF- enhances the VEGF receptivity of endo-
and IL-6 with tumor incidence and progression, thelial cells by increasing VEGF receptor expres-
nave T cells entering an established tumor are sion [175]. IL-17 also induces IL-6 and PGE2
more likely to be exposed to conditions favor- and enhances intercellular adhesion molecule
ing Th17 differentiation. Upon stimulation with (ICAM)-1 expression in broblasts. All these
TGF- and IL-6, CD8+ T cells not only lose their molecules were known to have a major role in
cytotoxic ability but are also induced to secrete angiogenesis and tumor invasion. IL-17 appears
IL-17 [165]. IFN- expressed by Th1 or CD8+ to stimulate production of IL-8 [176]. IL-8 sig-
naling promotes angiogenic responses in endo- of IL-17. Th17-polarized cells were found to be
thelial cells, increases proliferation and survival more effective than Th1 cells in eliminating large
of endothelial and cancer cells, and potentiates established tumors [182]. However, the Th17-
the migration of cancer cells and inltrating mediated tumor responses were highly dependent
neutrophils at the tumor site. Moreover, IL-17 on IFN--based mechanisms. Indeed, the effects
was found to induce IL-1 and TNF- in mac- of Th17-polarized cells were completely abro-
rophages and cytokines which can further syn- gated by the administration of IFN--depleting
ergize with IL-17 to activate neutrophil-specic Ab and not by IL-17- or IL-23-depleting Abs.
chemokine, thereby recruiting neutrophils to the Adoptively transferred IL-17-secreting CD8+ T
site of inammation [177]. cells also enhanced antitumor immunity resulting
One of the most important mechanisms under- in regression of B16 melanoma [183]. In addi-
lying IL-17 regulation of inammation which tion, IL-17 has been shown to inhibit the growth
orchestrates the tumor microenvironment is of hematopoietic tumors such as mastocytoma
through NF-B signaling, the master regulator of and plasmocytoma by enhancing CTL activity
the inammation [178]. IL-17R signaling results [184]. Different mechanisms have been proposed
in the activation of NF-B and regulates the activi- for the IL-17 enhancement of tumor-specic
ties of extracellular-regulated kinase 1 (ERK1), CTLs. IL-17 has been shown to induce IL-6 from
ERK2, c-Jun N-terminal kinase, and p38 mitogen- a variety of cells. Moreover, IL-17 stimulation
activated protein kinases [179, 180]. While the can induce IL-12 production from macrophages
IL-17-mediated cytokine expression is regulated [185]. Both IL-6 and IL-12 have been associated
primarily by NF-B, the same cytokines can fur- with induction of tumor-specic CTL. IL-17 pro-
ther stimulate NF-B-mediated transcription of motes maturation of DC progenitors as indicated
themselves in tumor cells and tumor-associated by increased expressions of co-stimulatory mol-
stromal cells, thereby creating a sustained chronic ecules, MHC-II antigens, and allostimulatory
inammatory state within the tumor microenvi- capacity [186]. This may lead to further improve-
ronment. In support of this notion, enhanced cervi- ment in T-cell priming by tumor cells producing
cal cancer growth elicited by IL-17 was associated IL-17. In addition, IL-17-transduced brosar-
with increased expression of IL-6 and macrophage coma cells induced tumor-specic antitumor
recruitment to the tumor sites [169]. Therefore, immunity by augmenting the expression of MHC
IL-17 might also function through IL-6 to promote class I and class II antigens [187]. These studies
tumor development. Chemokines can stimulate or were focused on the effects of exogenous IL-17 in
inhibit proliferation and chemotaxis of the blood established mouse tumor cell lines. A recent
vessel endothelial cells which serve the tumor. demonstration shows that tumor growth in subcu-
IL-17 has been shown to selectively enhance the taneous and lung tumor metastasis are enhanced
production of angiogenic chemokines such as in IL-17-decient mice [188]. The effect is
CXCL1, CXCL5, CXCL6, and CXCL8 from accompanied by reduced IFN- levels in tumor-
tumor cells and epithelial cells [168, 181]. In addi- inltrating NK cells and T cells.
tion, IL-17 is also known to inhibit angiostatic The evidence reviewed here demonstrates
chemokine secretion by broblasts [168]. Thus, that IL-17-secreting Th17 cells can either stim-
IL-17 may shift the local biologic balance between ulate or inhibit tumor growth and progression.
angiogenic and angiostatic chemokines toward a The benecial effects of IL-17 on upregulating
predominance of angiogenic chemokines in order host immune response may be present early in
to enhance the net angiogenic activity. inammation, but is eventually overcome by
increasingly large tumor burden. Clearly, as dis-
7.3.2.4 Antitumor Functions of IL-17 cussed above, many of the inammatory func-
Although IL-17 seemed to be a potential tumor- tions of IL-17 can benet the tumor. The shift
promoting cytokine, a considerable number of from benecial inammatory functions of IL-17
reports have described tumor-inhibitory effects to a detrimental one may depend on the tumor
108 M. Gopal
type and inammatory mediators in the tumor contrast to the antitumor role of IL-12, IL-23
microenvironment. The pro-tumor vs. antitumor upregulates inammatory processes, including
effects of IL-17 are thus functions of a number of matrix metalloproteinase expression and angio-
combinations of all these IL-17-induced inam- genesis and reduces inltration and the function
matory mediators and, perhaps, mediators which of CTLs, thus contributing to tumor growth
counter-regulate IL-17 production as well, oper- [196]. Indeed, the mice lacking IL-23/p19 are
ating in tandem. completely resistant to carcinogen-induced
tumor. The absence of tumor formation in these
mice correlated with the absence of various
7.3.3 IL-23 markers including IL-17, GR-1+, and CD11b+
myeloid cells which are indicative of tumor-
7.3.3.1 Overview associated inammation [196]. Recently, tumor-
IL-23 is a heterodimeric protein composed of two secreted lactic acid has been shown to activate
subunits IL-23p19 and IL-12p40. IL-23 is secreted the IL-23/Th17 pathway [159].
by activated DCs and macrophages. IL-23 binds In contrast, IL-23-overexpressing tumors
the IL-23R complex, composed of IL-23R and show reduced growth and metastasis [197201].
IL-12R1. Upon binding IL-23, IL-12R1, and The antitumor effects of IL-23 in these studies
IL-23R associate, marking the beginning of the were found to be mediated through enhancement
IL-23 signal-transduction cascade [189]. IL-23 of CD8+ T-cell response. In addition, intra-
plays an important role in bridging innate and tumoral injection of IL-23-overexpressing DCs
adaptive responses. Therefore, IL-23 has been resulted in a similar phenotype [201]. Articial
described as a key cytokine promoting inamma- overexpression of IL-23 could induce potent anti-
tion in peripheral tissues. The activity of IL-23 in tumor immunity through various mechanisms.
the regulation of tumor immunity is just begin- IL-23 can mediate myeloid inltration consisting
ning to be elucidated [190]. of DCs, macrophages, and granulocytes, which
instead may contribute to the inhibition of tumor
7.3.3.2 Pro- vs. Antitumor Functions growth and boost an immune reaction to these
of IL-23 immune-sensitive tumors. In addition, overex-
Belonging to the IL-12 family, IL-23 performs pression of IL-23 is likely to increase the sys-
both pro-tumor and antitumor functions. IL-23 is temic IL-23 levels that could lead to the growth
spontaneously produced by TAM in several and survival of memory CD8+ T cells.
mouse tumor models. Tumor-secreted PGE2
enhances the production of IL-23 and IL-1 in
macrophages and DCs while downregulating 7.3.4 IL-35
IL-12 production [191193]. While IL-12 pro-
duction is decreased, IL-23 production is 7.3.4.1 Overview
increased in tumors [194]. PGE2, together with IL-35 is a recently discovered IL-12 family cyto-
IL-23, favors the expansion of human Th17 cells kine composed of an IL-12 p35 subunit and an
from PBMCs; on the other hand, PGE2 enhances IL-12 p40-related protein subunit, EBI3 [202].
IL-17 production from memory CD4+ cells IL-35 is not constitutively expressed in tissues
induced by IL-23 [161]. The involvement of and is produced mainly by Tregs. IL-35 induces
IL-23 in the induction of Th17 was established the transformation of CD4+ effector T cells into
when investigators showed that IL-23 promotes Tregs, which in turn express IL-35 but lack the
the production of IL-17 by activated T cells expression of conventional Treg markers such as
[195]. Although IL-23 is not involved in the ini- Foxp3, TGF-, and IL-10 (Treg35 cells) [203].
tial differentiation of Th17 cells, it is crucial for The Treg35 cells generated in vitro can pre-
the function, survival, and propagation of this vent the development of autoimmunity in vari-
T-cell population in the inamed environment. In ous mouse models [204207]. Most recently, it
has been shown that human Tregs express and factor [214]. The function of IL-10 in cancer is
require IL-35 for maximal suppressive function. enigmatic. Depending on the experimental
Substantial upregulation of EBI3 and IL12A, model, IL-10 displays both immunosuppressive
but not IL10 and TGF-, was observed in acti- and immunostimulating activities. On the one
vated human Tregs compared with conventional hand, IL-10 promotes an antitumor CTL response
T cells [208]. leading to tumor regression. On the other hand,
IL-10 induces immunosuppression and assists in
7.3.4.2 Pro-tumor Functions of IL-35 the escape from tumor immune surveillance,
Evidence on the role of IL-35 in tumor immunity hence promoting tumor growth.
is beginning to emerge. IL-35 subunit EBI3 is
expressed in Hodgkin lymphoma cells, acute 7.3.5.2 IL-10-Mediated
myeloid leukemia cells, and lung cancer cells Immunosuppression in Cancer
[209211]. Small interfering RNA silencing of The cellular sources of IL-10 are Th2, Treg, Tr1,
EBI3 in lung cancer cells inhibits cancer cell pro- and Th17 cells; however, cytotoxic CD8+ T cells
liferation, whereas stable expression of EBI3 in can also produce IL-10, as can some subsets of
lung cancer cells confers growth-promoting DCs, macrophages, B cells, granulocytes, mast
activity in vitro [211]. High EBI3 expression in cells, keratinocytes, and epithelial cells. In addi-
human lung cancer cells was shown to be associ- tion, various cancer cells produce IL-10; among
ated with poor prognosis [211]. Recently, IL-35- those are multiple myeloma, melanoma, human
secreting Ag-specic Tregs have been observed colon carcinoma, lung cancer, oral squamous cell
in patients with prostate cancer [212]. Treg- carcinoma, renal cell carcinoma, non-Hodgkin
derived IL-35 was shown to inhibit antitumor lymphoma, and chronic lymphocytic leukemia
T-cell responses. In vitro generated Treg35 cells [15, 215]. Circulating concentrations of IL-10
accelerate the development of B16 melanoma were raised in patients with different cancer types
and prevent the generation of antitumor CD8+ and were associated with adverse disease stage or
T-cell responses [203]. In addition, T cells that with negative prognosis in those patients. It has
secrete IL-35 and have suppressive functions can been shown that serum levels of IL-10 increased
be induced in the tumor beds of melanoma and in parallel to clinical disease progression in
colorectal adenocarcinoma. Blockade of IL-35 patients with metastatic melanoma, as well as
has been shown to relieve suppression mediated colon cancer. Moreover, preoperative serum lev-
by Tregs [212]. Forced expression of IL-35 in els of IL-10 predicted the likelihood of colon
tumor cells leads to signicantly increased cancer recurrence [215, 216]. IL-10 can be
tumorigenesis in mice. IL-35 in the tumor micro- induced and sustained in the tumor microenvi-
environment signicantly increased the numbers ronment by a variety of cytokines. Macrophage-
of CD11b+Gr1+ myeloid cells in tumors and sub- derived IL-6 has been shown to induce production
sequently promoted tumor angiogenesis [213]. of IL-10 by cancer cells. Similarly, IL-6 in asso-
Furthermore, IL-35 renders tumor target cells ciation with TGF- can induce IL-10 production
more resistant to CTL destruction. in Th17 cells. However, TGF- alone can induce
IL-10, whereas IL-10 enhances the expression of
TGF- in a positive feedback circuit [217].
7.3.5 IL-10 TNF- promotes proinammatory reactions
while upregulating IL-10 in macrophages and
7.3.5.1 Overview monocytes as a negative feedback, thereby termi-
IL-10 is an important immunoregulatory cyto- nating the inammatory response. In addition,
kine produced by many cell populations. Due to IL-12 and IL-27 can also induce IL-10 produc-
its ability in inhibiting the production of IL-2 and tion from T cells [99, 218].
IFN- by murine and human Th1 cells, IL-10 was IL-10 inhibits NKG2D ligand expression on
initially named a cytokine synthesis inhibitory tumor cells and suppresses cytotoxicity mediated
110 M. Gopal
Fig. 7.3 IL-10-mediated

Tumor
tumor immunosuppression.
IL-10 can be induced in the
tumor microenvironment by
many cell types including
Th2 cells, Tr1 cells, Tregs,
DCs, TAM, and tumor cells. Th2
TAM
IL-10 has a multitude of
Treg
suppressive effects on the DC
antitumor immune response. Tr1 B
For example, IL-10 can
inhibit the maturation of DCs
T
and disrupt the differentia- CD8+
tion of CTLs and Th1 cells. Tr1
on IL-10 CTL activation
IL-10 can also inhibit the
izati
cytolytic activity of NK lar
1 po CTL
cells. In addition, IL-10 can Tr T
promote tumor growth Nave
through the promotion of Th T
IL-10-producing Tr1 cells 1 Nave
po
lar
iza
n
tio
io
n
at
NK activation
Th1
ur
at
m
NK
DC
NK
by NK cells. Furthermore, IL-10 induces HLA-G vaccine results in immune suppression and tumor
molecules that prevent the attack by NK cells progression, in line with a predominant inhibi-
[219]. These changes allow tumor cells to survive tory activity of IL-10 on DC-mediated antigen
from immunological attack by immune cells and to presentation. Moreover, IL-10-decient DCs are
grow exponentially. IL-10 can act as a negative reg- shown to be more effective in inducing protec-
ulator in the crosstalk between innate and adaptive tive antitumor immune response in mice [60].
antitumor immunity (Fig. 7.3): For instance, T cells Exposure of DCs to tumor cell lysates resulted
suppress NK and NKT cells by elaborating IL-10, in increased IL-10 production and expansion of
which ultimately leads to impaired activation of regulatory Tr1 cells. Tr1 cells have been shown
CTL and Th1 CD4+ T cells and tumor immune to down-modulate immune responses through the
privilege [220]. In vitro, IL-10 pretreatment can production of IL-10. In addition, IL-10 has been
convert different types of tumor cells to a CTL- shown to mediate the immunosuppressive activ-
resistant phenotype by decreasing the expression ity of Tregs [223]. Therefore, DCs that encounter
of HLA class I molecules on their surface [221]. tumor antigens in the presence of IL-10 in vivo
IL-10 acts on DCs and macrophages and inhib- acquire tolerogenic properties and subsequently
its the differentiation and the antigen-presenting induce T-cell tolerance to tumor antigens. In
properties of these cells. IL-10 inhibits essential addition, IL-10 signicantly suppresses other
steps in immune detection such as the expres- inammatory cytokines such as IL-1, IL- 6, and
sion of HLA-DR and co-stimulatory molecules, TNF expression in DCs. Moreover, inhibition
CD80 and CD86, on DCs. IL-10 also prevents the of IL-10 production by T cells or malignant cells
production of the Th1-polarizing cytokines IL-12 using anti-IL-10-/IL-10R-blocking Abs or anti-
and IFN- from DCs [222]. Administration of IL-10 antisense oligonucleotides improves anti-
IL-10 before and immediately after DC cancer tumor immune responses in animal models.
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Role of Chemokines
and Chemokine Receptors 8
in Cancer
Mathieu Paul Rodero, Christophe Combadire,

and Alexandre Boissonnas
Contents 8.6 Clinical Aspect ........................................... 133

8.6.1 Prognosis...................................................... 133
8.1 Introduction................................................ 121 8.6.2 CC Chemokines/Chemokine Receptors ...... 133
8.6.3 CXC Chemokines ........................................ 135
8.2 Chemokines and Chemokine Receptors .. 123
8.6.4 CX3C Chemokine Receptors....................... 135
8.3 Control of Tumor Cell Behavior ............... 125 8.6.5 Chemokine Circulating Expression ............. 135
8.3.1 Chemokines and Chemokine Receptor 8.6.6 Therapeutic Strategies ................................. 136
Alterations During Neoplastic
Transformation............................................. 125
8.3.2 Metastasis/Homing ...................................... 126 References ............................................................... 138
8.3.3 Senescence, Proliferation, and Survival....... 127
8.4 Control of Immune Cell Behaviors .......... 128
8.4.1 Chemokines Involved in T-Cell
Antitumor Immune Response ...................... 128
8.4.2 Chemokines in Innate Immune
Components ................................................. 130 8.1 Introduction
8.4.3 Chemokine and Tumor-Induced
Tolerance...................................................... 131 Living tissues are highly organized and dynamic
8.5 Alternative Tumor-Associated structures at the cellular level. Tissue renewal,
Physiological Functions of Chemokines... 132 remodeling, and repair, immunosurveillance, and
8.5.1 Angiogenesis................................................ 132
8.5.2 Fibrosis and Extracellular Matrix
cell-to-cell interaction and communication are
Remodeling .................................................. 132 examples of physiological processes relying on
the ne recruitment and displacement of numer-
ous cell types. This equilibrium is strictly depen-
dent on the principle of recruiting the right cell at
M.P. Rodero, PhD C. Combadire, PhD the right place and the right moment. One major
A. Boissonnas, PhD (*)
Sorbonne Universits, UPMC Univ Paris 06, component of this principle is the chemokine and
CR7, Centre dImmunologie et des Maladies chemokine receptor system. Chemokines (CKs)
Infectieuses CIMI-Paris, Institut National de la, for chemoattractant cytokines are small, secreted
Sant et de la Recherche Mdicale (INSERM), molecules historically dened on the basis of their
U1135, Centre National de la Recherche
Scientique (CNRS), ERL 8255, functional chemotactic activity [13]. They con-
Institut Universitaire de Cancrologie (UPMC-IUC), stitute a family of over 50 members which inter-
CHU Piti-Salptrire, 91 Bd de lhpital, act with about 20 dened corresponding/cognate
Paris 75013, France receptors (CKRs). This discrepancy highlights
e-mail: mathieu.rodero@upmc.fr;
christophe.combadiere@upmc.fr; the complexity of this system as several CKs can
alexandre.boissonnas@upmc.fr bind to a single receptor. Conversely, one receptor

122 M.P. Rodero et al.
Fig. 8.1 Fine modulation

of cellular recruitment by
chemokines. The chemokine
network is organized around Cell types
a
several levels of complexity. 1 2 3
(a) Most of the cell types
(1, 2, 3) express several
chemokine receptors and a
same receptor is found on
several cell types. Moreover,
different chemokines can
bind to a same receptor and
most of the receptors can
bind several chemokines CKR CK
with distinct afnity
(color gradient represent
differential afnity). This Affinity
apparent complexity allows
for the ne control of cell
population recruitment. b Mix cells CK gradient Fine recruitment
(b) The schematic
representation illustrates
the selective recruitment
of cell populations according
to the respective colored CK
gradient. The number of cell
recruited is related to the
afnity of the respective CK
for its receptor
can bind several different CKs. This redundancy Cancer constitutes a very complex pathology
associated with differential avidity of the CK in many aspects. Neoplastic cells result from the
for their CKR and the specic expression by the environmental, viral-induced, or inherited dereg-
different cell population contributes to the ne ulation of genes known as oncogenes or tumor
tuning of cell migration (Fig. 8.1) and explains suppressor genes. This primary modication
that a modest deregulation of the system can often leads to uncontrolled expansion of undif-
lead to severe pathological conditions. In addi- ferentiated cells for which the transcriptome and
tion, there is overwhelming evidence describing the proteome are highly modied in comparison
alternative functions of the CK/CKR couple in with the original cell. Nevertheless, it is impor-
hematopoiesis, reproduction, angiogenesis, and tant to note that tumor development does not
immune-associated functions such as cell activa- result from the simple expansion of neoplastic
tion, proliferation, effector function, and survival cell. Indeed, solid tumors (primary tumor as well
[4, 5]. Numerous reports from the past two as metastasis) are also constituted by a wide vari-
decades have validated the importance of the CK/ ety of stromal cells. Stroma is composed of non-
CKR network with its diverse range of physi- hematopoietic cells, such as healthy cells of the
ological properties and its involvement in various affected tissue, broblasts, or endothelial cells, as
physiopathological disorders [68]. well as hematopoietic cells. Hematopoietic cell
8 Role of Chemokines and Chemokine Receptors in Cancer 123
populations are mainly composed of innate specialized tissue and nally generate metasta-
immune cells, such as tumor-associated macro- ses. Inammation generated by neoplastic trans-
phages (TAMs), dendritic cells (DCs), natural formation contributes to the recruitment of
killers (NK cells), neutrophils, and partners of protumoral population and the production of
the adaptive immune response such as T and B growth factors as well as the recruitment of
lymphocytes. immune component with antitumor activity.
The relative importance of the stroma compared Thus, tumorigenesis is a dynamic process involv-
to tumor cells depends on the type of cancer [9], ing important tissue remodeling and angiogene-
but it is now well described that several stromal sis, recruitment and local migratory mechanisms,
cells are important predictive markers of cancer and survival and cell death for both tumor and
evolution (macrophages, regulatory T cells, and stromal cells in which the CK/CKR network has
endothelial progenitor cells). Even though the major implication.
stroma cannot be characterized properly in circu- The CK/CKR network appears to be a promis-
lating hematological tumors, leukocytes will have ing target in cancer therapy and has already been
an important impact on the expansion, survival, used in standard therapeutic approaches, as well
and potential homing of tumor cells to the specic as in immunotherapy. Numerous basic and clini-
tissue. This phenomenon is distinguishable from cal interventions rely on the development of ago-
the metastatic process where the tumor cells need nist or antagonist CKR in order to manipulate
to cross the endothelial barrier from a primary their critical biological function toward antitu-
tumor site and home to a distant tissue. The stroma mor activity.
contributes to the global organization and progres- In this chapter, the role of the CK/CKR net-
sion of the tumor known as tumor microenviron- work in these aspects of cancer development, as
ment through the production of growth factors, well as its potential application in the improve-
cytokines, CKs, exchange of nutrients, and tissue ment of cancer therapy, is described in detail.
remodeling and repair. In contrast, immune cells
are responsible for the control of tumor growth.
The concept of immunosurveillance proposed by 8.2 Chemokines and Chemokine
Burnett et al. [10] in the early 1970s has been Receptors
widely debated. Recently, Schreiber and colleagues
provided experimental evidence for the clinical Chemokines are small cytokines initially
emergence of cancer as a result of strong selection described for their chemotactic properties on leu-
and modeling of tumors by the immune system in a kocytes. During cell recruitment from the blood
process termed as tumor editing [11]. In this pro- to inamed tissues, CKs initiate the activation of
cess, neoplastic transformation occurs, and tumor circulating cells, promoting cell rolling, adhesion
cell expansion is detected by the innate and adap- to activated endothelium, and extravasation
tive immune systems, which either succeed in com- (Fig. 8.2). In tissues, CKs determine cell direc-
plete tumor elimination or maintain a state of tional migration, by establishing a concentration
equilibrium between tumor cell expansion and gradient (Fig. 8.3). Evidence from previous stud-
elimination. This phase leads to the immune selec- ies has shown that the control of cell mobility by
tion of tumor cell variants that develop immune CKs is implicated in developmental mechanisms
resistance and immunesuppressive mechanisms and cell homeostasis, as well as in the induction
resulting in tumor escape and cancer progression to and tuning of acute and chronic inammation
a clinical outcome. and control of the immune response. Numerous
Cancer is a complex process whereby undif- reviews have extensively described the CK clas-
ferentiated tumor cells expand locally in special- sication, structural organization, and their asso-
ized tissues, migrate in an active manner by ciated biological properties [12, 13]. CKs are
leaving the primary tumor site through the endo- subdivided in four subfamilies based on the
thelial barrier, establish in a distant and different number and spacing between conserved cysteine
Fig. 8.2 Chemokine-

associated extravasation
process. (a) Circulating cell a b
within the bloodstream.
(b) Chemokine presented by
proteoglycan on activated c d
endothelial cells, induce the
expression of adhesion
molecules implicated in the
slow rolling and the capture
process. (c) Once stuck to
the endothelium, cell exerts
crawling behavior on the
luminal side of the blood
vessel and (d) extravasates Endothelial cells Chemokine receptor
and migrates through the
tissue toward a chemokine Adhesion molecules Chemokine
gradient
Proteoglycan loaded with chemokine
a b c
Stromal cells Chemokines Migrating

Immune cells
immune cells
Inflammed cells
Fig. 8.3 Interstitial migration. (a) Upon activation, cells will migrate through the tissue toward the higher
(b) stromal cells will produce chemokines forming a concentration of chemokine
gradient within the tissue. (c) Tissue-inltrated immune
in the primary amino acids sequence [14]. CKRs is in reality a tool to tightly regulated leukocytes,
are seven transmembrane G-protein-coupled stem cells, and other cell types migrations during
receptor classied according to the CK family physiological and pathological condition.
they bind. As previously mentioned, most CKs It is now well established that CK function is
bind to several receptors, and most of the recep- not limited to cell migration. It has been clearly
tors can bind several CKs with different afni- demonstrated that CKs directly control cell
ties. Additionally, one cell subset can express proliferation, survival and senescence, as well as
different CKR and the same CKR is expressed by cytokine secretion and phagocytic properties
different cell subsets. This apparent redundancy (Fig. 8.4). It is the balance between these
Fig. 8.4 Control of cell

biology by chemokines.
Besides cell migration,
chemokines are implicated Inhibition
in multiple cellular functions Activation
including apoptosis,
proliferation, and
senescence. Chemokines
Cytokine secretion
are also directly implicated
in cell activation, cytokine
secretion, or phagocytosis
Apoptosis
Phagocytosis
Proliferation Senescence
migratory, secretion, phagocytic, survival, and resistant tumor variants. CK and CKR are not
proliferation signals which explains the central oncogenes per se; however, modulation in the
roles of CK in development, tissue homeostasis, production of CK or their receptors by tumor
repair, inammation, and immunity. cells is often the result of oncogenic modica-
tions and immune selection (Fig. 8.5). The rst
evidence came from a human papillary thyroid
8.3 Control of Tumor Cell cancer. The authors showed that RET (rearranged
Behavior during transfection)-tyrosine kinase rearrange-
ment promotes the secretion of numerous inam-
The biological property controlled by the CK/ matory cytokines, including CCL2, CCL20,
CKR recognition system is not restricted to che- and CXCL12, and increases the expression of
motactism. Several important processes involved CXCR4 [15]. Later studies have shown that Myc
in the behavior of tumor cells will be affected by overexpression in pancreatic cancer has been
these axes. In this section, the effect of CK/CKR associated with increased CK expression [16, 17].
expression on tumor cell behavior and cancer Nevertheless, the predictive outcome of onco-
progression is discussed. genic modications on the regulation of CK and
CKR expression is difcult to assess. While
RAS-RAF signaling pathway promotes CXCL8
8.3.1 Chemokines and Chemokine and CXCL1 transcription in pancreatic and ovar-
Receptor Alterations During ian cancer, it inhibits CCL27 transcription in skin
Neoplastic Transformation cancer [1820]. Similarly, Von Hippel-Lindau
tumor suppressor mutation in renal cancer [21]
Primary neoplastic transformation leads to strong and TP53 mutation in cancer stem cells promote
modication of the transcriptome and proteome CXCR4 expression [22] while downregulating its
which is mainly shaped by immune selection of expression in breast cancer cells [23].
Fig. 8.5 Oncogenes induce

altered chemokine and
chemokine receptor Inhibition
expression by tumor cells. Activation
Common oncogene mutations Sc
CCL27
are associated with Ras CXCL1/8
modication of chemokine Pc Oc
or chemokine receptor
CCL2/20
transcription, resulting in Pc
tumor promotion. RET/PTC Myc
PTc
rearranged RET tyrosine RET/PTC
kinase, VHL Von Hippel-
PTc
Lindau tumor suppressor
gene, Sc skin cancer, Pc Mutated
CXCR4
pancreatic cancer, Oc ovarian Tp53 cSC
cancer, HPTc human papillary Rc

thyroid cancer, cSC cancer VHL
stem cell, Rc renal cancer
Through modication in the prole of CKR gradients may induce tumor cell chemotaxis, but
expression, tumor cells will change their sensitiv- the direct implication of CKs in this specic pro-
ity to the microenvironment and acquire new cess is poorly documented. We can distinguish
migratory and homing capabilities. the indirect contribution of CK to the chemotaxis
activity of cancer cells through angiogenesis,
brogenesis, and matrix remodeling mediated by
8.3.2 Metastasis/Homing stromal cells.
CXCL12/CXCR4 is the major axis directly
The metastasis index is undoubtedly the major involved in tumor cell metastases. Overexpression
factor of prognosis and determines the therapeu- of CXCR4 in rat mammary adenocarcinoma
tic attitude. Metastasis denes the process enhances the motility of tumor cells in the pri-
through which tumor cells leave a primary site to mary tumor [26]. This receptor is widely involved
settle in a distant location and creates a new in the epithelial-to-mesenchymal transition
colony. This phenomenon is a characteristic of (EMT) process, which is a major step leading to
tumor malignancy including tumor invasion, metastasis [27, 28]. Few studies have reported the
intravasation, and homing to different sites. This implication of other CKs and CKRs such as
has to be distinguished from the potential sec- CCL18, CCL2, or CXCR7 [2931] through the
ondary localization of circulating tumor cells activation of EMT-implicated signaling path-
which only involves the homing mechanism. ways. IL8/IL8R axis might also favor mainte-
nance of the mesenchymal status of the tumor
8.3.2.1 Tumor Invasion cell [32]. Interestingly, the integration of multiple
The rst step of metastasis spreading relies on CKR axes adds complexity to the tumor invasion
either tumor cell or stromal cell-mediated brosis process. Indeed, overexpression of CXCR4 pro-
activity and the ability of tumor cells to acquire motes invasion. However, coexpression of
migration and intravasation capabilities, in order CXCR7 which binds the same ligand CXCL12
to leave the primary tumor site and reach the impairs invasion but favors angiogenesis and pri-
bloodstream. Chemotaxis of tumor cells is well mary tumor growth [26].
characterized [24]. This process requires a para-
crine loop between tumor cells and stromal cells, 8.3.2.2 Homing
such as macrophages shaping the microenviron- Once in the bloodstream, the tumor cell needs to
ment to favor metastasis [25]. Different chemical migrate to a site that will allow its engraftment,
Table 8.1 Metastases implantation of various cancer types based on their chemokine receptor expression
survival, and proliferation. In 2001, Muller et al. CXCL8 can result in senescence induction [37].
demonstrated for the rst time that the expression CXCR2 activation is thus able to act as a suppres-
of specic CKRs by tumor cells could predict the sor of malignancy in prostate and breast cancer
implantation of malignant cells in tissues express- [38, 39].
ing high levels of the receptor ligands [33]. Since Inhibition of tumor proliferation by CXCR2
then, several other studies have established asso- ligand is probably limited to tumor models and to
ciations between metastases, CKR expression, early stages of tumor development. Indeed, the
and implantation sites for various cancer types same CK axes display opposite effects in other
(Table 8.1). Consistently with their homeostatic tumor models. CXCR1 and CXCR2 activation by
functions, CCR7 expression by tumor cells is CXCL8 promotes the proliferation of gastric can-
associated with lymph node metastases; CCR10 cer, esophageal cancer, non-small lung cancer,
with skin metastasis; CX3CR1 with brain, liver, and melanoma cell lines [4043]. Other receptors
and bone metastases; CCR9 with intestine metas- of the CXC receptor family are involved in tumor
tases; and CXCR4 with bone and liver metastases cell proliferation. CXCR6 is involved in cell pro-
[3336]. liferation of pancreatic cancer cells [44], and
Overall, these observations show that CK CXCR4 is associated with tumor proliferation in
axes generate a complex relationship between numerous models, including ovarian, melanoma,
tumor cell and the environment and deserve glioma, renal, lung, and thyroid cancer cells [27,
further attention in preclinical studies as it 45]. Few studies have investigated the implication
represents an important target with clinical of CCRs in the control of tumor cell proliferation.
application. CCR6 favors colon tumor cell proliferation upon
CCL20 activation [46], and CCR9 favors
pancreatic cancer cell proliferations upon CCL25
8.3.3 Senescence, Proliferation, activation [47].
and Survival Another role of CK in tumor cell biology is
the ability to control tumor cell survival, essen-
Tumor expansion results in the capacity of tumor tially mediated through the CC receptor family.
cells to proliferate innitely without developing CCR10 activation promotes phosphatidylinositol-
senescent mechanisms. Several CKs have dem- 3-kinase-mediated protection from apoptosis of
onstrated the ability to activate signaling path- melanoma cells [48]. The same mechanisms are
ways in favor of this goal. observed in squamous cell carcinoma of the head
Cellular senescence is generally dened as an and neck after CCR7 activation [49]. CCR7
irreversible state of G1 cell cycle arrest in which engagement by CCL21 is also implicated in the
the cell is refractory to growth factor stimulation. prevention of apoptosis in NLCLC, through
Activation of CXCR2 by either CXCL1 or ERK-dependant activation pathways [50].
CK direct promotion of tumor cell survival is feature of this maturation is the downregulation
not limited to CC chemokines; CXCL12 through of peripheral tissue-associated CKR like CCR1,
CXCR4 activation promotes hepatoma, ovarian, CCR5, and CCR6 and the upregulation of CCR7.
and chronic leukemia tumor cells survival [51], Due to the constitutive expression of CCR7
and CXCR7 activation increases cell survival by ligand, CCL19, and CCL21 by peripheral lymph
reducing apoptosis [52]. nodes, this switch of CKR expression by APCs
Overall, these observations highlight extended promotes their migration toward the priming site.
functional contributions of the CK system to Once in the draining lymph node, APCs will
tumor development and reveal that they are not locate in the preferential area to present the tumor
merely restrained to chemotaxis. Ag to the CCR7 expressing naive lymphocyte.
8.4.1.2 Ag Presentation to T
8.4 Control of Immune Cell Lymphocytes
Behaviors Despite the fact that APCs display low dynamic
activity, nave lymphocytes have a high basal
As described previously, the immune system is mobility favoring scanning of thousand APCs per
known to shape the tumor through the tumor hour [55, 56]. This behavior requires CCR7
editing phenomenon. In this context, CKs are expression by T lymphocytes [57]. An additional
directly or indirectly implicated in the control of CKR-dependant mechanism favors the probabil-
immune cell activation, migration to the priming ity of encounter between APCs and T lympho-
site, and immune response induction. It is now cytes. Encounter of Ag-specic CD4+ or CD8+ T
clear that in most cases, the CK network is cells with an APC bearing their cognate Ag
shunted by the tumor, favoring its escape from induces the secretion of CC-chemokines by the
immunosurveillance and tumor progression. conjugate, namely, CCL19, CCL5, CCL3, and
Nevertheless, the production of some CKs pro- CCL4. These CKs will promote nave T-cell
motes the antitumor immune response and has scanning behaviors and attraction toward the
been associated with improved patient outcome, conjugate [5860], which is known to favor the
including lower recurrence rate or increased establishment of memory immune response, in
patient survival [53]. addition to the induction of polyclonal responses
against different tumor Ags [61].
CKs are also implicated in the improvement
8.4.1 Chemokines Involved in T-Cell of APC/T-cell adhesion mechanism as well as in
Antitumor Immune Response immunological synapse stabilization, promoting
T-cell priming (Fig. 8.6). CCR7 ligands secreted
Induction of antigen (Ag)-specic antitumor in the lymph node promote immunological syn-
immune response requires the uptake of tumor Ag apse formation by T cells [62]. CXCR4 and
by professional antigen-presenting cells (APCs) CCR5 expressed by T cells are recruited toward
and migration from the tumor site to the corre- the immunological synapses made with the
sponding draining lymph node, in order to present APC. This polarization results in desensitization
the processed tumor-Ag to T lymphocytes. These of T cells from external sources of CKs and
major immune functions can be divided into dif- improves synapse stability. A similar mechanism
ferent steps for which the CKR network has is observed during the interaction between tumor-
important regulatory implications [54]. inltrated lymphocytes (TILs) and tumor cells.
Indeed, the recruitment of CCR5 at the immune
8.4.1.1 Migration of APCs synapse formed between the TIL and the tumor
to the Priming Site cell results in defective responses to TIL toward a
Encounter with tumor Ag induces maturation of CCR5 gradient [63]. This mechanism allows for
APCs present in the tumor environment. One the modulation of the GO signals generated by
a b c
Immune synapse
Chemokine receptors TCR MHC Co-stimulatory molecules
Fig. 8.6 Control of cell polarization toward immune syn- sequestration of CKR leads to reduced sensitivity to dis-
apse. (a) T-cell scan for their cognate antigen-presenting tant CK gradient and may participate in the stabilization
cell. (b) Upon recognition, T cell will polarize chemokine of the immune synapse
receptors toward the immune synapse. (c) This
CKs, competing with the STOP signals medi- periphery; however, the underlying mechanisms
ated by the TCR-MHC interaction [64]. remain unclear. Several clues could help us specu-
late on the mechanism of trapping the TILs at the
8.4.1.3 Migration of Effector T tumor periphery. The recent contribution of real-
Lymphocytes to Tumor time imaging showed that dense peripheral extra-
Naive T cells, after clonal expansion and differ- cellular matrix might restrain TILs access to the
entiation into effector T cells, migrate toward the tumor parenchyma [68]. Whether specic niches
tumor site, implying that T cells downregulate of CKs are expressed on collagen bers is unclear
the expression of the CKRs implicated in the and needs further investigation. In addition,
retention at the priming site like CCR7. In addi- dynamic analysis showed that Ag-specic CTLs
tion, they upregulate various CKRs including are trapped in the network of tumor-associated
CCR1, CCR3, CCR5, and CXCR3 allowing their APCs restraining their inltration and probably
movement toward the tumor site [65]. Cytotoxic favoring immunosuppression [69, 70]. The role of
T lymphocytes (CTLs) recruitment to the tumor CKs in this trapping is not dened, but Ag expres-
site is consistent with this pattern of CKRs sion by APC at least induces stable engagement
expression and is mainly mediated by CCL3, between the CTL and the APC. In addition,
CCL5, CCL20, CXCL9, and CXCL10 [54]. experimental evidences showed that non-tumor-
Membrane-anchored CKs expression such as Ag-specic TIL cannot inltrate the tumor deeply
CXCL16 and CX3CL1 have also been shown to without the prior tumor cells destruction by
correlate with greater numbers of tumor- Ag-specic CTL. These results suggest that deep
inltrated lymphocytes and improved prognosis inltration of the tumor by TIL might be favored
in colorectal cancer [66, 67]. The antitumor effect by chemotactic agents secreted upon tumor cell
of the membrane-bound CK form vs. the soluble destruction by CTL or on extensive ECM remod-
form is yet to be clearly established. eling to allow their interstitial migration [71].
The control of TIL localization within the Overall, considering the numerous CKs
tumor is ill-dened. It is obvious that in most expressed by the various cell subsets of the tumor
cases, TILs are mainly found at the tumor microenvironment, it is very difcult to address
specic contributions of CK/CKR couple in the derived from the yolk sac or fetal liver [72].
interstitial migration and positioning of T lym- Within neoplastic tissues, it is suggested that
phocytes within the tumor parenchyma. The TAMs are mostly recruited from the periphery.
various properties of these molecules have dem- Nonetheless, knowledge of the relative propor-
onstrated that this positioning is controlled by tion of native resident macrophages remains a
sensitivity to the chemotactic gradient and the poorly investigated eld in oncology. CCL2, also
subsequent desensitization upon polarization called MCP-1 for monocyte chemoattractant pro-
toward the synapse or the downregulation of the tein-1, is probably the most frequently found
expression of CKRs. CC-CK in tumors involving recruitment of circu-
lating monocytes [73]. Interestingly, in a mela-
noma system where tumorigenesis is dependent
8.4.2 Chemokines in Innate on an external growth factor CCL2, there is a
Immune Components biphasic effect depending on its secreted quan-
tity. High amounts are associated with a massive
Innate immune cells constitute a rst barrier recruitment of TAM into the tumor with domi-
against tumor development. However, due to nant antitumor activity, while lower amounts
their plasticity and capacity to produce a myriad induce lower inltration into the tumor resulting
of cytokines, chronically activated innate immune in tumor promotion through the secretion of
cells are key modulators of cell activation and growth factor by the macrophages [74]. These
survival, as well as regulators of the ECM metab- results point out the importance of the ratio
olism. Several physiological processes necessary between protumor and antitumor macrophages
for tumor development, such as increased cell recruited into the tumor.
survival, tissue remodeling, angiogenesis, and Other CKRs implicated in TAM recruitment
suppression of antitumor adaptive immune are CX3CR1 and CCR1. In human glioblastoma,
responses, are regulated by innate immune cell the level of tumor inltration by microglial cells
inltrate in the tumor. is dependent on CX3CR1. Patients with a func-
Macrophages are the main stromal cell popu- tional mutation in the CX3CR1 gene associated
lation present in the tumor parenchyma. They can with impaired monocyte migration have a
account for more than 50 % of the tumor mass. reduced TAM inltration into the tumor [75].
The role of TAM in tumor development is criti- Injection of a thymoma tumor cell line (EL4)
cal, as these cells, depending on their state of with a liver tropism to mice results in an increased
activation, can display antitumor properties asso- inltration of the liver by immune cells, includ-
ciated with production of Th1 cytokine, high ing macrophages. In CCR1 KO mice, this recruit-
quantity of reactive oxygen species, and efcient ment during the rst stage of the tumor
Ag presentation or they could display protumor development is massively reduced [76].
properties mediated by the secretion of Th2 cyto- CXC chemokine receptors could also be
kine, proangiogenic factors, growth factors that implicated in TAM recruitment. In humans, IL-4
support tumor survival, and proliferation and the and IL-13, two cytokines secreted in the tumor
secretion of MMP which promote tumor invasion environment, sensitize monocytes to CXCL1 and
and metastases. Consistently, the impact of TAM CXCL8 by upregulating their receptors (CXCR1
on tumor development and metastases will and CXCR2). Thus, these cytokines indirectly
depend on the balance between M1 antitumor promote the recruitment of TAM into the tumor
macrophages and M2 protumor macrophages. through CXC chemokine receptors [77].
Depending on the tissue, resident macro- As previously discussed, CKs not only con-
phages are in a small proportion derived from trol leukocyte recruitment into the tumor but
the recruitment of circulating monocytes assur- also organize their localization within the
ing immunosurveillance and mainly origin from tumor. Lack of proper vascularization at the
self-renewal of interstitial resident macrophages center of the tumor induces the secretion of
several hypoxic factors like hypoxia-inducible 8.4.3 Chemokine and Tumor-Induced

factors (HIFs). HIFs promote the expression of Tolerance
CXCR4 by macrophages, favoring their recruit-
ment toward tumor hypoxic areas [78]. On the Recruitment of tolerogenic cells such as regula-
other hand, tumor environment decreases CKR tory T cells or immunosuppressive myeloid sub-
expression on monocytes. Indeed, macrophages sets is a feature or immune escape. Tumor cells
from tumor sites express low levels of CKR secrete ligands of CKRs expressed by immature,
[79]. Time-lapse imaging of TAMs in experi- regulatory or Th2 polarized cells. CCL22 and
mental murine model revealed that TAMs dis- CCL17 produced by tumor cells recruit mono-
play reduced displacement but intense cytes, as well as Th2 lymphocytes and regulatory
protrusive activity [69, 70]. Downregulation of T cells through CCR4 signaling [91]. This strat-
CKR might explain this retention at the tumor egy of immune escape has been also selected in
site. viral-induced oncogenesis process. HHV8 virus,
CKs do not only act on leukocyte attraction the pathogen of Kaposis sarcoma, encodes three
but are also implicated in their activation. viral CKs which bind to CCR3, CCR4, and CCR8
Induction of copper/zinc-superoxide dismutase involved in the recruitment of Th2 and regulatory
by CCL5/CCR5 activation causes tumor necrosis T cells [92].
factor-alpha and reactive oxygen species produc- Stromal cells produce CKs which promote the
tion by macrophages [80], promoting tumor recruitment of protumoral cells. Amongst others,
destruction. Inversely, in human monocytes, CC TAM produces CCL18 which is induced by IL10
chemokines induce the transcription of metallo- [93]. CCL18 favors the recruitment of nave T
proteinase, implicated in tumor invasion and cells through activation of an unknown receptor.
spreading. The fact that both TAM recruitment It is proposed that these nave T cells acquired
and activation are regulated by CK increases the tolerogenic properties in contact with the tumor
potential interest of targeting TAM for antitumor environment. CCR6+ immature lymphoid DCs
therapies. recruitment into the tumor is favored by the
NK cells represent another component of the secretion of CCL20 from both tumor cells and
innate immune system highly involved in antitu- TAM [94]. CCL5 recruits immature DCs as well
mor immune responses. NK cell recruitment to by binding CCR1 and CCR5 [95]. Immature DCs
the tumor is mainly mediated through the acquire tolerogenic properties in the tumor envi-
CXCL10-CXCR3, CX3CL1/CX3CR1, and ronment and participate in the immune tolerance
CCL3-4-5/CCR5 axes. High CX3CL1 quantity is loops against tumor Ags [96].
associated with increased NK cell recruitment Subversion of tumor immune component is
into the tumor in both human and mice [81, 82]. a central point of tumor outcome. The above
Similar phenomenon is observed with increased described implication of CK in cellular mech-
CCL5 and CCL3 expression by tumor cells in anisms should provide the basis to better
mouse models [83, 84]. CXCR3 is implicated in understanding the clinical implication of CK
the recruitment of human NK cells to breast can- network in cancer pathology. The regulation
cer tumor, which is mediated by CXCL10 secre- of the balance between immunogenic and
tion from tumor cells in response to IFN- tolerogenic components has deserved major
produced by the NK cells themselves [85, 86]. attention for a long time and is the basis of
Thus, CKs not only control NK cell recruitment immunotherapy which represents an apparent
but also regulate their antitumor properties. inexhaustible field of innovative anticancer
CX3CR1 activation by CX3CL1 results in strategies. Targeting the CK system in this
improved antitumor cytotoxicity of NK cells [87, goal is in the course of important investigation
88]. CCL3, CCL4 and CCL5 have been shown to through the development of pharmaceutical
activate NK cytotoxicity through induction of compounds able to stimulate or antagonize
degranulation [89, 90]. CKR axes.
8.5 Alternative Tumor- In contrast, ELR chemokine secretion is

Associated Physiological often associated with attenuation of angiogene-
Functions of Chemokines sis. ELR CXC chemokines are described by
their angiostatic properties. ELR CXC chemo-
8.5.1 Angiogenesis kine secretion is induced by IFN- and IFN-.
Through CXCR3 binding, these CKs mediate
One of the features of CKs is their dual role in the their angiostatic properties by inhibition of ELR+
angiogenic process. In the tumor environment, chemokine, VEGF, and FGF proangiogenic
there is increased production of proangiogenic effects in vitro [103]. Interestingly, the expres-
CK, while angiostatic CKs are downregulated. sion of CXCR3 is dependant of the cell cycle
In addition to a direct angiogenic effect of CKs, phase, limiting the angiostatic properties of ELR
this activity is indirectly potentialized by the CXC chemokines to the S/G2-M phase [104].
CK-induced recruitment of leukocyte display- This important association of CKs and angio-
ing angiogenic properties such as neutrophils or genesis within the tumor environment sets the
macrophages [97]. inhibition of ELR+ chemokine as a robust antitu-
CK from the CXC family are probably the mor therapy.
most described for their direct implication in
tumor-associated angiogenesis. CXCLs 1, 2, 3, 5,
6, 7, and 8 display angiogenic properties. All 8.5.2 Fibrosis and Extracellular
these CKs contain a specic amino acid sequence Matrix Remodeling
of glutamic acid-leucine-arginine (or ELR for
short) immediately before the rst cysteine of the The association of CKs in EMT leading to bro-
CXC motif (ELR-positive). This ELR sequence sis activity has been previously suggested by
absence from the other CXC chemokines is studies; however, there is no clear evidence that
responsible of the proangiogenic properties of CKs play a direct role in this process.
most of the CXC chemokine [98]. Fibrosis and extracellular matrix remodeling
ELR+ chemokines mediate angiogenesis are continuous processes present in the tumor
through binding to the CXCR2 receptor. ELR+ parenchyma reecting the intense dynamic and
chemokines are able to recruit endothelial pre- migratory activity of the neoplastic tissue. Two
cursor cells, induce cell proliferation, and pro- different types of migratory activity are dened,
mote maturation. These mechanisms could be namely, the amoeboid and mesenchymal migra-
negatively regulated by a decoy CKR expressed tion. The amoeboid migration does not require
by endothelial cells called duffy antigen receptor extracellular matrix (ECM) remodeling through
for CK (DARC). Unlike most of the other CKR, matrix metalloproteinases (MMPs) activity due
DARC is not linked to G protein, and its activa- to the ability of the cell to squeeze through the
tion does not induce calcium ux. DARC reduces ECM. The mesenchymal migration relies on pre-
angiogenesis by sequestering all the ELR+ CKs. vious proteolysis and degradation of the ECM to
One specicity within ELR chemokines is generate sufcient space for cell displacement.
attributed to CXCL12 which is the only ELR CK-mediated induction of MMP is mostly medi-
chemokine with proangiogenic activity. CXCL12 ated by CC chemokines; CCL5 and CCL9 pro-
mediates its proangiogenic effect by directly produced by mesenchymal stem cell promote tumor
moting the recruitment of endothelial progenitor cell invasion in a MMP-dependant manner [105,
cells [99, 100] or indirectly by promoting tumor 106]. CCL25 promotes MMP secretion in ovar-
angiogenesis through the recruitment of CXCR4+ ian cancer cells through CCR9 binding and favors
proangiogenic monocyte [78, 101] and through tumor cell invasion [107]. CCL21/CCR7 interac-
the secretion of vascular endothelial growth fac- tion favors MMP-9 secretion, tumor invasion,
tor (VEGF) by CXCR7 activation [102]. and metastases in colon cancer cells and in B-cell
chronic lymphocytic leukemia cells [108, 109]. correlation between functional variants and
At least, one CXC chemokine has been related to tumor risk or progression (Table 8.2).
MMP activity; thus, CXCL12 is implicated in The paragraphs below focus on the most com-
increased MMP2 activation and increased cell monly described polymorphisms, their functional
invasion in a pancreatic cancer cell line [110]. relevancies, and their subsequent prognostic
Studies have suggested that the extracellular value in tumor risk and/or progression.
matrix promotes tumor escape from the immune
system by trapping antitumor leukocytes at dis-
tance from tumor cell niches [111]. However, 8.6.2 CC Chemokines/Chemokine
tumor progression and metastases require degra- Receptors
dation of this extracellular matrix surrounding
the tumor. The main protagonists of these physi- 8.6.2.1 CCL2
ological activities are represented by mesenchy- A single-nucleotide polymorphism (SNP) in the
mal stem cell (MSC)-derived cell populations. CCL2 promoter, based on the substitution of an
CXCL12 is implicated in the recruitment of mes- adenine by a guanine in position 2518
enchymal stem cells (MSCs) from the bone mar- (A < 2518 < G), is associated with increased
row. Bone marrow-derived MSCs can account CCL2 secretion [113]. This polymorphism with
for up to 25 % of the cancer-associated bro- an allelic frequency close to 30 % is associated
blasts, the main source of brosis within the with an increased susceptibility to the develop-
tumor [112]. ment of breast, gastric, and oral squamous can-
There is ongoing evidence that targeting pro- cer. However, it is not associated with an
teolysis activity in combination with chemotaxis increased risk of developing hepatocellular and
would provide promising results in the strategy to prostate cancer, glioblastoma, and melanoma.
inhibit tumor cell invasion and metastasis. Despite this lack of association with the devel-
opment of melanoma, CCL2 polymorphism is
associated with increased Breslow index, sug-
8.6 Clinical Aspect gesting its link with melanoma progression
[114]. CCL2-2518G variant is also associated
CKs are implicated in several aspects of tumor with increased metastases development in naso-
development. Due to these pivotal roles in tumor pharyngeal and breast cancer. In the former
biology, CKs have been frequently associated case, the deleterious effect of the polymorphism
with tumor evolution and clinical outcomes and is observed only after radiotherapy [115].
have been highlighted for their potential use as Overall, the deleterious effect of the CCL2-
prognostic or diagnostic markers. Therefore, they 2518G allele-associated increase of CCL2
represent a promising target with a potential for a expression is consistent with the protumoral
diverse range of therapeutic strategies. effect of TAM in most tumors, as previously
described above.
8.6.1 Prognosis 8.6.2.2 CCL5

Conicting data arises from the study of the
Due to its importance across a wide range of CCL5 G < 403 < A polymorphism on cancer
physiological mechanisms, CK/CKR network risk. This mutation is thought to be responsible
alteration could impact tumor development. for the decreased secretion of CCL5 and is asso-
Correlative studies using genetic polymorphisms ciated with decreased risk for leukemia and gas-
provide essential information for prognosis. tric cancer in women [116], as well as an
Several functional polymorphisms in CKs or increased risk for prostate and pancreatic cancer
CKRs have been studied in order to establish [117]. This discrepancy could reect the balance
Table 8.2 Association between

chemokines and chemokine
receptor polymorphisms and
tumor risk and/or progression
Prog prognosis, + good indicator, poor indicator, = no association, * meta-analysis
between the antitumor effects of CCL5 through 8.6.2.3 CCR5

recruitment of cytotoxic CTL and the protumoral CCL5 main receptor (CCR5) is also subject to
effect of CCL5 through recruitment of immature another relevant polymorphism. A deletion of
DC. Nonetheless, there is no evidence supporting 32 base pairs named CCR5 delta 32 results in a
an association between CCL5 polymorphism and reading frame shift, associated with complete
tumor progression. defect in receptor expression. The impact of the
polymorphism in tumor risk and progression is ture make any interpretation challenging. Several
not well documented. Most studies conclude a meta-analyses have been performed in order to
lack of association; however, one report suggests gain some clarity, and despite some variation in
that CCR532 could be associated with higher the conclusion, it appears likely that the rare vari-
risks of the development of gallbladder cancer ant of CXCL8 promoter region is associated with
[118]. In melanoma, CCR532 is associated with increased risk of gastric and oral cancer [121123].
reduced survival of patients with grade 4 tumor
treated by immunotherapy strategies [119]. These 8.6.3.2 CXCL12
observations might reect the role of CCR5 in the CXCL12 is subject to a polymorphism in a 3
induction of T-cell priming and memory. untranslated region named CXCL12 3 G801A. The
rare variant is associated with increased secretion
8.6.2.4 CCR2 of CXCL12. Consistent with the protumoral effect
CCR2 V64I polymorphism has also been studied of CXCL12 mentioned above, studies essentially
for its implication in tumor risk and progression. report that CXCL12 801A variant is associated
There is no known effect of the genetic variation with an increased risk for several cancers (lung,
on the CCR2/CCL2-signaling pathway, but it is breast, oral, prostate, hepatocellular, and colorectal
associated with CCR5 instability, which could be cancer). It is also thought to favor tumor progres-
explained by stability alteration of the CCR2/ sion or metastases in lung cancer, hepatocellular
CCR5 dimer. Most of the studies conclude that carcinoma, colorectal cancer, and myeloid leuke-
there is an increased risk for people carrying the mia. The only three meta-analyses performed to
rare variant. This is the case for cervical, oral, date conclude that there is an increased risk for
bladder, prostate, and endometrial cancer. A recent breast and lung cancer, without any signicant
meta-analysis with 2,661 cancer patients and effect on other cancer types [124126].
5,801 healthy controls found an overall signicant
association between the CCR2-V64I polymor-
phism and cancer risk [120]. In the subgroup anal- 8.6.4 CX3C Chemokine Receptors
ysis stratied by cancer types, there was a
signicant association between this polymorphism The only receptor for the CX3C chemokine fam-
and the risk of bladder, cervical, and oral cancer. ily is CX3CR1, which is also subject to poly-
morphisms associated with cancer outcome.
Substitution of a valin by an isoleucine in position
8.6.3 CXC Chemokines 249 results in increased adhesion of the couple
CX3CR1/CX3CL1 and defective migration of
Two CXC chemokines, CXCL8 (also referred as CX3CR1+ cells. The rare variant is associated with
interleukin-8) and CXCL12 (SDF-1), have been increased risk of colorectal cancer, but not hepa-
intensively investigated for their association between tocellular cancer, melanoma, and glioblastoma.
polymorphisms and tumor risk and development. In this last case, the rare variant is associated with
improved patient survival after tumor biopsies and
8.6.3.1 CXCL8 decreased inltration of the tumor by microglial
CXCL8 T < 251 < A polymorphism is prob- cells [75]. This is consistent with the promotion
ably one of the most studied CK polymorphism of glioblastoma invasion by microglial cells [127].
in cancer. Its physiological effect and its impact
on CXCL8 expression remain to be elucidated.
There is an apparent discrepancy between stud- 8.6.5 Chemokine Circulating Expression
ies on these effects; however, this may reect
specicity depending on the cell type or the cell CK circulating levels have also been related to can-
activation status. The implication of CXCL8 poly- cer progression. A high concentration of CCL17 is
morphism in cancer risk and outcome remains associated with the progression of Hodgkin lym-
unclear. Unfortunately, controversies in the litera- phoma (HL) after treatment [128]. Interestingly,
Table 8.3 Current clinical trials evaluating the benets of targeting chemokines or chemokine receptors cancer
therapies
Inclusion criteria Phase Treatment
Colorectal cancer Phase I/II Chemokine-modulatory regimen
Stage IV adenocarcinoma of the lung Phase I/II GM.CD40L and CCL21
Metastatic castrate-resistant prostate Phase II Anti-CCL2 carlumab
cancer
Solid tumors Phase I Human monoclonal antibody against CCL2 (CNTO 888)
Colorectal cancer patients with hepatic Phase I CCR5 antagonist (Maravirok)
liver metastases
Previously treated peripheral T-cell Phase II Anti-CCR4 monoclonal antibody KW-0761
lymphoma (Mogamulizumab)
CCR4-positive adult T-cell Phase II Anti-CCR4 (KW-0761)
leukemia-lymphoma
High-grade glioma Phase I CXCR4 antagonist (Plerixafor/AMD3100) and
bevacizumab
Multiple myeloma previously treated Phase III Filgrastim with or without CXCR4 antagonist
with lenalidomide (plerixafor/AMD3100)
Non-Hodgkin lymphoma Phase III CXCR4 antagonist (Plerixafor/AMD3100) and G-CSF
Multiple myeloma Phase Ib Anti-CXCR4 (BMS-936564) alone or plus lenalidomide/
dexamethasone or bortezomib/dexamethasone
Multiple myeloma Phase I/IIA CXCR4 antagonist (BKT-140)
opposite effects are observed in melanoma, where been developed to target CKs or CKR as innova-
high CCL17 expression is associated with progres- tive strategies in cancer treatment. To date, there
sion-free survival in patients with immunotherapeu- is no molecule targeting macrophage release;
tic treatment [129]. Elevated concentrations of however, multiple clinical trials from phase I to
CXCL10 in the serum before treatment (monoclonal phase III are recorded at clinical trial.gov website
antibody therapy together with combination chemo- (Table 8.3). Some strategies aim to promote the
therapy) are associated with an increased likelihood production of CKs implicated in the recruitment
of clinical relapse and an inferior survival in patients of immune-competent cells to the tumor by injec-
with diffuse large B-cell lymphoma [130].Despite tion of IFN, celecoxib, and rintatolimod
numerous promising results, CK and CKR genes (NCT01545141). In another trial, patients with
and molecules are not currently used in clinical lung adenocarcinoma were directly injected with
settings to evaluate a patients risk of developing CKs implicated in the recruitment of antitumor
cancer or to predict tumor progression. This effector T cells, in combination with vaccination
could be explained in part by the nonhomoge- approach (NCT01433172). Inversely, another
neous distribution of the polymorphism variants trial aimed to inhibit the recruitment of protu-
amongst ethnic communities. Additionally, in moral leukocyte using an Ab against CCL2 in
most cases, CK and CKR gene polymorphisms order to control metastatic castrate-resistant pros-
are not singularly powerful predictive tools. Their tate cancer (MCRPC) (NCT00992186). However,
clinical utility is most likely to be dependent on this strategy failed as all the patients were
their association with other markers. removed from the study, due to progression of the
tumor despite anti-CCL2 treatment.
Another approach aimed to directly target
8.6.6 Therapeutic Strategies CKR expressed by neoplastic cells in order to
control tumor or metastases development. The
As discussed throughout this chapter, CKs are CCR5 antagonist, named maravirok, originally
implicated in all steps of the tumor development, commercialized for AIDS treatment, is under
invasion, and dissemination. Several tools have evaluation for its antitumor property in colorectal
cancer (NCT01736813). Promising results have immune system maturation and surveillance, and
been obtained with an anti-CCR4 Ab named tissue remodeling functions like angiogenesis or
KW-0761. Injection of KW-0761 in subjects brosis are shunted in most cases toward tumor
with CCR4-positive adult T-cell leukemia- promotion. The central role of the CK network
lymphoma resulted in the stabilization of tumor in these processes positions the CK system as an
progression in half of them. This molecule is now attractive target against tumor development, pro-
under evaluation in cutaneous T-cell lymphoma gression, and dissemination. Clinically, CK and
(NCT01728805) and in second-phase treatment CKR polymorphisms or serum levels are already
for peripheral T-cell lymphoma (NCT01611142). associated with susceptibility or prognostic mark-
CXCR4 antagonists are probably the most ers. Current investigations aiming at controlling
widely used molecules in trials targeting the CK tumor development by targeting the CK network
network. plerixafor is a FDA-approved CXCR4 are not limited to the direct effect on tumor cells.
antagonist for use in patients with non-Hodgkin For instance, it is proposed that CKs could modu-
lymphoma (NHL) and multiple myeloma. It is late the involvement of TAMs in tumor eradica-
used as a preconditioning regimen for its ability to tion or protection after chemotherapy suggesting
mobilize bone marrow resident hematopoietic that chemoattractant molecules could be used
stem cells and tumor stem cells toward circulation in combination with standard chemical chemo-
before chemotherapy. Plerixafor and other mole- therapies to favor tumor eradication through
cules targeting CXCR4 are now evaluated in sev- modulation of the TAM activity. Despite numer-
eral clinical trials from grades I to III phase in ous promising results, few molecules targeting
combination with other treatment, in various forms CKRs have received FDA approval. The CXCL12
of leukemia and myeloma. Evaluation of CXCR4 antagonism is already being used in patients
targeting in cancer therapies is not limited to blood with leukemia or myeloma to promote tumor
tumors. Plerixafor is currently being evaluated in a cell mobilization toward the bloodstream before
phase I trial in conjunction with bevacizumab for treatment, and the CCR5 antagonist maravirok
patients with high-grade glioma (NCT01339039). is currently being evaluated in colorectal cancer.
These low numbers of molecules targeting CKs
in the market could be explained by the relatively
8.7 Concluding Remarks recent discovery and characterization of the CKs.
In addition, the central role of CKs in most bio-
The advantages of targeting the CK network, logical functions would lead to potential numer-
through distinct strategies, have already been ous side effects. Given the phenomenal amount
demonstrated as well as its limitations. A new of progress made by the scientic and the medical
generation of clinical trials based on a combina- community, it is most likely that these challenges
tion of approaches from standard chemotherapies will be overcome. Several innovative technolo-
to innovative immunotherapies offer new per- gies allowing for more efcient and specic deliv-
spectives in CK network targeting strategies. ery of chemical compounds have been proposed
The 10 years following the discovery of the and optimized during the last few years, such as
majority of CKs were characterized by exten- Ab-coupled treatment and encapsulated or viral
sive investigations in the involvement of these delivered constructs. Targeting the CK network
molecules in the control of cellular trafck- using these tools will probably constitute the next
ing, specically leukocytes. Later on, scientists step in the development of a cancer therapy with
demonstrated that CKs do not only control cell minimal side effect.
migration but also cell proliferation, survival, and
activation state. It is now obvious that CKs act on Acknowledgment The authors wish to thank Neelam
a wider range of cell types rather than only leuko- Malik for the editorial assistance. M. R. is supported by
the program Emergence UPMC. This project has
cytes for which they were primarily characterized.
received funding from the European Unions Seventh
The complex physiological processes in which Programme for research, technological development and
CKs are involved such as tissue homeostasis, demonstration under grant agreement No 304810.
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122. Huang Q, Wang C, Qiu LJ, Shao F, Yu JH. IL-8-251A>T 129. Cornforth AN, Lee GJ, Fowler AW, Carbonell DJ,
polymorphism is associated with breast cancer risk: Dillman RO. Increases in serum TARC/CCL17 levels
a meta-analysis. J Cancer Res Clin Oncol. 2011; are associated with progression-free survival in
137(7):114750. advanced melanoma patients in response to dendritic
123. Wang N, Zhou R, Wang C, Guo X, Chen Z, Yang S, cell-based immunotherapy. J Clin Immunol. 2009;
et al. 251 T/A polymorphism of the interleukin-8 29(5):65764.
gene and cancer risk: a HuGE review and meta- 130. Ansell SM, Maurer MJ, Ziesmer SC, Slager SL,
analysis based on 42 case-control studies. Mol Biol Habermann TM, Link BK, et al. Elevated pretreat-
Rep. 2012;39(3):283141. ment serum levels of interferon-inducible protein-10
124. Gong H, Tan M, Wang Y, Shen B, Liu Z, Zhang F, (CXCL10) predict disease relapse and prognosis in
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Gene. 2012;509(2):22831.
The CD95/CD95L Signaling
Pathway: A Role in Carcinogenesis 9
Amlie Fouqu and Patrick Legembre

9.1 Introduction ................................................ 143
Apoptosis, or programmed cell death, plays a
9.2 TNF Receptor Family ................................ 144
9.2.1 TNFR1 Signaling Pathways ......................... 144
pivotal role in development, organ homeostasis,
9.2.2 TNF/TNFR: A Gold Mine and immunosurveillance. The term apoptosis was
for Therapeutic Tools ................................... 145 coined by Kerr et al. in 1972 [1] to describe the
9.3 CD95: A Death Receptor? ......................... 146 process of cell death associated with morphologi-
9.3.1 Structure/Function ........................................ 146 cal changes, including nucleus and cytoplasm
9.3.2 Type I/II Signaling Pathways ....................... 148 condensation and protuberances from the plasma
9.3.3 What Can We Learn from
membrane producing apoptotic bodies, so-called
CD95 Mutations? ......................................... 148
9.3.4 Regulation of the Initial Steps blebs, which are rapidly phagocytosed [1, 2].
of CD95-Mediated Signaling ....................... 150 Inhibition of this cellular process is observed in
9.3.5 Programmed Necrosis Also different pathologies, such as cancer and autoim-
Known as Necroptosis.................................. 152
munity, while amplication of the apoptotic sig-
9.3.6 CD95L, an Inammatory/Oncogenic
Cytokine? ..................................................... 152 nal was reported in neurodegenerative disorders
including Alzheimers and Parkinsons diseases
[3, 4], as well as infection by human immunode-
References ............................................................... 156 ciency virus (HIV).
The origin of the apoptotic signal has been
A. Fouqu, PhD used to distinguish two main signaling path-
Universit Rennes-1, Rennes, France ways. The intrinsic pathway stems from accu-
INSERM U1085, IRSET, Equipe Labellise Ligue mulation of DNA damage, deregulation of
Contre Le Cancer Death Receptors and Tumor mitochondrial function, or virus infection and
Escape, 2 Avenue du Professeur Lon Bernard, induces the release of proapoptotic factors from
35043 Rennes, France
the mitochondria, whereas extrinsic signals are
P. Legembre, PhD (*) transmitted by the binding of apoptotic ligands
Universit Rennes-1, Rennes, France
to death receptors present at the cell surface.
INSERM U1085, IRSET, Equipe Labellise Ligue Interconnections exist between these two
Contre Le Cancer Death Receptors and Tumor
signaling pathways: both leading to the activa-
Escape, 2 Avenue du Professeur Lon Bernard,
35043 Rennes, France tion of a family of cysteine proteases specic for
aspartic acid residues, called caspases [5]. The
CRLCC Centre Eugne Marquis, Avenue bataille
Flandres Dunkerque, 35042 Rennes, France apoptotic role of mitochondria is associated
e-mail: patrick.legembre@inserm.fr with reduction in its transmembrane potential

144 A. Fouqu and P. Legembre
and the loss of its extracellular membrane integ- 9.2 TNF Receptor Family
rity, leading to the release of different apopto-
genic factors in the cytosol. Among them, Death receptors TNFR1, Fas, DR3, DR4, DR5,
cytochrome c associates with the caspase-9/ and DR6 belong to the tumor necrosis factor
APAF1 complex to form the apoptosome and receptor (TNF-R) superfamily. These type I
trigger apoptosis [6]. transmembrane proteins share common fea-
These two signaling pathways share common tures, such as extracellular amino-terminal
features, and both require the aggregation of ini- cysteine-rich domains (CRDs) [19, 20], which
tiator caspases as their preliminary events. During contribute to ligand specicity [21], and pre-
interactions with respective ligands, members of association of the receptor at the plasma mem-
the death receptor superfamily recruit adaptor brane [2224] and a conserved 80 amino acid
proteins such as Fas-associating protein with a sequence located in their cytoplasmic tail called
death domain (FADD) [7, 8] or tumor necrosis death domain (DD), which is necessary for
factor (TNF) receptor 1-associated death domain DISC formation and initiation of the apoptotic
protein (TRADD) [9], resulting in the aggrega- signal [25, 26].
tion and activation of the initiator caspase-8 and
caspase-10 to form the death-inducing signaling
complex (DISC) [10]. In a similar manner, 9.2.1 TNFR1 Signaling Pathways
release of cytochrome c and ATP by mitochon-
dria promotes the formation of the apoptosome TNF- exerts its effects by binding to two recep-
with the cytosolic APAF-1, thereby aggregating tors, TNFR1 and TNFR2 [20]. Recently, pro-
and activating the initiator caspase-9, which in granulin was identied as a ligand of TNFR
turn cleaves caspase-3 [11]. with a higher afnity than TNF-. Progranulin
It should be kept in mind that death recep- antagonizes TNF- signaling and plays a criti-
tors CD95 [12], TNFR1 [13], DR4 [14], DR5 cal role in the pathogenesis of inammatory
[15], and DR6 [16] have been cloned based arthritis in mice [27]. TNFR1, a 55 kDa protein
on their ability to elicit apoptosis. Although expressed in almost all cell types, presents a DD
studies have revealed the ability of Fas/CD95, in its intracellular region; whereas TNFR2, a
DR4, and DR5 in triggering non-apoptotic sig- 75 kDa protein, is mainly detected in oligoden-
naling pathways even immediately after their drocytes, astrocytes, T cells, myocytes, thymo-
cloning [17, 18], most, if not all, studies have cytes, endothelial cells, and human mesenchymal
been focused on characterizing the molecu- stem cells [28]. Uncertainty remains on the
lar events leading to cell death. Accordingly, TNFR2 signaling pathway, which has been pre-
several agonistic molecules were developed in viously reviewed [28]. The CRD1 of CD95,
order to kill cancer cells, neglecting the impact TNFR1, and TNFR2 is involved in homotypic
of non-apoptotic signals in pathophysiological interactions, leading to pre-association of the
contexts. More recent data changed this vision receptor as a homotrimer in the absence of
by evaluating the biological role of death recep- ligand [23, 24, 29]. This domain has been desig-
tor-mediated non-apoptotic signaling path- nated as the pre-ligand binding assembly
ways in chronic inammatory disorders and domain (PLAD) [29]. Receptors of the TNFR
carcinogenesis. superfamily do not possess any enzymatic activ-
In this chapter, apoptotic signaling pathways ity on their own and rely on the recruitment of
induced by death receptors are discussed. adaptor proteins for signaling. Among these
Moreover, recent evidences pointing to the non- adaptor proteins, TRADD or FADD are instru-
apoptotic signals transmitted by the same recep- mental in the implementation of cell death pro-
tors are brought up, which may imply their cesses [710].
tremendous impact on tumor progression and the TNF is synthesized as a 26 kDa transmem-
design of therapeutic tools. brane type II protein (m-TNF) of 233 amino
9 The CD95/CD95L Signaling Pathway: A Role in Carcinogenesis 145
acids [30] which can be cleaved by the is recruited to TRADD by DD-DD interaction
metalloprotease TACE [31, 32] to release the and binds caspase-8 [2]. Noteworthy, when
17 kDa soluble form of the cytokine (cl-TNF). caspase-8 activity is inhibited or its expression
In contrast to cl-TNF, which only activates is extinguished, DISC is unable to trigger the
TNFR1, m-TNF can bind and activate both apoptotic signaling pathway, but TNFR1 or
TNFR1 and TNFR2 [33]. CD95 stimulation leads to the activation of
Activation of TNFR1 leads to the induction another cell death signal, namely, necroptosis
of cellular processes ranging from cell death [41, 42]. To prevent the induction of the
(apoptosis or necroptosis) to cell proliferation, necroptotic signal, caspase-8 cleaves and inac-
migration, and differentiation; the implementa- tivates RIP1 and RIP3 [43]. The ne-tuned
tion of such different cellular responses reects control of necroptosis by members of the apop-
the formation of different molecular complexes totic signaling pathway in the organism has
after receptor activation [28]. Binding of TNF to been elegantly conrmed by experiments
TNFR1 causes the formation of two consecutive showing that the embryonic lethality of mice
complexes. While the plasma membrane com- harboring the single KO of caspase-8 or FADD
plex (complex I) elicits a non-apoptotic signal- is rescued by an additional KO of the RIP3
ing pathway, a second, internalized, complex gene [4446].
(complex II or DISC) triggers cell death [2]. In
the presence of TNF, the adaptor protein
TRADD interacts with TNFR1 and recruits 9.2.2 TNF/TNFR: A Gold Mine
other proteins involved in the signaling of the for Therapeutic Tools
receptor, such as TRAF2, cIAP1, cIAP2, and
RIP1, to form complex I. At the plasma mem- Many studies on TNF demonstrated its pivotal
brane, this complex activates the NF-B signal- role in fueling inammation, a multistep process
ing pathway, which in turn promotes the that promotes autoimmunity (e.g., rheumatoid
transcription of antiapoptotic genes such as arthritis, ankylosing spondylitis, Crohns disease,
cIAP-1, cIAP-2, and c-FLIP [34]. The linear psoriasis, and refractory asthma) and cancer.
ubiquitin chain assembly complex (LUBAC) is Many TNF inhibitors, such as neutralizing mono-
also recruited to complex I via cIAP-generated clonal antibodies (mAbs) (e.g., iniximab,
ubiquitin chains [35]. HOIL-1, HOIP, and adalimumab, and golimumab), have been devel-
sharpin constitute the LUBAC complex. HOIL-1 oped to treat these chronic inammatory disor-
and HOIP add a linear ubiquitin chain by cata- ders, demonstrating that altering ligand/receptor
lyzing the head-to-tail ligation of ubiquitin [36] interactions with neutralizing mAbs is an invalu-
to RIP1 and NEMO (IKK) in complex I [37], able opportunity to treat certain chronic inam-
thereby activating NF-B. matory disorders. Other TNF- antagonists, such
TNF-induced caspase activation is mediated as etanercept, a TNFR2-immunoglobulin Fc
by a second, intracellular complex II, which is fusion protein, can improve the clinical course of
formed when complex I dissociates from the rheumatoid arthritis [47].
receptor, along with FADD and caspase-8 While ndings accumulate to decipher the
recruitment [2]. NF-B activation leads to molecular mechanisms involved in the induc-
c-FLIP overexpression, preventing formation tion of apoptotic and non-apoptotic signaling
of complex II. Contrariwise, when NF-B acti- pathways by TNFR1 and to elucidate how the
vation is blocked, c-FLIP, whose protein half- receptor can switch from one signal to the other,
life is short [38], is absent, and cells experience the mechanistic links involved in the implemen-
death [2]. RIP1 is deubiquitinated by enzymes tation of non-apoptotic signaling pathways by
such as Cezanne [39] and CYLD [40] and the CD95 remain elusive. However, recent ndings
complex composed of TRADD and RIP1 have revealed its proinammatory effects
moves to the cytosol to form complex II. FADD [4854].
mRNA 1 347 376 542 680 789 851 914 997 1022 1354 2689 nt
Exons 1 2 3 4 5 6 7 8 9
ORF 1 31 197 335 444 506 569 652 676 1008 nt
1 16 174 190 230 317 335 a.a.

Protein
1 2 3 4 5 6
SP TM DD
Fig. 9.1 CD95: mRNA to protein
9.3 CD95: A Death Receptor? 50 kDa by SDS-PAGE. The receptor is a type I

transmembrane protein harboring three CRDs.
In 1989, identication of the mAb APO-1 by Similar to the TNF receptor [29], CD95 is pre-
Peter Krammer et al. revealed the existence of a associated at the plasma membrane as a homotri-
52 kDa protein whose aggregation was able to mer, and this quaternary structure is mandatory
transmit an apoptotic signal in cancer cells [55]. for transmission of the apoptotic signals in the
This receptor was identied in 1991 by Nagata presence of CD95L [23, 24]. Homotrimerization
and colleagues and called Fas (CD95 or APO-1) of CD95 occurs mainly through homotypic inter-
[12]. Its ligand, FasL, was cloned in 1993 by the actions of the CD95-CRD1 [2224]. Binding of
same group and was found to be mainly CD95L or agonistic anti-CD95 mAbs to CD95
expressed at the surface of activated T lympho- alters both the conformation and the extent to
cytes [56] and natural killer (NK) cells [57]; which the receptor is multimerized at the plasma
however, its expression was also detected in dif- membrane. The intracellular region of CD95
ferent tissues in which the presence of acute or encompasses an 80 amino acid stretch designated
chronic inammation is undesirable including as the DD (Fig. 9.1), which consists of six anti-
the eyes [58] and testes [59]. In addition, two parallel -helices [67]. Upon addition of CD95L,
mouse models, in which either the level of CD95 CD95 undergoes conformational modication
expression was downregulated [due to an inser- of its DD, which induces a shift of helix 6 and
tion of a retrotransposon in intron 2 of the recep- fusion with helix 5, promoting both oligomer-
tor gene, these mice are called lymphoproliferation ization of the receptor and recruitment of the
(Lpr) [6062]] or the CD95L afnity for CD95 adaptor protein FADD [68]. A consequence of
was reduced [due to the germ line mutation the opening of the globular structure of CD95
F273L in CD95L, called generalized lymphop- is that the receptor becomes connected through
roliferative disease (gld), which decreases this bridge, which increases the magnitude of
CD95L binding to CD95 [63, 64]], have pro- its homo-aggregation. This long helix allows
vided some insight into the pivotal role played the stabilization of the complex by recruiting
by this interaction in immunosurveillance and FADD. Overall, the CD95-DD:FADD-DD crys-
immune tolerance [65]. tal structure provides some insights into the for-
mation of the large CD95 clusters observed using
imaging or biochemical methods in cells stimu-
9.3.1 Structure/Function lated with CD95L. In addition, it also conrms
that alteration in the CD95 conformation plays
The CD95 gene (APT-1) consists of nine exons, an instrumental role during signal induction [68]
with exon 6 encoding the transmembrane However, this elongated C-terminal -helix favor-
domain [66] (Fig. 9.1). CD95 can be resolved ing the cis-dimerization of CD95-DD was chal-
under denaturing conditions between 40 and lenged by Driscoll et al. who did not observe the
Apoptotic signaling pathway Type I Type II
Immune cell
Immune cell
CD95
mCD95L
DD DISC
FADD tBid Bid Caspase-8 FLIP/PEA-15
Mitochondrion
DISC
APOPTOSOME
Procaspase-8 Cytochrome c
Smac Apaf-1
Pro-caspase 9
XIAP Caspase 9
Caspase-8 c-IAP1, c-IAP2
Apoptosis
FLIP/PEA-15 Caspase-3/7 Caspase-3/7
DNA fragmentation
Nucleus
Fig. 9.2 Type I/II cells. Binding of transmembrane caspase activation leading to apoptosis. Type I cells are
CD95L to CD95 leads to DISC formation. DISC consists characterized by an efcient DISC formation, which
of FADD and procaspase-8. C-FLIP and PEA-15 bind to releases sufcient caspase-8 to directly activate caspase-3.
FADD and prevent caspase-8 recruitment. At the DISC By contrast, type II cells present a weak DISC formation,
level, aggregation of procaspase-8 promotes its auto- and the low amount of released caspase-8 activates the
cleavage and activation. Cleaved caspase-8 is then mitochondrion-dependent apoptotic pathway to amplify
released in the cytosol where it promotes the cascade of death signal
fusion of the last two helices at a more neutral pH the formation of a complex eliciting a sequence
(pH 6.2), compared to the acidic condition (pH of events different from the one occurring at
4) used in the initial study to resolve the CD95- physiologic pH.
DD:FADD-DD structure [68]. Consequently, at Once docked on CD95-DD, FADD self-
pH 6.2, association of CD95 with FADD pre- associates [71] and binds procaspases-8 and
dominantly consisted of a 5:5 complex, which procaspases-10, which are auto-processed and
occurred via a polymerization mechanism released in the cytosol as active caspases,
involving three types of asymmetric interactions which cleave many substrates leading to the
but without major alteration of the DD globular execution of the apoptotic program and cell
structure [69, 70]. It is likely that the low pH death. The complex CD95/FADD/caspase-8/
condition used in the study performed by Scott caspase-10 is called DISC (Fig. 9.2) [10]. Due
et al. altered CD95 conformation and resulted in to the importance of DISC formation in the fate
the formation of nonphysiological CD95:FADD of cells, it is not surprising that numerous cel-
oligomers [68]. Nonetheless, it cannot be lular and viral proteins were reported to ham-
excluded that a local decrease in the intracellular per the formation of this structure, such as
pH affects the initial steps of the CD95 signaling FLIP [72, 73] and PED/PEA-15 [74], which
pathway in vivo, through promoting the opening interfere with the recruitment of caspase-8/cas-
of the CD95-DD and eventually contributing to pase-10 (Fig. 9.2).
9.3.2 Type I/II Signaling Pathways are more addicted to this organelle compared to
type I cells (Fig. 9.2).
Following the discovery of CD95 and the rst To summarize, DISC formation and IAP
steps of its signaling pathway, Peter and col- amount are two cellular markers allowing a clear
leagues described that cells can be divided in discrimination between type I and type II cells.
two groups with regard to the kinetics through Even though IAP overexpression can account for
which they respond to CD95-mediated apoptotic the mitochondrion dependency observed in type
signals, the magnitude of DISC formation and II cells, it remains unclear why DISC formation
the role played by the mitochondrion in this is hampered in type II cells and/or enhanced in
pathway [75]. DISC formation occurs rapidly their type I counterparts. Recently, high activity
and efciently in type I cells releasing a large of the lipid kinase phosphoinositide 3-kinase
amount of activated caspase-8 in the cytosol, (PI3K) or downregulation of its neutralizing
while type II cells have difculty forming this phosphatase, phosphatase and tensin homologue
complex, and the amount of active caspase-8 is on chromosome 10 (PTEN), was found in type II
insufcient to directly activate the effector cas- cells, while this signal is blocked in type I cell
pase-3 and caspase-7 [75]. Nonetheless, type II lines [87, 88]. The PI3K signaling pathway was
cells experience cell death upon CD95 engage- reported to prevent the aggregation of CD95 [89],
ment and are even more sensitive to the CD95- probably by retaining the receptor outside of
mediated apoptotic signal compared to type I lipid rafts [87, 90]. PEA-15, also known as PED,
cells [7577]. This discrepancy can be partly is a protein containing a death effector domain
explained by the fact that the low amount of acti- (DED) that has been shown to inhibit the CD95
vated caspase-8 in type II cells is sufcient to and TNFR1 apoptotic signals (Fig. 9.2) [74].
cleave BID, a BH3-only protein, which consti- Activation of PI3K and its downstream effector,
tutes the molecular link between caspase-8 acti- serine-threonine kinase Akt, leads to phosphory-
vation and the apoptotic activity of mitochondria. lation of PEA-15 at serine 116 [87, 90]; this post-
Indeed, after cleavage by caspase-8, truncated translational modication promotes its interaction
BID (tBID) translocates to mitochondria, where with FADD, ultimately inhibiting DISC forma-
it triggers the release of proapoptotic factors tion [91, 92].
(Fig. 9.2) [78, 79]. Although CD95 stimulation Notably, the existence of type I and type II
activates the mitochondrion-dependent apop- cells is not only an in vitro observation, but has
totic signal in type I and type II cells, it seems been identied physiologically in human body.
that only type II cells are addicted to this signal CD95-mediated apoptotic signal cannot be
as they display a higher amount of the caspase-3 altered in thymocytes or activated T cells express-
inhibitor XIAP compared to type I cells [80]. ing a Bcl-2 transgene, conferring to their type I
Among the inhibitor of apoptosis protein (IAP) nature [93], whereas hepatocytes expressing the
family, XIAP, c-IAP1, and c-IAP2 inhibit cas- same transgene resist CD95-induced apoptosis
pase-3, caspase-7 [81, 82], and procaspase-9 and thus behave as type II cells [94, 95].
[83] activity by direct binding, thereby prevent-
ing access to substrates. Furthermore, XIAP can
function as an E3 ligase whose activity is 9.3.3 What Can We Learn
involved in the ubiquitination of active caspase- from CD95 Mutations?
3 and its subsequent degradation through the
proteasome [84]. To detach XIAP from caspase- Germinal mutations in APT-1 have been reported
3 and restore the apoptotic signal, cells require in patients developing a syndrome termed
the release of SMAC/DIABLO (second mito- autoimmune lymphoproliferative syndrome
chondria-derived activator of caspase/direct type Ia (ALPS, also called Canale-Smith syn-
IAP-binding protein with low PI) by the mito- drome) [9698]. ALPS patients show chronic
chondrion [85, 86], explaining why type II cells lymphadenopathy and splenomegaly, expanded
Mutation/a.a.
1 2 3 4 5 6 7 8 9 10 11121314
CD95L interactions
Extracellular
Intracellular
P201
E202
1
2 R234
D244
Q257K 3 V251
(Beneteau et al, Cancer Research 2007)
4
Y275F
Internalization (Lee et al, EMBOJ, 2006) 5
A285
6
1297D
Open conformation (scott et al, Nature, 2009)
Fig. 9.3 Distribution of somatic and germinal mutations within CD95 protein sequence
populations of double-negative / lympho- of various histological origins (reviewed in [54]).

cytes (CD3+CD4CD8), and often develop auto- Extensive analysis of CD95 mutations and their
immunity [96, 97, 99, 100]. In agreement with distribution in APT-1 reveals that, with some
the notion that CD95 behaves as a tumor suppres- exceptions, most are gathered in exons 8 and 9
sor, ALPS patients display an increased risk of encoding the CD95 intracellular region (Fig. 9.3)
Hodgkin and non-Hodgkin lymphoma [101]. [105]. Remarkably, most of these mutations are
Predominance of post-germinal center (GC) lym- heterozygous, mainly localized in CD95-DD,
phomas in patients exhibiting either germ line or and lead to inhibition of the CD95-mediated
somatic CD95 mutations can be explained by the apoptotic signal. Indeed, in agreement with the
fact that, inside germinal centers of the secondary notion that CD95 is expressed at the plasma
lymphoid follicles, the CD95 signal plays a piv- membrane as a pre-associated homotrimer [23,
otal role in the deletion of self-reactive maturat- 24], formation of heterocomplexes containing
ing B lymphocytes [102], in addition to the fact wild-type and mutated CD95 prevents FADD
that APT1 belongs to a set of rare genes (i.e., recruitment and abrogates the ignition of the
PIM1, c-myc, PAX5, RhoH/TTF, and Bcl-6) sub- apoptotic signal in a dominant manner.
ject to somatic hypermutation [103, 104], which Extensive analysis and positioning of various
may affect biological function. In addition to CD95 mutations described in the literature seem
post-GC lymphomas, signicant amounts of to highlight mutation hot spots in the CD95
mutations in the CD95 gene were found in tumors sequence (Fig. 9.3). Among these hot spots,
arginine 234, aspartic acid 244, and valine 251 develop resistance to CD95L-expressing immune
account for a signicant amount of the docu- cells. Indeed, the plasma membrane is a hetero-
mented CD95 mutations. Indeed, among the 189 geneous lipid bilayer comprising compacted or
mutations annotated in the 335 amino acids of liquid-ordered domains, called microdomains,
CD95, 30 (~16 %) are localized on these three lipid rafts, or detergent-resistant microdomains
amino acids (Fig. 9.3). Strikingly, the pivotal role (DRMs). These domains are described as oat-
played by these amino acids in stabilization or ing in a more uid or liquid-disordered 2-D lipid
formation of intra- and inter-bridges between bilayer and are enriched in ceramides [109]. It
CD95 and FADD may explain these hot spots. has been elegantly shown that while CD95 is
For instance, both R234 and D244 contribute to mostly excluded from lipid rafts in activated T
the homotypic aggregation of the receptor and lymphocytes, TCR-dependent reactivation of
FADD recruitment [67]. Nevertheless, the these cells leads to rapid distribution of the death
observation of death domain hot spots is in receptor into lipid rafts [110]. This CD95 com-
contradiction with the study of Scott and col- partmentalization contributes to reducing the
leagues demonstrating that the region of the apoptotic threshold leading to the clonotypic
CD95-DD interacting with the FADD-DD elimination of activated T lymphocytes through
extends over a disperse surface through weak activation of the CD95-mediated apoptotic sig-
binding afnity [68]. nal [110]. Similarly, the reorganization of CD95
Most ALPS type Ia patients affected by into DRMs can occur independent from ligand
malignancies do not undergo loss of heterozy- upon addition of certain chemotherapeutic drugs
gosity (LOH), which formed the hypothesize that (e.g., rituximab [111], resveratrol [112, 113],
preservation of a wild-type allele may contribute edelfosine [87, 114, 115], aplidin [116], perifo-
to carcinogenesis [106, 107]. In the same line, it sine [115], cisplatin [117]). The molecular cas-
was demonstrated that expression of a unique cades that underlie this process remain elusive.
mutated CD95 allele blocks the induction of Nevertheless, a growing body of evidence leads
apoptotic signals, while it fails to prevent non- us to postulate that alteration of intracellular sig-
apoptotic signals such as NF-B and MAPK naling pathway(s), such as the aforementioned
[106, 107], whose induction promotes invasive- PI3K signal [87, 90], may change biophysical
ness in tumor cells [105, 108]. In addition, muta- properties of the plasma membrane, such as
tions found in the intracellular CD95-DD exhibit membrane uidity, which in turn may facilitate
a higher penetrance of ALPS phenotype features CD95 clustering into large lipid raft-enriched
in mutation-bearing relatives compared to extra- platforms, favoring DISC formation and induc-
cellular mutations. These results suggest that tion of the apoptotic program [118].
unlike DD mutations, CD95 mutations localized
outside the DD somehow prevent the apoptotic 9.3.4.2 Posttranslational Modications
signal but may fail to promote non-apoptotic Accumulation of CD95 mutations is not the only
pathways, which may contribute to disease mechanism by which malignant cells inhibit the
aggressiveness. extrinsic signaling pathway. Posttranslational
modications in the intracellular tail of CD95,
such as reversible oxidation or covalent attach-
9.3.4 Regulation of the Initial Steps ment of a palmitic acid, were reported to alter the
of CD95-Mediated Signaling plasma membrane distribution of CD95 and
thereby its subsequent signaling pathway. For
9.3.4.1 Lipid Rafts instance, S-glutathionylation of mouse CD95 at
In addition to CD95 downregulation or expres- cysteine 294 promotes clustering of CD95 and its
sion of the mutated allele of the receptor, the distribution into lipid rafts [119]. This amino acid
plasma membrane distribution of CD95 repre- is conserved in the human CD95 sequence and
sents an additional pathway for tumor cells to corresponds to cysteine 304 (or C288 when
subtraction of the 16 amino acid signal peptide is steps play a critical role in the implementation
taken into consideration [12, 120]). Interestingly, of apoptotic signals.
Janssen-Heininger and colleagues emphasize Of note, similar to S-nitrosylation, both the
that death receptor glutathionylation occurs aforementioned S-glutathionylation at C304
downstream of caspase-8 and caspase-3 activa- (C288) and palmitoylation at C199 (C183) pro-
tion whose catalytic activity damages the thiol mote the partition of CD95 into lipid rafts and
transferase glutaredoxin 1 (Grx1), an enzyme enhance the subsequent apoptotic signal. Further
implicated in the denitrosylation of proteins investigation is required to address whether these
[119]. The consequence of Grx1 inactivation is posttranslational modications are redundant and
the accumulation of glutathionylated CD95, occur simultaneously in dying cells or are elic-
which clusters into lipid rafts, sensitizing cells to ited in a cell-specic and/or in a microenvironment-
the CD95-mediated apoptotic signal. Based on specic manner. Understanding the molecular
these ndings, caspase-8 activation occurs prior mechanisms controlling these posttranslational
to aggregation of CD95 and redistribution into modications would be of great interest in order
lipid rafts, both of which are requisite to form the to identify the mechanism by which tumor cells
DISC and subsequently activate larger amounts block them, leading to their resistance to the
of caspase-8. In agreement with these observa- extrinsic signaling pathway.
tions, activation of caspase-8 was reported to
occur in a two-step process. First, an immediate 9.3.4.3 CD95 Internalization
and small amount of activated caspase-8 (<1 %) Using a powerful magnetic method to isolate
is generated when CD95L interacts with CD95 receptor-containing endocytic vesicles, it has
that orchestrates acid sphingomyelinase (ASM) been shown that CD95 promptly associates with
activation, ceramide production, and CD95 clus- endosomal and lysosomal markers when incu-
tering, which in turn promote DISC formation bated with an agonistic anti-CD95 mAb [126]. In
and the outburst of caspase-8 processing essen- addition, expression of a CD95 mutant in which
tial to mount the apoptotic signal [121]. the DD-located tyrosine 291 (Y275) is changed
S-Glutathionylation consists in a bond to phenylalanine does not seem to alter the capac-
between a reactive Cys-thiol and reduced gluta- ity to bind FADD but compromises CD95L-
thione (GSH), a tripeptide consisting of glycine, mediated CD95 internalization occurring through
cysteine, and glutamate; its attachment to the an AP-2/clathrin-driven endocytic pathway
protein will alter its structure and function in a [126]. More strikingly, expression of the
manner similar to the addition of a phosphate internalization-defective CD95 mutant Y291F
[122]. S-Glutathionylation is not the only post- abrogates the transmission of apoptotic signals,
translational modication of CD95 on a cysteine. but fails to alter the non-apoptotic signaling path-
S-nitrosylation of cysteine 199 (corresponding to ways (i.e., NFkB and ERK), and even promotes
C183 after subtraction of signal peptide sequence) them (Fig. 9.3). These ndings provide insight
and 304 (C288) in colon and breast tumor cells into the presence of a region in the DD, interact-
also promotes the redistribution of CD95 into ing with AP2 and promoting a clathrin-dependent
DRMs, the formation of the DISC, and the trans- endocytic pathway in a FADD-independent man-
mission of the apoptotic signal [123]. ner. Regarding the role of palmitoylation in
Two reports have brought into light that receptor internalization, the interplay between
covalent coupling of a 16-carbon fatty acid lipid alteration and the AP2/clathrin-driven inter-
(palmitic acid) to cysteine 199 (C183) elicits the nalization of CD95 remains to be elucidated.
redistribution of CD95 into DRMs, the formation
of SDS-stable CD95 microaggregates resistant to 9.3.4.4 Ca2+ Response
denaturing and reducing treatments, and internal- It has been recently demonstrated that CD95
ization of the receptor [124, 125]. Although their engagement evokes a rapid and transient Ca2+
order remains to be ne-tuned, these molecular signaling, which stimulates the recruitment of
protein kinase C-2 (PKC-2) from the cytosol to ing enzyme that is also cleaved and inactivated
the DISC [127]. This kinase transiently brakes by caspase-8 [135].
DISC formation, providing a checkpoint before Overall, these ndings suggest that the apop-
the irreversible commitment to cell death [128]. totic machinery controls the necrotic one. This
These ndings raised the following questions: concept has been recently established in vivo by
what are the Ca2+-dependent molecular mecha- double-KO experiments [4446, 136]. The KO of
nisms transiently inhibiting DISC formation, and FADD or caspase-8 is deleterious in mice mainly
do tumor cells use this signal to escape the by the fact that these two apoptotic factors are
immune response and/or resist chemotherapy? benecial in inhibiting a RIP1-/RIP3-dependent
necrotic signal; thus, their loss unleashes the
necroptotic program and leads to embryonic
9.3.5 Programmed Necrosis Also lethality. Yet, most studies on necroptosis have
Known as Necroptosis focused on the TNF signaling pathway, whereas
the mechanism by which CD95 can elicit this cell
In 1998, inhibition of caspase activity was shown death pathway, and how the switch in this recep-
to sensitize broblastic L929 cell line to TNF- tor occurs between non-apoptotic, apoptotic, and
mediated necrotic cell death [42]. With respect to necroptotic signals remains unclear. Importantly,
CD95 signal, Tschopp et al. showed that FADD the impact of each cell death on antigen presenta-
and RIP1 participate in the implementation of a tion, and on the efciency of immune response
non-apoptotic signaling pathway, which leads to after elimination of infected or transformed cells,
a necrotic morphology without chromatin con- remains unclear.
densation and with loss of plasma membrane
integrity [41]. Of note, BID cleavage was not
observed in this necrotic signal. While FADD 9.3.6 CD95L, an Inammatory/
plays a crucial role in both apoptotic and necrotic Oncogenic Cytokine?
pathways, RIP1 recruitment to CD95 occurs
independently of this adaptor protein. Indeed, 9.3.6.1 A Ligand to Create Immune
yeast two-hybrid experiments showed that RIP1 Privileges
can bind directly to the CD95 DD, while this The transmembrane CD95L (CD178/FasL) is
interaction is lost when a bait corresponding to present at the surface of activated lymphocytes
mutated CD95-DD (replacement of Val 238 to [64] and NK cells [137] where it orchestrates the
Asn) is used [129]. In addition, RIP3 (RIPK3, a elimination of transformed and infected cells. In
member of the RIP kinase family) is an indis- addition, CD95L is expressed on the surface of
pensable factor for the induction of the necrotic neurons [138], corneal epithelia and endothelia
signaling pathway [7880]. A growing body of [58, 139], and sertoli cells [59] to prevent the
evidence supports the existence of necroptosis inltration of immune cells and thus to prohibit
(programmed necrosis). In addition, identica- the spread of inammation in these sensitive
tion of necrostatin, a chemical inhibitor of organs (i.e., brain, eyes, and testis, respectively),
necroptosis [130], which specically inhibits commonly called immune-privileged sites.
RIP1 kinase activity [131], has accelerated the The description of physiological immune privi-
pace of discovery in this eld of cell death. lege was followed by tumor-mediated immune
Interplays exist between apoptosis and necropto- privilege, since two groups reported that the
sis; for instance, caspase-8, a potent inhibitor of ectopic expression of CD95L by malignant cells
necroptosis for both CD95 and TNFR1 [132], participated in the elimination of inltrating T
plays a critical role in necroptosis by its ability to lymphocytes and thus could play a role in the
process and inactivate RIP1 and RIP3 [133, 134]. establishment of a tumor site whose access was
At least for TNF signaling, the necrotic signal denied to immune cells [140, 141]. However,
relies on the activity of CYLD, a deubiquitinat- these observations are controversial since
ectopic expression of CD95L in allogenic for CD95 binding, thus acting as an antagonist of
transplant of -islets [142, 143] and in tumor cell the death signal [155, 156]. It has been recently
lines [144] led to a more rapid elimination of demonstrated that this metalloprotease-cleaved
these cells than control cells, due to increased CD95L (cl-CD95L) actively participates in the
inltration of neutrophils and macrophages aggravation of inammation and autoimmunity
endowed with antitumor activity. in patients affected by systemic lupus erythema-
tosus (SLE) by inducing the non-apoptotic
9.3.6.2 At Least Two Different Ligands NF-B and PI3K [51, 53] signaling pathways
and Two Different Signals (Fig. 9.4). Unlike transmembrane CD95L,
Among the weapons at the disposal of immune induction of the PI3K signaling pathway by its
cells, transmembrane CD95L contributes to the metalloprotease-cleaved counterpart occurs
elimination of pre-tumor cells. Therefore, pre- through the formation of a complex devoid of
tumor cells that escape the immunosurveillance FADD and caspase-8 which recruits the src
will be shaped to develop resistance to CD95, a kinase c-yes instead [53, 148]; this unconven-
process termed immunoediting [145]. In other tional receptosome was designated motility-
words, imprinting of the immune system on pre- inducing signaling complex (MISC) [53, 157]
tumor cells will select malignant cells with (Fig. 9.4). Even though experiments by the
increased resistance towards the CD95L-induced authors did not detect any trace of caspase-8 in
signal. As previously mentioned, these altera- the MISC, this enzyme has been shown to partici-
tions of the CD95 signal not only block the pate in cell migration. The protease activity of
CD95-mediated apoptotic signal but also procaspase-8 can be abolished by its phosphoryla-
mote the transmission of non-apoptotic signals tion at tyrosine 380 by src kinase [158]. This
by CD95L, which may play a critical role in car- posttranslational modication was observed in
cinogenesis [106108, 146]. In agreement with cells stimulated with EGF and in colon cancer
this hypothesis, a complete loss of CD95 expres- cells exhibiting constitutive activation of src;
sion is rarely observed in malignant cells [147]. from a molecular standpoint, this modication
Accumulating evidence indicates that the does not alter caspase homodimerization or
apoptotic ligand CD95L behaves as a chemoat- recruitment in DISC [158]. Moreover, the EGFR-
tractant for neutrophils, macrophages [50, 143, driven phosphorylation of caspase-8 at Y380
144], T lymphocytes [53], and malignant cells in turns out to be a potent inducer of the PI3K sig-
which the CD95-mediated apoptotic signal is naling pathway by recruiting the PI3K adaptor
nonproductive [108, 148]. Nonetheless, the bio- p85 alpha subunit [159]. Ultimately, caspase-8
logical role of CD95L has to be claried due to phosphorylation triggers cell migration.
the fact that pathophysiologically the ligand is Nonetheless, it is noteworthy that CD95-induced
present in at least two forms with different stoi- migration and invasion do not appear to require
chiometries. Indeed, CD95L is a transmembrane an intact DD (reviewed in [160]), suggesting that
cytokine whose ectodomain can be cleaved by either the caspase-8-dependent mode of cell
metalloproteases such as MMP3 [149], MMP7 migration occurs as an alternative signal for death
[150], MMP9 [151], and ADAM-10 (A disinteg- receptors or that it only participates in non-death
rin and metalloproteinase 10) [152, 153] and receptor-induced cell motility. It would be inter-
released as a soluble ligand in the bloodstream. esting to address this question in the future. To
Based on the data demonstrating that a hexameric date, it can only be surmise that phosphorylation
CD95L represents the minimal level of self- of caspase-8 at Y380 upon EGFR stimulation
association required to signal apoptosis [154] and may prime certain cancer cells to become unre-
that cleavage by metalloproteases releases an sponsive to the apoptotic signal triggered by
homotrimeric ligand [154, 155], this soluble cytotoxic CD95L and meanwhile promote cell
ligand has long been considered as an inert ligand migration, an essential event in the course of
competing with its membrane-bound counterpart cancer cell metastasis (Fig. 9.4).
Pro-motile signaling pathway
Caspase Caspase
independent dependent Immune cell
MISC
PI3K
C-yes
Metalloprotease
Cleaved CD95L PIP2 PIP PIP
3 2 Caspase
inactivation
? Y
? P Src kinase
AKT p110
p85
?
P
? MA se
a YP
kin
EGF
k
N F- Procaspase-8 EGFR
Cell motility
Transcription survival
inflammation
Nucleus
Fig. 9.4 CD95 triggers an unconventional PI3K signal- Panel: It was reported that procaspase-8 can be phosphor-
ing pathway. Left panel: In the presence of cl-CD95L, ylated by the tyrosine kinase src upon EGFR stimulation.
CD95 triggers MISC formation. This complex is devoid This posttranslational modication not only blocks the
of FADD and caspase-8, but, instead, recruits the src catalytic activity of caspase-8 but also promotes the
kinase c-yes that implements the PI3K signaling pathway. recruitment of the p85 subunit of PI3K. We surmise that
CD95 engagement is also capable of NF-B and MAPK this caspase-8 phosphorylation may favor the non-apop-
activations through a yet unknown mechanism. Right totic signals induced by CD95
It is noteworthy that in a similar manner, a with the ability to contain the spread of inamma-
decrease in the plasma membrane level of CD95 or tion or impede carcinogenesis at least in patholo-
expression of a mutated CD95 allele, as observed gies involving increased soluble CD95L such as
in ALPS patients and malignant cells, inhibits cancers (e.g., pancreatic cancer [162], large gran-
the implementation of the apoptotic signal but ular lymphocytic leukemia, breast cancer [157],
does not affect the transmission of non-apoptotic and NK cell lymphoma [163]) or autoimmune
signals, such as NF-B, MAPK, and PI3K [106, disorders (e.g., rheumatoid arthritis and osteoar-
107, 147], suggesting that these signals may stem thritis [164], graft-versus-host-disease (GVDH)
from a different domain than CD95-DD or rely [165, 166] or SLE [53, 167]). Altogether, these
on different thresholds to be elicited. In summary, studies support the notion that the death function
although the CD95/CD95L interaction can elimi- of CD95 may correspond to its day job, while
nate malignant cells by implementation of the the receptor may act as a night killer by fuel-
DISC or can promote carcinogenesis by sustaining inammation in certain pathophysiological
ing inammation and/or by inducing metastatic contexts.
dissemination [50, 51, 53, 108, 147, 148, 161], Strikingly, while the soluble form of CD95L
the molecular mechanisms underlying the switch generated by MMP7 (cleavage site inside the
113
between these different signaling pathways ELR115 sequence, Fig. 9.5) induces apoptosis
remain enigmatic. An important question to be [150], its counterpart processed between serine
addressed is how the magnitude of CD95 aggre- 126 and leucine 127 does not [51, 53, 155].
gation controls the formation of death- vs. To explain this discrepancy, one may speculate
motility-ISCs. Addressing these questions will that the different quaternary structures of the
lead to the development of new therapeutic agents naturally processed CD95L underlie the
CD95-interacting amino-acids
Self
assembly domain
P205 Y218 F275L(gld like)
1 82 102 137-183 281
hCD95L TM
STALK REGION
110 120 130 139 218
103
QLFHLQKELAELRESTSQMHTASSLEKQIGHPSS-RGS YPQDLMMEGK
Cleavage sites: 1 oxidation 2 3 Oxidation
MMP3/7/9 & ADAM10 Cis-Interaction
Fig. 9.5 CD95L: metalloprotease cleavage sites and domains
implementation of a death- vs. non-death- Overall, these ndings emphasize that it will
inducing signaling complexes and downstream be of great interest in the future to nely charac-
signals. In agreement with this notion, soluble terize the quaternary structure of the naturally
CD95L bathed in the bronchoalveolar lavage processed CD95L from the sera of patients
(BALs) of patients suffering from acute affected by cancers or chronic/acute inamma-
respiratory distress syndrome (ARDS) undergoes tory disorders, to better understand the molecular
oxidation at methionines 224 and 225 (Fig. 9.5), mechanisms implemented by this ligand and thus
which enhances the aggregation level of the solu- predict its subsequent biological functions.
ble ligand followed by its cytotoxic activity
[168]. The same authors observed that the stalk
region of CD95L, corresponding to amino acids 9.4 Concluding Remarks
103136 and encompassing the metalloprotease
cleavage sites (Fig. 9.5), participates in the multi- Apoptosis is a fundamental process contributing
merization of CD95L, which accounts for the to tissue homeostasis, immune response, and
damage of the lung epithelium in ARDS [168]. development. CD95, also called Fas, is a member
Of note, in ARDS BALs, additional oxidation of the tumor necrosis factor receptor (TNF-R)
occurs at methionine 121 (Fig. 9.5), which in superfamily. Its ligand, CD95L, was initially
turn prevents the processing of CD95L by detected at the plasma membrane of activated T
MMP7, and explains why this cytotoxic ligand lymphocytes and natural killer (NK) cells where
keeps its stalk region [168]. Nonetheless, preser- it contributes to the elimination of transformed
vation of this region in soluble CD95L raises the and infected cells. Given its implication in
question that whether an unidentied MMP7- immune homeostasis and immune surveillance
independent cleavage site exists in the juxta- combined with the fact that various lineages of
membrane region of CD95L, near the plasma malignant cells exhibit loss-of-function muta-
membrane, or the ligand detected in ARDS tions, CD95 was initially classied as a tumor
patients corresponds to the full-length CD95L suppressor gene. Nonetheless, in different patho-
embedded in exosomes [169, 170]. Indeed, this physiological contexts, this receptor is able to
peculiar exosome-bound CD95L can be transmit non-apoptotic signals and promote
expressed by human prostate cancer cells (i.e., inammation and carcinogenesis. Although the
LNCaP), and evokes apoptosis in activated T different non-apoptotic signaling pathways
lymphocytes [171]. (NF-B, MAPK, and PI3K) triggered by CD95
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in normal human T lymphocytes. J Exp Med.
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20. Locksley RM, Killeen N, Lenardo MJ. The TNF and
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MHC Class I Molecules and Cancer
Progression: Lessons Learned 10
from Preclinical Mouse Models
Irene Romero, Ignacio Algarra,

and Angel M. Garcia-Lora
Contents 10.4.2 Immunotherapy as a Treatment Against

Metastatic Progression Derived
10.1 Introduction ............................................ 161 from Primary Tumors with
10.2 MHC-I Cell Surface Expression Different MHC-I Expression ..................... 170
on Tumor Cells and Primary 10.5 Concluding Remarks ............................. 170
Tumor Growth........................................ 162
10.2.1 Studies in GR9 Tumor Model: References ............................................................. 171
H-2 Antigen Surface Expression
and Tumorigenic Capacity ......................... 164
10.3 MHC-I Expression and Metastatic
Progression ............................................. 166
10.3.1 MHC Class I Expression on Primary
Tumor Cells May Determine
Spontaneous Metastatic Capacity .............. 166
10.3.2 Different MHC-I Surface Expression 10.1 Introduction
on GR9 Tumor Clones Determines Their
Spontaneous Metastatic Capacity .............. 167 Major histocompatibility complex (MHC) is
10.4 Immunotherapy as a Treatment composed of a set of molecules that play a pivotal
Against Cancers Expressing Different role in the immune response against different
MHC-I Surface Expression ................... 169 pathogens and tumors cells. These molecules
10.4.1 Immunotherapy as a Treatment
Against Primary Tumors with were described in mice for the rst time by Gorer
Different Levels of MHC-I Expression ..... 169 while performing transplantation studies with
tumor cell lines injected in inbred strains of mice
[1]. In the middle of the 1950s, Jean Dausset
described the HLA system in humans which is
equivalent to the mouse H-2 complex [2]. MHC
I. Romero, PhD A.M. Garcia-Lora, PhD (*) class I (MHC-I) molecules comprise the classical
Servicio de Analisis Clinicos & Inmunologia,
(class Ia) HLA-A, HLA-B and HLA-C antigens
UGC Laboratorio Clinico,
Hospital Universitario Virgen de las Nieves, in humans and H-2 K, H-2 D, and H-2 L in mice
Av de las Fuerzas Armadas, 2, Granada, 18014, Spain and the nonclassical (class Ib) -E, -F, and -G in
e-mail: iren_romero@hotmail.com; humans and Qa and Tla antigens in mice [3].
angel.miguel.exts@juntadeandalucia.es
Their structure is quite similar in human and
I. Algarra, PhD mice, forming a trimolecular complex consisting
Departamento de Ciencias de la Salud, Facultad de
of a 45 kDa highly polymorphic heavy chain,
Ciencias Experimentales, Universidad de Jaen,
Jaen, Spain peptide antigen, and the nonpolymorphic 12 kDa
e-mail: ialgarra@ujaen.es 2-microglobulin (2m) light chain [4]. HLA/H-2

162 I. Romero et al.
class I molecules are expressed on the surface of heavy chains or 2m in tumor cells will have a
nucleated cells [5]. It is estimated that there are profound effect on the recognition and killing of
up to 250,000 of each MHC-I molecule on the those tumor cells by T lymphocytes [16, 17]. In
surface of a somatic cell [6]. this context, a new phase has been proposed into
MHC-I molecules bind antigens in the form of the tumor evolution, called the immunoblindness
peptides, generated from endogenous proteins, phase, which comes after the three phases of
present on the cell surface to CD8+ T cells. In immunoedition process [18]. During this phase,
tumor cells, MHC-I molecules present tumor- CTLs lose control over tumor cells, since losing
associated antigens (TAAs) to T cytotoxic lym- MHC-I surface expression makes them invisible.
phocytes (CTLs) activating cell proliferation, Our research group has a long and well-
cytokine production, and target cell lysis. These established history identifying and dening the
TAAs are generated from degraded foreign HLA class I altered phenotypes present in human
endogenous proteins by the antigen presentation tumors. In fact, the data accumulated indicate
machinery (APM). This process is carried out by that alterations in HLA class I expression are
a large number of proteins and accessories mol- commonly found in most human tumors [19, 20].
ecules [79]. Correct functioning of these APM Seven different altered HLA class I phenotypes
components gives rise to cells with normal surface have been dened in a large variety of human
expression of the MHC-I molecules [10, 11]. Any tumors, and the molecular mechanisms that have
defect in these processes will lead to non- been found to underlie these alterations in MHC-I
expression of MHC-I molecules on the cell sur- expression are multiple [21]. These defects can
face. These MHC-I-decient tumor cells might occur at any step required for MHC synthesis,
be recognized by natural killer (NK) cells [12]. assembly, transport, or expression on cell surface.
In this chapter, we will focus on analyzing the Only some of these defects can be recovered by
role of MHC-I antigens in cancer immunosurveil- cytokines or other agents, while others remain
lance in murine tumor models without obviating unrecovered. Thus, MHC alterations can be clas-
the great contributions done in human tumor sied into two main groups: reversible defects
models; the authors laboratory is the reference (regulatory or soft) and irreversible defects
to the ndings described. (structural or hard) [22, 23].
Many studies in human and experimental
tumors have reported variations in MHC-I antigen
10.2 MHC-I Cell Surface cell surface expression [2427]. These variations
Expression on Tumor Cells have been associated with important changes in
and Primary Tumor Growth tumor behavior and metastatic colonization [28,
29]. The crucial role of MHC-I in local tumor
For over 30 years ago, our group of investigators growth and metastasis has also been demon-
is working on human and mouse preclinical strated in many different murine tumor models.
tumor models in an attempt to dene the mecha- The rst detection of MHC-I lack in mouse
nisms through which tumor cells evade immune tumors was described in 1976; loss of one H-2 Kk
system. We have found that tumor cells develop private specicity was reported in Gardener
sophisticated molecular and biological mecha- lymphoma derived from a C3H mouse [25].
nisms which allow them to escape immunosur- Following these studies, different groups reported
veillance. Among the mechanisms studied, MHC altered expression of MHC molecules in other
alteration is one of the most important and fre- tumors, i.e., the absence of some H-2d molecules
quent mechanisms, possibly playing a relevant in a methylcholanthrene-induced sarcoma (MCG4)
role in the tumor-host scenario [1315]. Any in a BALB/c mouse [30]; loss of Kk antigen (Ag)
alteration affecting the surface expression of expression in a particular AKR tumor cell line
MHC-I molecules, as the expression and function designated K36.16, this tumor cell line showed
of APM components, the expression of MHC-I resistance to killing by AKR anti-MuLV CTLs
10 MHC Class I Molecules and Cancer Progression: Lessons Learned from Preclinical Mouse Models 163
in vitro [31]; loss of the products of the H-2 Ld coworkers have studied an MuLV-induced AKR
locus in a BALB/c brosarcoma [32]; and tumor in which the expressed H-2 K and H-2 D
absence of H-2 Ds Ags in SJL/J lymphomas [33]. Ags are differentially induced by IFN- [42]. In
Another eld in the study of MHC-I Ags in the spontaneous BALB/c line 1 murine carci-
murine tumors originates from transfection of noma, it has been shown that the induction of
MHC-I molecules in MHC-I-decient murine MHC-I antigen expression by IFN- and DMSO
tumors. The transfection and cell surface expres- differ at the molecular level. A point mutation in
sion of one H-2 k gene product in the AKR lym- the D1 region of the Dd promoter diminished
phoma cell line K36.16, a subline of K36 IFN- responsiveness, but did not alter induction
(H-2 Kk-negative) lymphoma, inhibited the syn- of Dd molecule by DMSO. Thus, DMSO appears
geneic growth of this tumor [34, 35]. Studies to regulate MHC-I transcription through multiple
with the methylcholanthrene (MCA)-induced regions of the MHC-I heavy chain promoter by
T10 sarcoma demonstrated that the transfection mechanisms distinct from IFN- [43]. Studies
of Kk or Kb gene into H-2 K-negative parental with mutant phenotypes have led to the descrip-
cells reduced tumorigenicity and abolished the tion of factors controlling the folding, the intra-
formation of metastasis in syngeneic mice [36]. cellular transport, and surface expression of class
Similar results were obtained in other experimen- I molecules [44].
tal models [37]. In all these studies, absence of Components of APM are important elements in
MHC-I molecules has been interpreted as a factor the MHC-I cell surface expression. Alteration in
which selects immunodecient variants and rep- the Ag presentation pathway may serve as an eva-
resents a major escape mechanism from T cell sive mechanism rendering tumors unrecognizable
recognition. The reconstitution of H-2 class I by host immunosurveillance mechanisms. Certain
expression has demonstrated that even MHC-I murine tumor cell lines, such as the chemical-
molecules on tumor cells are responsible for reg- induced CMS-5, EL4, MCA102, and MCA205
ulation of NK susceptibility. Restoration of these cells, with decient expression and/or function of
molecules by transfection with 2m gene resulted multiple APM components, in particular the pep-
in a strong decrease in susceptibility to NK lysis tide transporters (TAPs) and tapasin, show reduced
in S3 cell line, a negative variant for H-2 Db and levels of MHC-I surface expression accompanied
Kb of the murine thymoma EL4 [38]. by low immunogenicity, hence evading T cell-
The differential expression of H-2 class I K, mediated immune recognition in vivo [45]. In the
H-2 class I D, and H-2 class I L molecules is B16 melanoma, MHC-I-decient phenotype has
another event present in some tumors. Studies on been attributed to the downregulation or loss of the
AKR-derived B cell lymphomas (H-2k) have expression and function of multiple APM com-
shown that Dk molecules are processed slower ponents [46]. In other studies, it has been shown
than Kk molecules, with a half-time of 45 h [39]. that inoculation of C57BL/6 mice with a mixture
Other studies have shown that Ld Ags are of TAP-1-positive and TAP-1-negative tumor cell
expressed at levels three to four times lower than lines, generated from a transformed murine bro-
Dd or Kd Ags [40]. This is in line with the studies blast line, produced tumors exclusively composed
that show that in BALB/c S49 lymphoma sublines, of TAP-1-negative cells, indicating an in vivo
there is a locus-specic regulation for Kd, Dd, and selection for TAP-decient cells. Thus, loss of
Ld surface molecules [41]. The differential TAP function can allow tumor cells to avoid T cell
expression of these molecules on the cell surface immunity producing tumor cells with increased
could be a mechanism used by the tumor cells to tumorigenicity [16]. In the APM-decient mouse
escape from immunosurveillance. Therefore, lung carcinoma cell line CMT.64, re-expression
these studies all together could add to our knowl- of TAP-1 after infection with TAP-1 adenovirus
edge about tumor biology [39]. Some examples vector led to an increase of MHC-I cell surface
of this locus-specic regulation have been docu- expression and increased susceptibility to specic
mented in other tumor models. Green and CTLs [47].
In addition, there are examples of tumor outcome of the MHC-I expression in the primary
progression associated with increased expression tumor [55].
of MHC Ags. For instance, one H-2 class
I-decient cell line from RBL-5 lymphoma
(RMA-S), isolated after mutagenization and sev- 10.2.1 Studies in GR9 Tumor Model:
eral cycles of selection by lysis of MHC-I- H-2 Antigen Surface
positive cells, was rejected in syngeneic C57BL/6 Expression and Tumorigenic
mice. In contrast, the H-2-positive wild-type cell Capacity
line (RMA) was highly tumorigenic [48]. The
transfection of this H-2 class I-decient mutant Since the generation of the GR9 tumor model in
(RMA-S) with TAP-2 gene led to a marked the 1980s, our knowledge about the role of
increase in tumor outgrowth potential in vivo. MHC-I molecules in the tumor scene has
This occurred despite restored antigen presenta- increased dramatically [28, 29, 56, 57, 58]. GR9
tion and sensitivity to CTLs and was found to be tumor model is a subcutaneously induced meth-
due to escape from NK cell-mediated rejection. ylcholanthrene (MCA) brosarcoma in BALB/c.
These data suggest that a defect in the machinery The original tumor mass was directly adapted to
responsible for processing and loading of pep- tissue culture without any in vivo passage in
tides into MHC-I molecules is sufcient to ren- syngeneic or allogeneic mice to avoid immu-
der cells sensitive to elimination by NK cells noselection [56]. Forty-three cell lines were
[49]. These data are in accordance with the miss- obtained after cloning using a phase contrast
ing self hypothesis [12] in which NK cells are microscope and limit dilution, adapted to tissue
able to distinguish class I-expressing and class culture and criopreserved. The GR9 brosarcoma
I-decient tumor cells. These cells are able to kill tumor and the GR9-derived clones have been
TAP-decient RMA-S cells (H-2 class I nega- extensively studied and characterized by our
tive) more efciently compared to RMA cells group. The H-2 class I phenotype of the different
(MHC-I positive). NK cells refrain from killing cell lines were analyzed (Fig. 10.1) [13, 56, 59].
when target cells express self MHC-I molecules GR9 cell line presents surface expression of the
[50]. Similar results have been obtained after three H-2 class I molecules (Kd, Dd, and Ld), and
IFN- treatment in murine H-2-negative YAC-1 it is composed of tumor clones with a great het-
lymphoma cell line. In this case, re-expression of erogeneity in H-2 phenotype which could be
H-2 antigens abrogated NK lysis of the cells [51]. classied in four groups: highly positive clones
In other tumors including EL4 lymphoma [12, (D8, A7, G2), middle positive clones (B10, B7,
48] and murine tumor cell lines expressing B3), low positive clones (B6, C11, C5, G10), and
human papilloma virus (HPV) 16-derived E6/E7 very low/negative clones (B9, B11) (Fig. 10.1)
oncoproteins TC-1 (MHC-I-positive) and MK16 [13, 56, 59]. Transcriptional analysis of the H-2
(MHC-I-negative) variants, NK cells appear to be class I heavy chains, 2m, and APM components
an effective tool against MHC-I-decient cells genes showed a correlation between the expres-
[52, 53]. In this case, immunization with the sion of these genes and the surface expression of
MHC-I-negative (MK16), but not with TC-1 MHC-I molecules [59]. A coordinate transcrip-
(MHC-I-positive), cell line inhibits the growth of tional downregulation of H-2 Ld heavy chain, cal-
MHC-I-negative tumors. NK cells are responsi- reticulin, LMP-2, and TAP-1 has been found in
ble for this immunity, although IFN- production B11, B7, and C5 clones in comparison with A7
by CD4+ and CD8+ T cells cannot be excluded clone. In all instances, H-2 class I Kd, Dd, and Ld
[54]. The heterogeneity of MHC-I expression in molecules of all tumor cell lines could be recov-
tumor cell population and the balance of the ered after IFN- treatment [59]. This data indi-
MHC-restricted CTLs and MHC-unrestricted NK cates that tumor cells have reversible (soft)
cells immune mechanisms determine the nal defects underlying MHC alterations [23, 60].
Fig. 10.1 GR9 brosarcoma tumor model. Cell clones are adapted to tissue culture from the primary tumor and clas-
sied according to MHC-I surface expression
More recently, we have shown that the tumor competent BALB/c mice showed an inverse
suppressor gene Fhit is involved in the coordi- correlation between the MHC-I phenotype of
nated transcriptional regulation of various APM tumor clones and their local tumorigenic capacity
components and/or MHC-I heavy chains [58]. [59, 61]. Comparing local tumor growth after
Transcriptional levels of Fhit are signicantly subcutaneous injection of 6.25 105 cells of A7,
lower in tumor clones with low expression of B7, C5, and B11, we found that all cell lines grew
MHC-I molecules. Results have shown that in vivo locally. A7 and B7 showed similar growth
transcriptional level of Fhit in A7 clone is 1.4 rate, but different from C5 and B11. Thus, local
higher than those found in B7 clones and 3.6 and tumors of mice injected with C5 and B11 cell
3.2 time higher than that expressed in C5 and clones began to grow at day 8 and were removed
B11 clones [59]. at days 23 and 28, respectively. In contrast, the
The intratumoral heterogeneity in H-2 class I other two clones, A7 and B7 cells, began to grow
expression presented in GR9 cell lines is not an later at days 14 and 16 postinjection, respec-
unusual case since other MCA-induced tumors tively; the primary tumor was removed at day 39.
obtained in our laboratory (GRB7.1, GRB7.2, Clones with high MHC-I expression are very
and GRIR5) presented similar levels of H-2 class immunogenic in local tumor growth experiments;
I heterogeneity. These differences have a strong in contrast, clones with decreased MHC-I expres-
inuence on in vivo tumor behavior in immuno- sion grew rapidly in vivo when injected subcuta-
competent mice [13]. Local tumor growth of neously. The behavior is totally opposite in
different clones of GR9 in syngeneic immuno- spontaneous metastatic capacity (see following
section). In brief, results clearly show that in this a specic tissue [63, 7375]. The other alternative
tumor model, an inverse correlation between extensively used, GRAFTs, recapitulate all steps
MHC-I surface expression on tumor clones and of secondary colonization by spontaneous visceral
local tumorigenic capacity exists. Moreover, metastasis. In these models, tumors or tumor cell
these differences in local tumor growth were lines are transplanted into mouse, generating a
associated with an immune response, since the primary tumor that will be excised to prolong
clones progressed similarly in irradiated synge- survival of hosts, thus increasing the possibility
neic BALB/c mice [61]. of distant spontaneous metastases [7679].
Experimental metastasis assay also is the other
common test to investigate biological behavior of
10.3 MHC-I Expression tumor cells in vivo. In experimental metastasis
and Metastatic Progression assays, tumor cells are directly injected into
blood circulation to spread to organs. We consid-
Metastatic progression is a complex process dur- ered that spontaneous metastases assay resem-
ing which cancer cells leave the heterogeneous bles all sequential steps associated with the
primary tumor to spread to secondary sites. Thus, metastatic cascade, from primary local tumor to
pathogenesis of cancer metastases involves a set secondary colonization. In contrast, experimental
of sequential events initiated when tumor cells metastasis assay is a bypass in the metastatic cas-
acquire an invasive phenotype [6264]. These cade, evading the rst steps: local primary tumor
invasive tumor cells detach from matrix, invade growth, migration, and extravasation into blood
the tissue, and migrate toward blood or lymphatic and/or lymphatic vessels. Our research group has
vessels to nally get access to the systemic circu- compared the behavior of different tumor cell
lation. However, most tumor cells are destroyed lines in experimental and spontaneous metastases
after extravasation into circulation by the immune assays, nding that it is opposite. Tumor cell
system or hemodynamic forces, and only a small lines with high spontaneous metastatic ability
proportion eventually extravasate and arrive at showed very low experimental metastatic capacity
the new site [65, 66]. This last step requires com- [59]. In consequence, we think that experimental
plex interactions between tumor cells and distant metastasis assays should not be used as a model
tissue microenvironment [67, 68]. Some in vitro for studying metastatic advance disease.
model systems have contributed to the study of
individual steps of metastatic cascade [69, 70].
However, the major limitation of these models is 10.3.1 MHC Class I Expression
that they do not incorporate the complex inter- on Primary Tumor Cells May
play between host and tumor cells; therefore, it is Determine Spontaneous
necessary to work with in vivo models. One of Metastatic Capacity
the most common problems about cancer research
and treatment is difculty reproducing metastatic During the late 1970s, heterogeneity in metastatic
human disease using in vivo models. Preclinical potential of tumor populations was demonstrated
tumor models must mimic the fundamental steps by Fidler and Kripke, using a mouse malignant
associated with the metastatic cascade [71, 72]. melanoma [80]. Great difference between the abil-
Three main types of models in vivo have been ity of clones from B16 cell line was observed in
employed to approximate the situation observed terms of developing metastatic colonies in vivo.
in patients with advanced metastatic disease: This fact suggests that a heterogeneous population
genetically engineered mouse models (GEMM), composed the primary tumor where there were
transplantable tumor model systems (GRAFT) or nonmetastatic and metastatic tumor cells. Later
spontaneous metastasis assays, and experimental research on various cell lines including clones
metastasis assays. At rst, an oncogenic altera- with different metastatic potentials isolated in
tion is introduced (deletion or overexpression) in tumor cell populations of BALB/cfC3H mammary
adenocarcinoma [81], methylcholanthrene [82], or 10.3.2 Different MHC-I Surface

ultraviolet-light-induced brosarcomas [83, 84] Expression on GR9 Tumor
supported these ndings. However, Haywood and Clones Determines Their
McKhann were the rst to suggest the possible Spontaneous Metastatic
inuence of the MHC-I genes on metastatic Capacity
capacities of tumor cell populations [85]. They
compared metastatic capacity of ve methylcho- In our laboratory, the GR9 brosarcoma murine
lanthrene-induced sarcomas, nding that tumors model was used to assess whether levels of
more metastatic had quantitatively more H-2 sur- MHC-I surface expression on primary tumor
face expression. These results, as well as later evi- cells exert inuence on their spontaneous meta-
dences observed by other groups, showed that the static capacity. Four cell clones (A7, B7, C5, and
level of MHC-I expression was implicated in the B11) with different MHC-I surface expressions
metastatic capacity of the tumor cells. Three dif- were chosen for spontaneous metastasis assays
ferent spontaneous tumors originated in mouse, (Fig. 10.1). Results showed signicant differ-
Lewis lung carcinoma (3LL), B16 melanoma, and ences in metastatic capacity between these clones
BW T lymphoma, have been used by Eisenbachs [59]. For example, A7 clone with a strong H-2
research group to show whether metastasis disease class I surface expression was highly metastatic,
is inuenced by MHC-associated mechanisms. generating metastases in 90 % of the hosts and
They worked with different tumor cell variants of resulting in 150 metastases per animal. Clones
these tumors, nding that metastatic ability with intermediate or low H-2 class I expression,
directly correlated with surface expression levels as B7 or C5, presented lower metastatic capacity,
of the H-2 D Ags and inversely with the of H-2 K 50 and 20 %, respectively. In contrast, MHC-I-
Ags [8689]. Moreover, H-2 K-negative/D-positive negative B11 clone did not present spontaneous
clones with high metastatic ability reverted their metastatic capacity, and the B11 tumor-bearing
metastatic phenotype, inducing H-2 K-restricted mice remained free of metastasis at the end of the
CTLs when transfected with the H-2 K gene [87, assays for more than 24 months. In brief, cell
90, 91, 92]. In brief, these results support that the clones with high surface expression of H-2 class
metastatic phenotype is associated with H-2 D sur- I molecules were also highly metastatic, but those
face expression and loss of H-2 K surface expres- clones with low or negative H-2 class I expres-
sion in primary tumor cells. In this context, Kazav sion were weekly or nonmetastatic (Fig. 10.2).
et al. using T10 sarcoma (H-2 b H-2 k) [induced Our experimental evidences support the idea that
by methylcholanthrene in a (C57BL/6J X C3HeB/- levels of MHC-I surface expression of primary
FeJ) mouse] reported that expression of MHC-I tumor cells directly correlated with spontaneous
increased the metastatic capacity of tumor cells metastasis ability and inversely with local onco-
[93, 94]. Several clones of T10 sarcoma presented genicity, as it was shown above [59] (Fig. 10.2).
differential expression of H-2b and H-2k haplo- Consequently, extrapolation of oncogenic and
types: H-2b x H-2k positive and only H-2b positive. metastatic behavior of tumor cells in vivo is not
Metastatic clones characterized to express both always possible, because they may be completely
parental haplotypes and nonmetastatic clones only opposite.
showed expression of H-2b haplotype [95]. Analysis of MHC-I cell surface expression on
Furthermore, metastatic potential in this tumor spontaneous metastases derived from these bro-
system was only acquired when H-2 Dk-Ags were sarcoma clones displayed that in all cases the
expressed on the surface of tumor clones. metastases presented the same or lower MHC-I
Moreover, T10 clones expressing only H-2 Dk- surface expression than the original clone [59]. In
Ags were more metastatic than clones expressing consequence, metastatic progression promoted a
both H-2 Db and H-2 Dk-Ags, while clones downregulation in MHC-I surface expression.
merely expressing H-2 Db Ag were nonmetastatic Analysis of leukocyte subpopulations in tumor-
[95, 96]. bearing mice revealed a distinct behavior among
Fig. 10.2 Schematic

representation of the
dissemination and invasion of
GR9 primary tumor cells.
MHC-I-positive tumor cells
from GR9 primary tumor
presented a high spontaneous
metastatic capacity, whereas
MHC-I-negative tumor cells
presented a weak spontane-
ous metastatic capacity
different clones. A7 and B7 produced immuno- In contrast, this clone is highly metastatic using
suppression characterized by decrease in T lym- nu/nu BALB/c mice, ranging 57 per mouse [28,
phocytes and increase in Treg cells [29]. In 97]. Moreover, metastases were H-2 class I nega-
contrast, B11 tumor-bearing mice developed a tive in immunocompetent hosts and H-2 positive
strong immunostimulation characterized by an in immunodecient hosts. Thus, we observed that
increase in T lymphocytes, dendritic, and macro- H-2 phenotype of spontaneous metastases was
phages cells (unpublished observations). In brief, inuenced by immunological state of the hosts.
A7 and B7 cells progressed to metastatic disease GR9 brosarcoma cell line, composed of dif-
suppressing the immune response, whereas that ferent cell clones, presented intermediate levels
B11 clone promoted an immune response which of H-2 Kd, H-2 Dd, and H-2 Ld molecules.
avoided metastatic progression. The other GR9 Analysis of spontaneous metastases assay with
tumor clone studied was B9, with H-2-negative GR9 tumor cells revealed that GR9 cells have
surface expression and with weak spontaneous high spontaneous metastatic capacity; 90 % of
metastatic capacity (01 metastasis per mouse). tumor-bearing mice develop metastases, ranging
19 per animal. GR9 produced strong immuno- molecules or by myeloid-derived suppressor

suppression in tumor-bearing mice. Interestingly, cells (MDSCs) [100, 101].
96 % of metastases derived from GR9 clone Several murine tumor models have been used
showed downregulation of MHC-I surface to evaluate the application of different immu-
expression. These results suggest that MHC-I- notherapies to recover MHC-I surface expres-
positive clones, as A7 or B7, produced immuno- sion in MHC-I-decient tumor cells, in order
suppression, favoring the growth of MHC-I low to promote an antitumor immune response. In
or negative clones. MHC-I-negative B16 melanoma cells, intratu-
Other experimental evidences from our moral electroporation of IL-12 cDNA promoted
tumor model also support the idea that in GR9 an increase in their MHC-I surface expression,
brosarcoma tumor, the amount of MHC-I Ags mediated by IFN-, leading to the eradication of
also affects NK cell cytotoxicity [98]. Since NK established melanomas by activation of CTLs
cells have been recognized as one of the main [102]. In cervical carcinoma cells, administration
host immunological mechanisms against metas- of synthetic oligodeoxynucleotide-bearing CpG
tasis disease, this notion seems imperative [99]. motifs (CpG-ODNs) upregulated MHC-I surface
In our system, tumor clones with no or low expression causing tumor regression mediated
expression of MHC-I molecules were found to by CTLs [103]. Other studies also have reported
be sensitive to NK mediated lysis, while clones that CpG-ODNs immunotherapies delayed the
with high levels of MHC-I expression were rela- growth or inhibited minimal residual tumor dis-
tively resistant [98]. ease of both MHC-decient and MHC-positive
tumors [104, 105]. Moreover, combination of
dendritic cell-based vaccines with CpG gener-
10.4 Immunotherapy ated inhibition of tumor growth in MHC-positive
as a Treatment Against and MHC-negative tumors [106]. CpG-ODN
Cancers Expressing Different 1585 only produced regression of MHC-decient
MHC-I Surface Expression tumors, principally activating NK cells [105]. In
other assays, depletion of T(reg) cells avoided
10.4.1 Immunotherapy the growth of recurrent tumors after surgery of
as a Treatment Against MHC-negative and MHC-positive tumors [107].
Primary Tumors with Different In all these assays, the action against MHC-I-
Levels of MHC-I Expression decient tumors was mediated by NK or NK1.1+
cells [108]. Previous to the application of immu-
As mentioned above, MHC-I molecules present notherapy, MHC-I-decient tumor cells may
TAA to CTLs; therefore, MHC-I surface expres- be treated with agents to upregulate MHC-I
sion on tumor cells may play an important role in surface expression. Epigenetic mechanisms
the outcome of immunotherapies as anticancer are frequently implicated in MHC-I downregu-
treatments. During treatment with vaccines con- lation of tumor cells; as a result, application of
taining peptides derived from TAAs, MHC-I- agents as 5-azacytidine (5AC) or trichostatin A
positive surface expression on tumor cells could increase MHC-I surface expression [109,
presenting these TAAs is crucial to make this 110]. Treatment of 5AC with CpG-ODN or with
immunotherapy effective. As a consequence, IL-12 showed additive effect against MHC-I-
before the application of immunotherapies, decient tumors, being the immune response
MHC-I surface expression on tumor cells must mediated by CD8+ T cells [111]. Other chemo-
be analyzed. Furthermore, two immunosuppres- immunotherapies, based on ifosfamide derivative
sive mechanisms have been described recently CBM-4A together with IL-12, also led to signi-
showing evasion of tumor cells from CTLs attack, cant inhibition in the growth of MHC-I-decient
mediated by expression of noncognate MHC-I tumors [112].
10.4.2 Immunotherapy as a Treatment chemotherapy. In the control group, mice

Against Metastatic Progression injected with A7 tumor cells and treated with
Derived from Primary Tumors saline solution, a high number of spontaneous
with Different MHC-I Expression metastases in all mice were observed (Fig. 10.3)
[29]. In brief, the two immunotherapy protocols
Immunotherapy has also been used as an anti- and the one chemo-immunotherapy protocol
metastatic treatment against spontaneous metas- eradicated metastasis completely and cured the
tasis derived from primary tumors with different mice, whereas chemotherapy treatment reduced
MHC-I expressions. As mentioned above, stud- the number of metastases partially. When the
ies performed by Eisenbachs et al. showed an same four treatment protocols were applied
inverse correlation between H-2 K tumor cell against spontaneous metastases generated from
surface expression and spontaneous metastatic B7 brosarcoma clone (intermediate MHC-I
capacity [86, 89, 90, 113]. Tumor cell lines expression level and with lower spontaneous
derived from H-2 K-low or H-2 K-decient pri- metastatic capacity than A7 clone), the antimeta-
mary tumors presented high spontaneous metastatic effect was not as effective (Fig. 10.3).
static capacity, which was reverted by transfection PSK, PSK + docetaxel, and docetaxel promoted
of tumor cells with H-2 K gene [86, 114, 115]. partial reduction in the number of metastases,
Moreover, injection of the H-2 K-transfected whereas that CpG + irradiated autologous B7
tumor cells that protect against metastatic disease cells treatment did not produce any antimeta-
originated from H-2 K-low or H-2 K-decient static effect (unpublished data). In the case of
tumors. Furthermore, therapy with IFN--treated spontaneous metastases derived from GR9 bro-
tumor cells or with H-2 K-transfected tumor cells sarcoma, neither treatment had any antimeta-
promoted upregulation of H-2 K surface expres- static effect. Analysis of lymphocyte
sion and protected against metastatic dissemina- subpopulations in different assays showed that
tion from parental tumor cells [113, 115]. An growth of local tumors promotes strong immu-
additional effect was reached when tumor cells nosuppression in the three cases. However, this
were jointly transfected with IFN- and alloge- immunosuppression was completely reverted by
neic MHC class I genes [116]. immunotherapies in the case of A7-injected
In GR9 murine tumor model, the inuence of mice, partially reverted for B7-injected mice,
MHC-I cell surface expression on primary and remained unchanged in GR9-injected mice
tumors has been investigated with respect to the [29]. All these results suggest that immunothera-
success of immunotherapy as antimetastatic pies may be potential antimetastatic treatments
treatment. A7 is a brosarcoma clone with strong against primary tumors with high MHC-I cell
spontaneous metastatic capacity. Four treat- surface expression.
ments were used: two immunotherapies (CpG +
irradiated autologous A7 cells, and PSK) [117],
one chemotherapy (docetaxel), and one chemo- 10.5 Concluding Remarks
immunotherapy (PSK + docetaxel). A7 tumor
clone was injected subcutaneously in BALB/c In tumor cells, MHC-I molecules may present
mice, and the primary tumor was excised when peptides derived from tumor-associated antigens,
the large tumor diameter reached 10 mm. which are new proteins expressed or overex-
Treatment began 1 week after tumor removal, on pressed in tumor cells. Presentation of these new
a weekly basis during 6 weeks; 1 week after the peptides may allow recognition and destruction
last dose, mice were euthanized and autopsy was of tumor cells by CD8+ T lymphocytes. Loss of
performed. Interestingly, all mice treated with MHC-I expression on tumor cells is a widespread
each immunotherapy or chemo-immunotherapy and frequent mechanism developed to escape
appeared metastases-free (Fig. 10.3) [29]. In from immunosurveillance. Alteration in MHC-I
contrast, partial reduction in the number of in both human and murine experimental tumors
metastases occurred in the mice treated with has been widely reported. Results show an
Fig. 10.3 Immunotherapy as an antimetastatic treatment ate level of MHC-I expression), immunotherapy accom-
against tumors with different MHC-I expression. plished partial reduction in the number of spontaneous
Immunotherapy was completely effective in inhibiting metastases. In the case of GR9 brosarcoma, immuno-
spontaneous metastatic progression in A7 tumor clone therapy had no antimetastatic effect
(MHC-I highly positive). For B7 tumor clone (intermedi-
inverse correlation between MHC-I expression

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Role of Plasmacytoid Dendritic
Cells in Cancer 11
Michela Terlizzi, Aldo Pinto,
and Rosalinda Sorrentino

11.1 Introduction .............................................. 177
Dendritic cells (DCs) are highly specialized
11.2 Localization and Trafcking
Patterns of Plasmacytoid
antigen-presenting cells (APCs) essential to
Dendritic Cells (pDCs) ............................. 178 generate immune responses [1], recognizing, pro-
cessing, and presenting danger signals to the
11.3 Plasmacytoid Dendritic Cells
(pDCs) Phenotype ..................................... 179 adaptive immune system. It is now clear that DCs
are not a unique homogeneous cell population,
11.4 Activation of pDCs ................................... 180
but rather a pool of subsets with different origins,
11.5 pDCs: Bridging the Gap Between phenotypes, and functions [2, 3]. However, two
Innate and Adaptive Immunity ............... 183
are the most important DC subsets: myeloid-
11.6 pDCs and Human Diseases ...................... 184 derived dendritic cells (mDCs) and plasmocytoid
11.6.1 Role of pDCs in Human dendritic cells (pDCs). mDCs reside in an imma-
Infections .................................................... 184
11.6.2 Role of pDCs in Autoimmune ture state in peripheral tissues where they behave
Diseases ...................................................... 185 as sentinels to actively capture and process anti-
11.6.3 Role of pDCs in Cancer ............................. 186 gens (Ags). Following exposure to proinamma-
11.7 Potential Therapies: tory cytokines or pathogen-derived products
Clinical Signicance ................................. 189 (pathogen-associated molecular patterns:
11.8 Concluding Remarks................................ 189 PAMPs), they undergo a maturation process and
migrate to the draining local lymph nodes via the
References ................................................................. 190
afferent lymphatics [4]. In contrast, pDCs do not
reside in peripheral tissues during homeostasis,
but are encountered in the peripheral blood and
lymphoid organs [1, 5]. The hallmark of pDCs is
their unique capability to produce large amounts
of interferon- and interferon- (type I IFN) in
response to viruses [6]. Furthermore, pDCs can
differentiate into mature DCs when stimulated by
M. Terlizzi, PhD A. Pinto R. Sorrentino, PhD (*) viruses [7, 8]. Thus, pDCs represent key effectors
Department of Pharmacy (DIFARMA), in innate immunity and the ideal cell population
University of Salerno, Via Giovanni Paolo II, 132,
in connecting innate and adaptive immunity [6].
Fisciano, Salerno 84084, Italy
e-mail: mterlizzi@unisa.it; pintoal@unisa.it; Their discovery dates back to more than 50 years
rsorrentino@unisa.it ago when Lennert and Remmele [9] identied a

178 M. Terlizzi et al.
previously unrecognized rare cell types with cells [16, 18]. In particular, pDCs accumulate in
plasma cell-like morphology in the paracortical inammatory sites, e.g., lymphoid hyperplasia of
area of reactive lymph nodes. Later data revealed the skin [11], cutaneous systemic lupus erythe-
that these cells express both T-cell and monocyte matosus (SLE), psoriasis vulgaris (basal epider-
markers and, therefore, designated plasmacytoid mis and papillary dermis, but not normal skin),
T cells or plasmacytoid monocytes [2, 3, 10]. In contact dermatitis, and allergic mucosa [19].
the 1980s, pathologists became increasingly pDCs also inltrate ascites associated with pri-
aware of this enigmatic cell, and its tissue accu- mary and malignant melanoma [20, 21], head
mulation was shown to be restricted to lymphoid and neck carcinoma [22], and ovarian carcinoma
organs aficted by reactive or neoplastic disor- [23]. Recruitment into these sites suggests that
ders [3, 4], as well as skin-associated lymphoid pDCs may contribute to the ongoing inamma-
tissue [11, 12]. However, despite an increasing tory response through the release of cytokines
interest in these cells, their functional signi- and chemokines and lead to the activation of lym-
cance has still remained enigmatic. phocytes [24] or, alternatively, to the induction of
tolerogenic responses [25].
An intriguing question is how do pDCs enter
11.2 Localization and Trafcking LNs and inammatory sites? Chemokines are
Patterns of Plasmacytoid important regulators of DC trafcking in vivo.
Dendritic Cells (pDCs) Similar to mDCs, blood pre-pDCs (an immedi-
ate precursor of pDCs) undergo maturation and
The development and molecular regulation of upregulate functional CCR7 after activation
pDCs is still under investigation. FMS like tyro- with microbial stimuli or CD40 ligation, thereby
sine kinase 3 ligand (Flt3L) is the main growth acquiring responsiveness toward CCL19 and
factor that induces the differentiation of common CCL21 expressed by HEVs and LN constituents
myeloid progenitor cells into both mDCs and [26, 27]. Furthermore, pDCs express L-selectin
pDCs [13]; however, the E22 transcription fac- (CD62L), which recognizes corresponding
tor is uniquely required for pDC differentiation ligands (peripheral lymph node addressin
[14]. During steady-state conditions, mouse [PNAd]) on HEVs [18]. These observations may
pDCs reside in lymphoid organs and blood, as account for the localization of pDCs around
well as the liver, lung, and skin; nonetheless, their HEVs and in T-cell-rich areas of LNs during
proliferation rate is very low [15]. Human pDCs pathological conditions. pDCs also express
reside in primary, secondary, and tertiary lym- ligands for VCAM-1, an inducible molecule on
phoid organs (aggregates/follicles lymph nodes endothelial cells which may enhance migration
(LNs), tonsils, spleen, thymus, bone marrow, and to draining LNs [25]. Pre-pDCs express several
Peyers patches [16], in addition to the liver and additional chemokine receptors, e.g., CCR2,
blood [17]. They can migrate from lymphoid CCR5, and CXCR3 [28, 29]. Nevertheless,
organs toward T-cell-rich areas of secondary unlike mDCs, they marginally respond to the
lymphoid tissues through high endothelial corresponding ligands (MCP-1; RANTES,
venules (HEV) and toward the marginal zone of MIP-1, and MIP-1; Mig [CXCL9], IP-10
the spleen [18]. In contrast, during pathological [CXCL10], and I-TAC [CXCL11], respec-
conditions, pDCs leave the bone marrow or the tively). Instead, they migrate efciently follow-
circulation and inltrate inamed tissues where ing the recognition of CXCR4 ligand SDF-1/
they can sense danger signals, both PAMPs and CXCL12, which is expressed on dermal endo-
endogenous danger signals (danger-associated thelial cells, in LN-derived HEVs, and in malig-
molecular patterns: DAMPs), leading to the nant cells [25]. Although relatively inactive on
release of large amounts of type I IFNs [16, 18]. their own, CXCR3 ligands produced by Th1
In this scenario they generate protective immunity cells can enhance the responsiveness of pre-
as type I IFNs can activate mDCs, B, T, and NK pDCs to SDF-1 by 20- to 50-fold [26, 29].
11 Role of Plasmacytoid Dendritic Cells in Cancer 179
During microbial infection or inammation, the [16, 18, 34]. Human pDCs are CD4+, CD45RA+,
induction of CXCR3 ligands might drive the IL-3R (CD123)+, immunoglobulin-like tran-
recruitment of immature pDCs to tissues respon- script factor (ILT)-3+, ILT-1low/, Siglec-H+, and
sible for SDF-1 production. In tonsils and in CD11clow/ cells (Table 11.1) [18]. Two additional
psoriatic skin, epithelial cells expressing SDF-1 surface markers for human pDCs are represented
have been associated with the expression of by BDCA-2 and BDCA-4 that correspond to the
CXCR3 ligands [29]. However, pDCs lose their murine mPDCA-1, restricted to the peripheral
responsiveness to SDF-1 once differentiated blood and bone marrow-derived pDCs [18].
[28]. Interestingly, pDCs express cutaneous BDCA-2 is a C-type lectin transmembrane gly-
lymphocyte-associated antigen (CLA), which coprotein which can internalize Ags for present-
binds to E-selectin on dermal endothelial cells ing to T cells. Some data show that triggering
and which may enhance their recruitment to BDCA-2 can potently inhibit in vitro induction of
cutaneous inammatory lesions [30]. IFN-/IFN- expression in pDCs by viruses [35].
Adenosine has recently been identied as a On the other hand, BDCA-4 does not have a sub-
potent chemotactic factor for immature pDCs via stantial effect on pDC function, but can be used
an A1 receptor-mediated mechanism [31]. Upon for the purication of pDCs by magnetic selec-
maturation, the receptor is downregulated, result- tion (Table 11.1).
ing in loss of migratory function. In turn, the A2a In addition, recent evidence demonstrated that
receptor is upregulated, through which adenos- CD9+ Siglec-Hlow pDCs secrete IFN- when
ine reduces the production of proinammatory stimulated with TLR agonists, induce CTLs, and
cytokines [31]. Thus, adenosine, as a resultant of promote protective antitumor immunity. By con-
tissue injury from the degradation of the trast, CD9neg Siglec-Hhigh pDCs secrete negligible
increased release of ATP, as well as SDF-1 and amounts of IFN-, induce Foxp3+ CD4+ T cells,
CXCR3 ligands, facilitates the recruitment of and fail to promote antitumor immunity [36].
immature pDCs from blood to inammatory Although newly formed pDCs in the bone mar-
sites, but subsequently limit their contribution to row are CD9+ and are capable of producing
an inammatory response upon maturation after IFN- after aggregating in peripheral tissues,
an encounter with virus, bacteria, or activated T they lose CD9 expression and the ability to pro-
cells [31]. duce IFN-. Therefore, recognition of the pDC
Local maturation upregulates CCR7, allow- surface markers is actually very important not
ing pDCs to migrate to LNs in response to CCL19 only to distinguish pDCs from mDCs and other
and CCL21 and resist apoptosis [32]. At this site, cell types but also to identify their function and to
pDCs could potentially present peripherally allow researchers to isolate them. To date, Bdca2-
acquired Ags to T cells. Recently, IL-18 produced DTR [37] and Siglec-H-DTR models [38] are the
by mDCs in inamed sites was shown to attract recently developed appropriate murine models
pre-pDCs and modulate their function to skew Th used to study the role of pDCs in the pathogene-
cells toward Th1 cells [33]. sis of various diseases. These mouse models
allow the study of pDCs in pathophysiological
conditions through the depletion of pDCs by
11.3 Plasmacytoid Dendritic Cells diphtheria toxin (DT) using the human diphtheria
(pDCs) Phenotype toxin receptor (DTR) that is driven by the BDCA2
promoter, as the mouse receptor for DTR binds
pDCs are a rare cell type representing only 0.5 % several orders of magnitude more weakly to
of circulating cells in healthy individuals [16]. DT. However, many studies have also been con-
They are round-shaped cells characterized by a ducted by using specic depleting antibodies
prominent endoplasmic reticulum [18]. Mouse (Abs), such as 120G8 Ab [39], BST-2 Ab [40],
pDCs manifest most of the morphological and and mPDCA-1 [41] in vivo. All these Abs bind to
phenotypical features of their human counterpart the same surface marker (BST-2 or CD317).
Table 11.1 Markers currently identied on pDCs

Marker Structure/function Ligand Effect of activation
BDCA-2/BDCA-4 Associated with FcRl to form ITAM Upon ligation, they inhibit
a signaling receptor complex TLR activation and release
of type I IFN
CD4 A glycoprotein expressed on It recognizes the TCR- Activation of pDCs
the surface of T-helper cells, MHC class II complex and
monocytes, macrophages, is required together with
and dendritic cells the CD3 zeta chain for the
recognition of antigens
CD 123 The IL-3 receptor (70KD) is IL-3 Amplication
composed of a ligand specic of inammation
alpha subunit and a signal-
transducing beta subunit shared by
the receptors for interleukin 3
(IL3), colony-stimulating factor 2
(CSF2/GM-CSF), and interleukin
5 (IL-5)
IL-T3 Characterized by its cytoplasmic Fc receptor Tolerance induction
ITIM domain
IL-T7 Characterized by its cytoplasmic IFN I Inhibition of release of Type I
ITIM domain and is also IFN (negative feedback)
expressed on B, T, and NK cells
CD-11 c A heterodimeric integral ICAM-2 and VCAM-1 Induces cell activation; it is
membrane protein composed of an an adhesion receptor that is
alpha chain and a beta chain. It is implicated in phagocytosis of
present only on mouse, but not latex beads and bacteria in
human, pDCs the absence of complement. It
plays an important role in the
inammatory response and
can lead to the production of
proinammatory cytokines
after an APC response
TLR-7 An intracellular endosomal Single-stranded RNA Upregulation of CD40,
pattern recognition receptor CD80, CD86, and CCR7.
Induction of high levels of
Type I IFN. Does not induce
IL-12p70 production
TLR-9 An intracellular endosomal Unmethylated CpG Upregulation of CD40,
pattern recognition receptor oligonucleotides from CD80, CD86, CD83,
bacterial DNA HLA-DR, and CCR7.
Upregulation of Type I IFN,
IL-6, TNFa, IL-8, and IP-10.
Does not induce IL-10
secretion
Ab-depletion models seem to be less specic 11.4 Activation of pDCs

than DTR models, but still very efcient in pDC
depletion, thus allowing the investigation of the Plasmacytoid dendritic cells are highly
role of pDCs during steady state and pathological specialized at sensing nucleic acids via the intra-
conditions. The limitation of Ab-mediated pDC cellular pattern recognition receptors, Toll-like
depletion stands on the role of some molecules, receptors (TLR) 7, and TLR9 [16, 34]. pDCs
such as BST-2, which is also expressed by stro- and mDCs have a different repertoire of TLR
mal and other immune cells after an inamma- expression [16, 18, 34]. Human and mouse
tory stimulus [40]. mDCs can express TLR1, TLR2, TLR4, TLR5,
TLR7, and TLR8, while pDCs selectively interferon regulatory-factor 7 (IRF-7) activation
express high levels of TLR7/TLR8 and TLR9 [43]. MyD88 also leads to TRAF-6-mediated
[42]. TLRs are a family of receptors associated NF-B and MAP-kinases (MAPKs) activation,
with the innate immune response [43]. In par- essential for the transcription of proinammatory
ticular, TLR7 recognizes single-stranded RNA cytokines, chemokines, and costimulatory mol-
enriched with guanosine or uridine from viruses, ecules [43, 51].
synthetic imidazoquinolines, and guanosine The exposure of pDCs to TLR7 or TLR9
analogs [43]. On the other hand, TLR9 is acti- ligands can lead to the production of type I IFN
vated by unmethylated CpG oligodeoxynucleo- and proinammatory cytokines, such as TNF-,
tide (CpG-ODN) motifs typical of viruses and and chemokines, such as IL-8 (CXCL8) [1, 16,
bacteria [43]. Interestingly, the response of 18]. Constitutive expression of IRF7, which is
human pDCs is dependent upon the class of syn- different from mDCs in which induction is
thetic CpG-ODN used to stimulate them. needed, renders pDCs high producers of type I
Stimulation with CpG-A (D)/2216 ODN induces IFN [1, 16, 18], regulating T-cell immunity,
sustained high IFN- production by pDCs, but leading toward a Th1 and cytotoxic T lymphocyte
minimal upregulation of cell surface maturation polarization and activation of mDCs, NK cells,
markers including CD80, CD86, and major his- and B cells [1, 16, 18]. Remarkably, IFN- mod-
tocompatibility complex class II (MHC-II) [44, ulates several aspects of the immune system,
45] has no effect on B cells (which also express including pDC survival [52], mDC differentia-
TLR9). On the other hand, stimulation with tion, modulation of Th1 and CD8+ T-cell
CpG-B (K)/2006, a strong B-cell activator, responses, cross-presentation, upregulation of
results in increased expression of costimulatory MHC and costimulatory molecules, activation of
and Ag-presenting molecules and higher IL-8 NK cells, and induction of primary Ab responses
and TNF- secretion, but lower levels of IFN- [53]. However, a recent study found that type I
production by pDCs. Two distinct pathways of IFN negatively controls pDC turnover in that an
IFN-/IFN- production have been identied overproduction of type I IFNs can lead to the
regarding stimulation with CpG-A vs. CpG-B death of pDCs during steady-state conditions
[45]. pDCs constitutively express IRF-7 and and viral infections [50]. pDC activation can
synthesize high levels of IFN- in response to also lead to the production of IL-12p70, IL-1,
CpG-A, which also triggers an autocrine feed- and IL-6 [54]. Furthermore, recent discovery
back loop involving the IFN receptor-dependent found that pDCs may mediate the release of
pathway [42]. In contrast, IFN-/IFN- induc- IL-10 [26]; however, another group [55] showed
tion by CpG-B is independent of the IFN-/ that these cells do not directly produce IL-10
IFN- receptor loop [45, 46]. Recently, CpG-C, (Fig. 11.1).
a new class of CpG ODN in which structural ele- Moreover, it was recently demonstrated that
ments of CpG-A and CpG-B have been com- pDCs produce high amounts of granzyme B [56],
bined, has emerged. This sequence activates B which is effective only in combination with perfo-
cells and induces IFN- production by pDCs rins mainly produced by cytotoxic T lymphocytes
[47]. Furthermore, non-CpG-containing ODNs (CTLs). This further connects pDCs to adaptive
have been shown to bind human TLR9 [47, 48] immunity. Additionally, in the absence of an ef-
and to stimulate pDCs [49]. cient adaptive CTL immunity, pDCs can behave
TLR7 and TLR9 are very sensitive to different as killing DCs due to the release of tumor necrosis
stimuli; the rst triggers ssRNA viruses and the factor-related apoptosis-inducing ligand (TRAIL)
latter responds to DNA viruses [50]. TLR7 and and to the induction of DR5 expression, a TRAIL
TLR9 activation recruits a cytoplasmic adaptor, receptor, on the cell target [37, 56].
myeloid differentiation primary response gene A diversity of C-type lectin receptors (CLRs)
88 (MyD88), which is able to assemble a mul- has been identied on DC subsets, includ-
tiprotein signal-transducing complex-inducing ing DC-SIGN (CD209), DEC-205 (CD205),
Stimuli (i.e. DNA/RNA from virus and/or bacteria: CpG /Imiquimod)
pDCs
ENDOSOME P
IRF-8 P IFN-
IRF-7 IFN- Activation of :
DNA RNA
IFN- mDCs, NK, B cells,
IFN- Th1 and Tc
TRAF-3
TLR 9 IRF-7 NUCLEUS
TLR 7
MyD88 CD-80
CD-86 Cross-Presentation
TRAF-6 CD-40 to T cells
IRF-5
BTK
IRAK-4
IRF-4
MAPKs
TAK-1 IL-6
TNF Pro-Inflammatory
NF-kB Patterns
Fig. 11.1 The recognition of stimuli, such as DNA or signalling pathways that lead to the expression of cyto-
RNA motifs from viruses and bacteria, by pDCs via TLR7 kines such as IL-6 and TNF-, costimulatory molecules
and/or TLR9, induces the activation of MyD88-dependent such as CD80, and the synthesis/release of type I IFN
langerin (CD207), mannose receptor (CD206), face of TLR activation by microbes. On the
BDCA-2, and dectin-1. CLRs typically recog- other hand, it has been shown that dectin-1 col-
nize carbohydrate-rich structures on microbes laborates with TLR2 in inducing proinamma-
and self-antigens [35]. They have been impli- tory cytokine secretion in murine macrophages
cated in cell adhesion and regulation of signaling and DCs [59]. Whether BDCA-2 has any con-
events (e.g., BDCA-2), migration and homing nection to TLRs in pDCs remains to be eluci-
(e.g., DC-SIGN), Ag uptake and processing for dated. However, early reports have shown that
MHC-II presentation to T cells (e.g., DC-SIGN, secretion of type I IFNs by pDCs in response to
BDCA-2, langerin, and mannose receptor), cell- inuenza virus (most likely triggering TLR7/8)
cell transmission of pathogens (e.g., DC-SIGN), or to complexes of plasmid DNA and anti-DNA
and tolerance to self-antigens (e.g., DEC-205). Abs (possibly stimulating both FcR and TLR9)
pDCs express BDCA-2 and BDCA-4, dectin-1, is signicantly inhibited by ligation of BDCA-2
and possibly DEC-205 but lack DC-SIGN and with anti-BDCA-2 Ab [35]. It is worth noting
langerin, found on CD34+ and monocyte-derived that BDCA-2 is downregulated after pDCs mat-
DCs and Langerhans cells (LCs), respectively uration and that mature pDCs secrete less IFN-/
[57]. The physiologic function of CLRs on IFN- in response to viral stimuli than immature
pDCs remains unknown. Anti-BDCA-2 Abs are pDCs do [60, 61]. BDCA-2 has an intracellular
rapidly internalized and efciently presented to domain of 21 amino acids without known motifs
T cells, suggesting a role in Ag capture and pre- implicated in signal transduction; however, liga-
sentation [35]. Interesting relationships between tion induces Src family protein-tyrosine kinase-
CLRs and TLRs have been documented. In dependent intracellular calcium mobilization
mDCs, interaction of DC-SIGN with lipoara- and protein-tyrosine phosphorylation of intra-
binomannan secreted by mycobacteria inhibits cellular proteins [35]. BDCA-4 (neuropilin-1) is
lipopolysaccharide (LPS)-induced DC activa- also upregulated in blood mDCs after overnight
tion through TLR4 [58]. This mechanism may culture and may participate in DC-lymphocyte
permit pathogens to evade immune responses interactions [62].
and perpetuate tolerance to self-antigens in the
a
Th1
IFN I polarization
IFNAR
mDCs
pDCs
NK cells
IL-12p70 CTL response
Stimulus
IFN I PLASMACELL
b IFNAR
B cell
pDCs
IL-6 IL-6R
Stimulus YY Y Y
CD27 YY
CD70 IMMUNOCOMPLEXES
CD80 or
c
CD86
or PDL1 T Reg-cell
pDCs Immune-suppression
CTLA-4
IDO or PD-1
Stimulus
T cell Death
KYNURENINES
Fig. 11.2 (a) Activated pDCs produce high amounts of to differentiate into plasma cells via the activation of
Type I IFNs which both amplify its own production in an IFNAR, IL-6R activation, and the interaction of CD70-CD27
autocrine manner via the expression of IFNAR on them- on B cells; (c) pDCs can lead to immunosuppression via
selves and induce the release of other proinammatory both direct interaction with Treg (CD80 or CD86+CTLA-4
cytokines such as IL-12p70 from mDCs and NK cells that or PD-L1+PD1) and the release of IDO-induced kynuren-
lead to Th1 and CTL polarization; (b) pDCs induce B cells ines metabolites which induce Th1 cell toward death
11.5 pDCs: Bridging the Gap proinammatory cytokines such as IL-12p70

Between Innate from mDCs and NK cells [63] (Fig. 11.2a).
and Adaptive Immunity Activation of mDCs diverts the immune environ-
ment toward a Th1-like bias, during which IFN-
The production of type I IFNs by pDCs represents production facilitates Th1 differentiation [16, 18,
the bridge between the innate and adaptive 63], long-term T-cell immunity [18, 63], and a
immune system. Type I IFN (IFN- and IFN-) is CTL-mediated response [64], as well as prolifer-
an important component of the innate immunity, ation and survival of T cells [63, 64]. Moreover,
especially during viral infections [16, 18]. through the production of IL-6 and type I IFNs,
In contrast to mDCs, pDCs produce high amounts pDCs induce B cells to differentiate into plasma
of type I IFNs upon activation [16, 18], which cells which are immunoglobulin (preferentially
both amplify its own production in an auto- IgG and IgM)-producing cells (Fig. 11.2b). In the
crine manner and induce the release of other process of B-cell activation, a key role is played
by the CD70 receptor expressed on pDCs, as it Taken together as a whole, these data suggest
can induce the differentiation and the prolifera- that pDCs represent a key effector cell in both
tion of IgG-producing B cells [65] (Fig. 11.2b). innate and adaptive immunity regulation [1].
In addition, activated pDCs can undergo other
important phenotypic changes that induce them
to change their phenotype toward an mDC phe- 11.6 pDCs and Human Diseases
notype [1]. The upregulation of MHC and T-cell
costimulatory molecules enables pDCs to engage A wide spectrum of human diseases including
and activate nave T cells [6668]. There have infection, autoimmunity, and cancer are associ-
been many controversies regarding the role of ated with accumulation of pDCs in lymphoid and
pDCs to prime T cells and cross-present Ags peripheral tissues strictly correlated to the reduc-
[68]. The expression of MHC and T-cell costimu- tion of these cells in the peripheral blood [21].
latory molecules is not as high as in mDCs, and For many of these diseases, compelling evidence
this is why pDCs are less efcient than mDCs at supports a pathogenic role of pDCs, mainly
priming T cells [69]. Moreover, the repertoire of related to either the increase or reduction of pro-
Ags that can be presented by pDC-derived MHC inammatory or antiinammatory functions of
molecules is more restricted than those of mDCs pDCs. Alternatively, pDC accumulation might
because not all of these Ags reach the endocytic exert an adjuvant immune function, as in viral
compartment into pDCs [68, 69]. However, some infection, and in imiquimod-treated cancers,
pDC receptors such as BDCA2, Siglec-H, and where they seem to encounter an antiviral and
DCIR are able to bind Ags, mediate endocytosis, antitumor activity. In many other pathologies,
and process and present to T cells [68, 69]. information available is still limited, and pDC
Interestingly, activated pDCs can also pro- biology is largely unknown.
mote Th2-like immune responses [63] underlin-
ing their functional plasticity. There is evidence
that IFN- stimulates the differentiation of pDCs 11.6.1 Role of pDCs in Human
into Th1-polarizing pDCs, whereas in the absence Infections
of IFN- but only in the presence of proinam-
matory signals, pDCs can also stimulate Th2 pDCs have been most extensively studied during
polarization/differentiation [70]. Moreover, some HIV and chronic viral hepatitis, particularly hep-
authors reported that CpG-activated pDCs exert a atitis C virus (HCV) infections. The emerging
strong immunosuppression and induce the differ- picture suggests an important role for pDCs in
entiation of allogeneic CD4+CD25+ T cells into these infections; however, the exact mechanism
CD4+CD25+ regulatory T cells in tumor condi- and consequences of pDC activity are controver-
tions [50, 55]. Very interestingly, pDCs can sial at present [75]. pDCs can respond to HCV
directly or indirectly recruit Treg cells via PD-L1/ and particularly to HCV-infected hepatocytes
PD-1 axis [71] (Fig. 11.2c), release of immuno- which induce pDCs to signal via an endocytosis-
suppressive cytokines, such as IL-10 [55, 71], and IRF7-dependent mechanism, but not via the
and the membrane tolerogenic inducible costim- NF-B pathway, implying a non-full functional
ulator ligand (ICOS-L) [72]. response of pDCs that contribute to the evasion
pDCs can also synthesize large amounts of of immune responses by HCV [76]. In contrast,
functional indoleamine 2,3-dioxygenase (IDO), other studies demonstrated normal pDC func-
which requires autocrine release of type I IFN, tionality in chronic HCV infection [77]. The res-
upon TLR9 and CD200R ligands stimulation olution of this controversy would establish pDCs
[16]. IDO-derived metabolites promote T-cell either as a weak link of anti-HCV immune
death [55, 73] and suppresses T-cell immunity in response or as a potentially powerful effector
normal and pathological settings. In the same type that can be harnessed for immunotherapy of
manner, reduced tryptophan amounts can lead to chronic HCV.
the release of regulatory cytokines, such as IL-10 Similarly, pDC dichotomy is observed in HIV
[74], associated with a tolerogenic environment. infection, in which some authors assume that
pDCs can be infected with the HIV and/or lupus erythematosus (SLE) [84]. In psoriasis,
respond to it with robust IFN secretion [78], early skin lesions are highly inltrated by
while others reported impaired activity of pDCs activated pDCs, corresponding with decreased
in HIV infected patients [79, 80]. Interestingly, numbers of circulating pDCs [85]. Blocking IFN
pDCs are progressively depleted from the blood production by pDCs using anti-BDCA-2 Ab
of infected patients, either through infection- inhibited the development of skin lesions in a
induced death or due to redistribution to lym- xenograft mouse model, providing causal proof
phoid organs. The key unresolved question is of pDC function in the disease [85]. Gilliets
whether HIV-induced pDC activation is bene- group [86] identied the activating stimulus for
cial or harmful for the host. On one hand, IFN pDCs as complexes of self-DNA with the antimi-
secretion by pDCs was shown to inhibit viral rep- crobial peptide LL-37. This and possibly other
lication in T cells and promote pDC and cDC homologous proteins promote the aggregation of
maturation, leading to the killing of infected T released cellular DNA and RNA into large com-
cells. In this context, it is likely that HIV may plexes that efciently activate pDCs [86, 87].
have evolved mechanisms to suppress pDC acti- Although the origin of these immunostimulatory
vation, e.g., through BDCA-2 ligation [81], complexes and the consequences of pDC activa-
which disables pDC functions as APCs and type I tion remain to be elucidated, the major role of
IFN-producing cells. On the other hand, the same pDCs in psoriasis is well established. Similarly,
functions of pDCs may exacerbate T-cell deple- lupus patients show a decrease in circulating
tion, e.g., by disseminating HIV to uninfected pDCs and the accumulation of activated, IFN-
CD4+ T cells or by bystander T-cell killing. Most producing pDCs in affected tissues such as the
importantly, elevated IFN response by pDCs may skin [88]. The hallmark of lupus is the production
contribute to chronic immune activation and of antinuclear Abs and immune complexes of
faster T-cell depletion [82]. It is plausible that the such Abs with endogenous nucleic acids were
function of pDCs in HIV infection changes from shown to activate pDCs through TLR7/9 [89, 90].
protective to pathogenic as the disease progresses. These complexes may be delivered into the endo-
At the early stages of infection, IFN production somal compartment of pDCs via Fc receptor II
and virus cross-presentation by pDCs may help (FcRII) [89, 91], and their stimulatory capacity
limit virus spread and mount cytotoxic T lympho- can be augmented by the nuclear DNA-binding
cyte responses; whereas as the virus replication protein HMGB1 [92]. In addition, self-DNA
escapes control, IFN secretion may drive poly- forms complexes with LL-37 and other antimi-
clonal T-cell hyperactivation and depletion [77]. crobial peptides released by neutrophils, and the
The eventual loss, redistribution, or functional resulting complexes induce IFN secretion in
impairment of pDCs at the late stages of infection pDCs through TLR9 [92]. Notably, TLR-
would contribute to immunodeciency. Thus, the activated pDCs become resistant to glucocorti-
role of pDCs in HIV and HCV infections high- coids, which could underlie the limited efcacy
lights the power and the danger of pDC activation of these drugs in lupus [93, 94]. The direct causal
and reveals another strategy of immune system relationship between pDC-derived IFN and lupus
subversion by these viruses. progression/severity is hard to establish in the
human system and should await for elucidation in
animal models. Nevertheless, the likely connec-
11.6.2 Role of pDCs in tion between the formation of nucleic acid-con-
Autoimmune Diseases taining immune complexes, pDC activation, and
IFN secretion and the pronounced IFN signature
Several autoimmune diseases are associated of the disease makes a strong case for the pDC as
with elevated levels of type I IFNs, implying a a major player in lupus pathogenesis [77].
potential role for pDCs in cytokine production Overall, the aberrant conversion of self-nucleic
[83]. To date, the strongest evidence for pDC acids into ligands for TLR7/TLR9 on pDCs (via
involvement has been accumulated from the immune complex formation, antimicrobial
study of two diseases: psoriasis and systemic peptide binding, and other mechanisms to be
discovered) may represent a common pathogen- in the inamed skin of tumor-bearing mice
esis step in psoriasis, lupus, and possibly other facilitated pDC recruitment [56].
autoimmune diseases such as Sjgrens syn- Once recruited, pDCs seem to be important
drome [95]. players in cancer immunoediting as their capac-
The activity of pDCs in viral and autoimmune ity to bring together the innate and the adaptive
diseases might teach us how and why pDCs highly immunity. In particular, it seems that critical role
populate cancerous masses playing a pivotal role is played by type I IFNs. Endogenously produced
for the tumor immune microenvironment. IFN-/IFN- was required for the prevention of
the growth of primary carcinogen-induced sar-
coma [99]. In this study, host hematopoietic cells
11.6.3 Role of pDCs in Cancer were critical targets of IFN-/IFN- during the
development of protective antitumor responses
Recent studies have shown that the density and [99]. pDCs have been widely described as profes-
location of immune cells in primary tumors can sional type I IFN-producing cells; therefore, the
predict patient survival [96], supporting the higher presence of pDCs in the tumor mass might
notion that monitoring local immune response directly link pDCs to cancer immunoediting in
might represent a critical step in predicting that pDCs may behave as antitumor cells.
patient prognosis and likely the response to anti- However, other reports showed opposite activi-
tumor strategies [97]. pDCs have been found in a ties of pDCs in cancer. Animal studies demon-
variety of neoplasms; nonetheless their function strated that tumor-associated pDCs (TApDCs)
is still unknown. Solid tumors, such as head and are defective in type I IFN production but instead
neck, breast, ovarian, lung cancer, and skin secrete immunosuppressive factors responsible
tumors, are populated by non-active pDCs [97]. for tumor progression [100, 101]. Similar to what
Clinical studies have suggested a direct correla- described for viral infections and autoimmune
tion between reduced numbers of circulating diseases, the dichotomy of pDCs in cancer might
pDCs and higher presence of these cells into underlie their phenotype and maturation state.
malignant masses [1, 97]. Although the causal
relationship is still under investigation, recent 11.6.3.1 Antitumor Activity of pDCs
results from mouse models are starting to dene Type I IFNs are pleiotropic cytokines with a dem-
the specic role(s) of pDCs in tumor masses. The onstrated clinical benet to cancer patients and
mechanism that induces the recruitment of pDCs have recently emerged as the connection bridge
to the tumor site is not clear. Circulating pDCs between tumor cells and the immune system
express multiple chemotactic receptors such as [102]. pDCs produce large amounts of type I
CXCR4 and ChemR23 being the only biological IFNs upon TLR7 and TLR9 stimulation. Drobits
active receptors in healthy donors [28]. CXCR4 et al. showed that the intratumoral stimulation of
binds CXCL12, widely expressed in tissues and pDCs with imiquimod renders these cells cyto-
which most likely represents the main axis for toxic and contributes to tumor regression inde-
pDC accumulation in human tumors [25]. pendently from conventional adaptive immune
CXCL9, CXCL10, and CXCL11, which bind mechanisms, but via the production of TRAIL
CXCR3, present on pDCs, are all IFN-inducible and granzyme B secretion by pDCs via IFNAR1
proteins and might be involved in pDC inltra- signaling [56]. However, the role of TApDC-
tion [98]. In addition, cytokines such as CXCL10, derived granzyme B in the absence of perforins
CXCL12, and chemokines, such as CCL2, are not produced by pDCs still remains to be
released by tumor and stromal tumor-associated elucidated.
cells, such as cancer-associated broblasts Another mechanism that may underlie the
(CAFs), allowing pDCs to migrate from the antitumor activity of TApDCs is their antigen-
circulation to the injured tissue [23]. Accordingly, presenting activity. Although in their immature
Drobits et al. demonstrated that CCL2 produced state, TApDC are still capable to internalize Ags
in vivo and to activate CD4+ T cells [103]. TLR9 ligand, did not lead to the same results as
The immature state of pDCs is reected in that observed by Liu et al. [106]. Activation of pDCs
they have altered cytokine production in response through CpG had the opposite effect in that pDC
to TLR-9 ligands in vitro, while preserving activation increased the recruitment of Tregs and
unaltered response to TLR7 ligands [104], which limited the inammatory cell inux to the lung,
instead seem to have potential antitumor activity. thereby establishing an immunosuppressive envi-
To date, imiquimod is in phase III clinical trial ronment enabling tumor growth [1, 105, 109].
against melanoma. In contrast to these results, The same was observed in another mouse model
systemic administration of CpG favored pDC- of breast cancer in which in vivo depletion of
induced lung tumor progression [105], as also pDCs delayed tumor growth showing that
observed in a mouse model of breast cancer TApDC provide an immune-subversive environ-
[104]. Similar to the data showed by Drobits ment, most likely through Treg activation thus
et al., Mercier et al. proved that, although CpG favoring breast tumor progression [110]. The dis-
did not alter TApDC activity, the intratumoral crepancy in these data and the one from Liu et al.
administration of a TLR7 ligand led to TApDC [106] could be a result of tissue-specicity and
activation and displayed a potent curative effect route of CpG administration which is very impor-
in a type I IFN-dependent manner [56]. In addi- tant in determining the tumor microenvironment,
tion, Liu et al. [106] demonstrated that the intra- which in turn strongly inuences immune cell
tumoral activation of pDCs via CpG could induce phenotype. Moreover, in the absence of a specic
NK cell-dependent tumor regression in a mela- stimulus, pDCs in the tumor mass have been
noma animal model. Remarkable is that TLR9 associated with the development and mainte-
expression and responsiveness is impaired by nance of the immunosuppressive microenviron-
tumor-derived components [107]. ILT7 on pDCs ment [111]. Similar to mice, human pDCs in
binds BST-2 expressed by tumor cells and their tumor masses are in their immature phenotype;
interaction inhibits type I IFN production by nonetheless, a thorough study has never been
pDCs, disabling TLR9-dependent signaling path- conducted on the role of these cells in human
ways [108]. Moreover, tumor-derived TGF- and tumor microenvironment. Nevertheless, it is clear
TNF- have been identied as the main in vivo that pDCs play a fundamental role in the tumor
mechanisms blocking type I IFN production by microenvironment. The specic depletion of
pDC in tumors through inhibition of IRF7 signal- pDCs induced lung tumor regression with a con-
ing complex, leading to a negative impact of comitant Th1 polarization that arrested tumor
defective pDCs in breast cancer through Treg progression [105]. On the other hand, stimulation
expansion [109]. of TLR7, rather than TLR9, can subvert the
Taken altogether, these data supported the immunosuppressive activity of TApDCs. TLR7-
rationale to use TLR7 ligands to restore TApDC dependent pathway induced melanoma regres-
activation in both breast and skin cancer. sion in mice [56] through the transformation of
However, it still remains to be determined how pDCs into tumor-killing cells able to produce
the activation of TLR7 and TLR9, which is granzyme B and TRAIL. Likewise, another
MyD88-dependent, on pDCs, can behave differ- group revealed that human pDCs can kill mela-
ently according to the tissue specicity and on noma cells in vitro under imiquimod and IFN-
the route of administration. stimulation [112]. While pDCs can produce high
levels of granzyme B, their role as cytotoxic
11.6.3.2 Pro-tumor Activity of pDCs immune cells remains to be determined as they
Several evidence have shown the prevailing lack the pore-forming perforin [112]. On the
immunosuppressive activity of pDCs due to both other hand, it has been proposed that under IL-3
of the impairment in type I IFN production and and IL-10 exposure, pDCs release abundant
the release of pro-tumor factors [1]. Stimulation granzyme B, which in turn is capable of
of lung tumor-bearing mice with systemic CpG, a blocking T-cell proliferation, thus suggesting a
new potential mechanism for tumor-immune induced by IFN- and inhibits IFN- production
evasion [112]. by human pDCs, indicating that the ILT7L-ILT7
Several mechanisms have been postulated interaction between cancer cells and pDCs may
for the immunosuppressive nature of tumor- cause impairment of pDCs in the tumor microen-
associated pDCs: (1) release of tolerogenic fac- vironment, possibly leading to immunosuppres-
tors, (2) ILT-7 expression, (3) PD-L1 expression, sion and poor prognosis of cancer patients as
(4) Siglec-H activity, and (5) induction of a Th2- observed in preclinical studies [119]. Moreover,
like environment. Tolerogenic factors produced under tumoral conditions pDCs can also direct
by tumor cells, such as PGE2 [113] and TGF- mDC phenotype toward a more immature state,
[109], can alter type I IFN signaling pathway. as already reported for human lung cancer [16,
Tumor-derived PGE2 and TGF- act synergisti- 70, 105]. However, the underlying mechanism is
cally to block IFN- and TNF- secretion by still not dened.
pDCs [16, 109]. Opposite to IFN- and TNF-, To date, pDCs can directly interact with Treg
IL-6 and IL-8 production are enhanced in PGE2- via the PD-1/PD-L1 axis [55] (Fig. 11.2c), pav-
and TGF--treated pDC [114]. Both IL-6 and ing the road to another mechanism of action of
IL-8 promote immune-cell survival and chemo- the newly approved monoclonal Ab, anti-PD-1
taxis but also enhance tumor cell proliferation for cancer immunotherapy.
and angiogenesis [115, 116]. Moreover, PGE2 is Moreover, Ag targeting to pDCs via Siglec-H
crucial for the secretion of other immunomodula- inhibits Th1 cell-dependent immunity [103]. The
tory factors such as SDF-1, the ligand for administration of CpG increased Siglec-H
CXCR4, which is upregulated on both human expression on pDCs recruited to the lung of
pDCs and tumor environment [117]. Thus, pDCs tumor-bearing mice, further supporting their
can be retained in the tumor tissue via PGE2- implication in the inhibition of Th1 cell expan-
induced sensitization for SDF-1 [29]. In further sion [105].
support, PGE2- and TGF--mediated retention of pDCs activated by IL-3 and CD40 ligand
pDCs in the tumor tissue is accompanied by the (CD40L) promote the differentiation of naive
suppression of the lymph node-homing receptor, CD4+ and CD8+ T cells into Th2 cells and aner-
CCR7 [113]. PGE2-exposed pDCs are unlikely gic IL-10-producing CD8+ regulatory T cells,
to present Ags and to prime T cells in the regional respectively [123]. This state of anergy is medi-
LNs. Concomitantly, suppression of CD40 ated by IL-10, either directly (by interaction
expression and the overexpression of CD80/86 with cytotoxic T lymphocytes, CTLs) or indi-
on pDCs enhances and even promotes Treg acti- rectly (by inhibition of DCs) [114]. Since the
vation via the negative regulatory receptor cyto- tumor microenvironment is Th2-like, pDCs par-
toxic T-lymphocyte antigen-4 (CTLA-4) [118, ticipate in this scenario by further augmenting
119] (Fig. 11.2c). immunosuppression.
Another potential mechanism for pDCs favor- Overall, these effects may allow pDCs to
ing tumor immune escape is the release of IDO- establish a reduced inammatory pattern but, at
derived metabolites [119] from both pDCs the same time to favor tumor progression/estab-
(Fig. 11.2c) and tumor cells, inducing Treg dif- lishment, as observed in asthma [124], virus
ferentiation and Th1 cell apoptosis [55, 74]. Most infection [125], and cigarette smoke exposure
human tumors overexpress IDO [120], explain- [70]. To note, the aforementioned studies describe
ing the elevated tryptophan catabolism in cancer the role of pDCs which are not activated by a spe-
patients. Interestingly, the activation of IDO in cic stimulus; then, it seems obvious that the
either cancerous cells or regulatory DCs can be activation of pDCs at the tumor site is a limiting
sufcient to promote tumor immune escape step in tumor regression. Therefore, the dichot-
[121]. Some cancer cells, such as lung cancer- omy of pDCs in cancer may rely on the stimula-
derived cells, highly express ILT7L, which can tion/activation of pDCs with specic stimuli as in
bind to ILT7 that is on pDCs [122]. ILT7L is the case of imiquimod.
11.7 Potential Therapies: Clinical tin, drugs that are highly used in the clinical
Signicance antitumor practice, had a much effective activity
against lung tumor progression due to the induc-
Secreted factors by tumor cells, such as TGF-, tion of proinammatory pDCs, activated by tumor
VEGF, and IL-10, may inhibit pDCs functions cell death. This latter study was conducted on
with the resulting prevailing of the suppressive mouse models. Therefore, clinical correspon-
immune response dictated by the same pDCs and dence could prove the potential antitumor activity
adaptive immune cells. On the contrary, other of proinammatory pDCs resulting in tumor
studies reported tumor-inltrating pDCs as func- regression. In addition, previous studies on the
tional and fully competent APCs. Production of role of pDCs as antitumor cells only after intratu-
IFN- renders TApDCs as antitumor cells. In this moral activation of these cells by means of imiqui-
context, the activation of intratumoral pDCs by mod and CpG could underlie the same mechanism
means of imiquimod (TLR7 ligand) and/or CpG of action. In other words, several endogenous
(TLR9 ligand) has been successfully used in the molecules (DAMPs) that participate to the sterile
clinic to treat basal cell carcinoma and melanoma inammation have been described as potential
[1]. TLR signaling on pDCs can be used to induce TLR ligands. Similarly, we could speculate that
type I IFNs and possibly protect pDCs from tumor cell death can induce the release of DAMPs
tumor-derived inhibitory factors (such as TGF- which activate pDCs in a TLR7- or TLR9-
and IL-10), as well as support T-cell-mediated dependent manner leading to type I IFN produc-
antitumor immune response. However, this prac- tion by pDCs. This prevails and allows the gap
tice can only refer to the activation of TApDCs between the innate and the adaptive immunity to
in loco, as mouse models showed that systemic overcome tumor-mediated immunosuppression.
administration of CpG rendered pDCs immuno- In this scenario, Aspord et al. demonstrated that
suppressive, favoring lung and breast tumor pro- stimulation of PBMCs from HLA-A*0201+
gression [1, 101, 105, 109, 110]. donors by HLA-A*0201 matched allogeneic
Many therapeutic trials have been designed to pDCs pulsed with tumor-derived peptides trig-
potentiate CTL responses. Myeloid-derived gered high levels of antigen-specic and func-
dendritic cells-based vaccines succeeded in induc- tional cytotoxic T lymphocyte responses; this
ing specic T cells in patients, but without suf- resulted in melanoma regression in a humanized
cient clinical efcacy [126]. A potential mouse model [127]. This semi-allogeneic pDC
explanation of this failure may underlie the role of vaccine was more effective than conventional
pDCs at modulating tumor immune-environment mDC-based vaccines, endowing a strong potential
and, more specically, mDCs activity [105]. for clinical application in cancer treatment [127].
Animal studies on several diseases, such as
asthma, virus infection, and cigarette-exposed and
lung cancer models, revealed that pDCs can ham- 11.8 Concluding Remarks
per the activity of mDCs [105]. In particular, the
presence of high levels of pDCs in tumor masses In the last decade several studies provided evi-
was associated with immature mDCs incapable of dence that pDCs actively participate in a wide
mounting an effective adaptive immune response spectrum of human diseases including infection,
against cancer. Specic ablation of pDCs ren- autoimmunity, and cancer. In particular, human
dered mDCs active and prone to induction of a neoplasms are populated by pDCs which pres-
CTL response against tumor cell proliferation ence is related to a poor prognosis. However, the
[105]. Therefore, we speculate that pharmacologi- role of tumor-associated pDCs (TApDCs)
cal manipulation of pDC phenotype could result remains controversial. Various studies indicate
in successful antitumor therapy together with the that pDCs play an immunosuppressive role and
conventional strategies. In support, our unpub- facilitate tumor progression in both animal mod-
lished data showed that doxorubicin or oxalipla- els and humans. In contrast, others found that the
presence of activated pDCs results in tumor 12. Toonstra J, van der Putte SC. Plasmacytoid monocytes
in Jessners lymphocytic inltration of the skin. A
regression in mice. Given these ndings, it is
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Cancer Immunoediting:
Immunosurveillance, Immune 12
Equilibrium, and Immune Escape
Alka Bhatia and Yashwant Kumar
Contents
12.1 Introduction
12.1 Introduction .............................................. 195
12.2 Cancer Immunoediting The immune systems regulation of the cancerous
with Its Three Es: Reection process is a long-known fact. However, the role
of the Dual Role of Immunity
in Cancer................................................... 196 played by it in malignancies has been a matter of
12.2.1 Immune Elimination: Evidences debate. The history of cancer immunity dates
For and Against .......................................... 197 back to 1909 when Paul Ehrlich proposed the
12.2.2 The Equilibrium Phase: The Most concept of immunosurveillance in cancers for the
Controversial and the Least
Understood Phase ...................................... 200 rst time [1]. However, due to lack of experimental
12.2.3 Immune Escape: The Best evidence, this concept fell into disrepute. In 1957
Studied Phase ............................................. 201 Burnet and Thomas argued that indeed, the
12.3 Tumor Antigens and Cancer immune system ghts and eliminates certain
Immunoediting ......................................... 203 cancers and the frequency of malignancy would
12.4 The Tumor Microenvironment have been much higher if immunity was not there
During Cancer Immunoediting .............. 204 [2]. In 1970s, several experiments were con-
12.5 Clinical Relevance of the
ducted in athymic mice to prove immunosurveil-
Immunoediting Process lance in cancers; however, the results were not as
in Cancer................................................... 205 expected, which was thought to be due to the
12.6 Concluding Remarks ............................... 206 presence of residual immunity in the animals
used for these studies [35]. Consequently, the
References ............................................................... 206
experiments done again on animal models with
specic molecular immune defects revealed more
A. Bhatia, MD (*)
frequent development of carcinogen-induced
Department of Experimental Medicine and tumors in these immunodecient animals [6].
Biotechnology, Post Graduate Institute of Medical However, more recently, the recognition of the
Education and Research, Chandigarh 160012, India dual nature of the part played by immune system
e-mail: alkabhatia@ymail.com
in malignancies has led to the modern concept of
Y. Kumar, MD, DNB cancer immunoediting. Since then, immunoedit-
Department of Immunopathology, Post Graduate
ing in cancer has served as the foundation stone
Institute of Medical Education and Research,
Chandigarh 160012, India of most of the work being carried out in cancer
e-mail: dryashwant@ymail.com immunity [7,8].

196 A. Bhatia and Y. Kumar
12.2 Cancer Immunoediting tumors. The clue to the tumor-editing role of the
with Its Three Es: Reection immune system came from the experiments of
of the Dual Role of Immunity Robert Schreibers group on spontaneous and
in Cancer 3-methylcholanthrene (MCA)-induced tumors in
129/SvEv mice (Fig. 12.2) [6]. The concept of
The cancer immunoediting theory states that immunoediting was introduced by Dunn et al. in
tumors are sculpted by the immune system, result- 2002 to explain the antitumor as well as pro-tumor
ing in the selective growth of the variants which features of our immune response at different stages
are better equipped to ght the immune system of cancer [3]. Since then, many studies conducted
(Fig. 12.1). This selective growth advantage con- over a period of time have demonstrated the edit-
ferred on tumors is a consequence of a number of ing of tumors by host adoptive cells, leading to
genetic and epigenetic events occurring within the their complete reprogramming. A more recent
Innate and adoptive

ELIMINATION antitumor immunity
surveillance Spontaneous
destruction of
Immuno-
transformed cells
TILs associated with

good prognosis
I M M U N O E D I T I N G
Adoptive immunity
Genetic and epigenetic changes
EQUILIBRIUM Cellular dormancy

C A N C E R
Angiogenic dormancy
Immunosuppressive
microenvironment
Immune system
sculpts tumors
Decades
Selection of less
immunogenic variants
? Therapy
Resistance to immune
attack
ESCAPE Clinical symptoms
Fig. 12.1 Cancer immunoediting process with its three the events from equilibrium phase may proceed either
Es of elimination, equilibrium, and escape. Please note toward escape or back to the elimination phase, the revers-
that although in many cases the sequence is followed, in ibility of the escape phase with or without therapy to other
others one or the other phase may be skipped. Although two phases is questionable
12 Cancer Immunoediting: Immunosurveillance, Immune Equilibrium, and Immune Escape 197
a MCA
100g subcutaneously
129/SvEv
Wild RAG-2-/-
Late tumors Early tumors
RAG-2-/-STAT1-/- STAT1-/- n=11/57 n= 30/52 IFNGR1-/-
(RKSK) n=13/18 n=17/20 n= 12/20
b Spontaneous tumors
RKSK (11.9-17.5 months) Wild (15.5-21 months) RAG-2-/- (15.0-16.1 months)

Malignancy, n= 6/11 Malignancy, n=0/11 Malignancy, n=6/12
tumors, n=11/11 tumors, n=2/11 tumors, n= 12/12
c MCA
Wild RAG-2-/-
Sarcoma Sarcoma
RAG-2-/-
RAG-2-/- Cell lines Cell lines
Progressive
Progressive
growth
growth
Immunocompetent Immunocompetent
n=17/17 sarcoma n=12/20, 8 rejected
Less immunogenic More immunogenic
Fig. 12.2 Mice experiments by Shankaran et al. [6] demon- RAG-2/ mice and were injected into immunocompe-
strating surveillance and sculpting roles of immune system. tent and RAG-2/ mice. Progressive tumor growth was
(a) Immunodecient (RAG-2//IFNGR1/STAT1/ or noted in immunodecient mice transplanted with sarcoma
combined RAG-2/ STAT1/, RkSk) mice developed cells derived from wild or RAG-2/ mice. The immuno-
tumors earlier than wild type and with greater frequency on competent mice transplanted with sarcoma cells from
subcutaneous injection of MCA, thus necessitating the wild mice also showed progressive tumor growth; how-
presence of intact T, NKT, and B cells for prevention of ever, many mice transplanted with sarcoma cells derived
chemically induced tumors. (b) Spontaneous tumor devel- from RAG-2/ mice rejected the transplanted tumor
opment was also observed to be higher in RAG-2/ and cells. This occurred due to sculpting of sarcoma by the
RkSk mice as compared to unmanipulated 129/SvEv wild- immune system in wild mice, thus rendering it less immu-
type mice. Moreover, the later merely developed benign nogenic. Tumors from the immunodecient mice which
tumors and no malignancy was noted. (c) Furthermore, were not edited were more immunogenic and thus were
cells were taken from MCA-induced tumors in wild and rejected by immunocompetent mice
study has linked processes such as epithelial mes- 12.2.1 Immune Elimination:
enchymal transition in tumor cells, which result in Evidences For and Against
an invasive phenotype, to the immunoediting pro-
cess through the involvement of cytokines such as The immune elimination phase of cancer immu-
TNF- and TGF- [9]. Cancer immunoediting is a noediting is sine qua non of the original
broad concept which includes three Es of elimi- immunosurveillance process. It envisages the
nation, equilibrium, and escape which together destruction or eradication of cancer by the host
sum up to all the events occurring during an immune system and is believed to occur when a
immune response to cancer [3]. cell gets transformed by overcoming its intrinsic
Table 12.1 Timeline of events depicting evolution of cancer immunity from immunosurveillance to immunoediting
Study Hypothesis/observation/experimental evidence Results
William B Cooley Injected cultures of heat-inactivated bacteria or Demonstrated marked regression
(1891) [10] bacterial culture supernatants into cancer patients of tumors and prolonged survival
after the treatment
Paul Ehrlich Immune system protects the host from malignancy Gave birth to the idea of immune
(1909) [1] control of malignancies
Burnet Immune system must be removing the carcinogenic Formal emergence of
and Thomas events arising out of ongoing evolutionary genetic immunosurveillance hypothesis
(1957) [2] remodeling taking place in an individual
Several groups Induced immunodeciency by thymectomy or No consensus regarding
(19651973) heterologous antilymphocyte serum or immunosurveillance
pharmacological agents. Immunodecient animals
are more prone to develop cancers
Stutman O The methylcholanthrene (MCA)-induced cancer Rejection of immunosurveillance
(1975) [11] incidence in immunodecient nude athymic mice hypothesis
was not higher than the control mice
Kaplan et al. IFN- and perforin decient animals were more Resurrection of immunosurveillance
(1998) [12] prone to MCA-induced tumors as compared to in cancer
controls
Shankaran et al. Experiments in RAG-2 null mice (lacking T, B, Denitive evidence of existence
(2001) [6] and NKT cells) revealed higher incidence of both of cancer immunosurveillance
MCA-induced sarcomas and spontaneous epithelial
tumors in these animals
Dunn et al. Concept of cancer immunoediting to explain the Coined the term immune elimination
(2002) [3] tumor sculpting role of immune system as a part of broader concept of cancer
immunoediting with 3 Es of
elimination, equilibrium, and escape
tumor suppressor mechanisms, before being quietly by the immune system without us ever
able to establish a full-blown tumor. Although being aware about it. Spontaneous regression
the existence of such a phenomenon has been has been reported in some tumors including
hypothesized since long, the early experiments cutaneous melanoma, retinoblastoma, osteosar-
carried out on nude mice models which are only coma, etc., in humans [15]. Studies have shown
partially immunodecient failed to prove it. that both innate as well as adaptive immune
The denitive experimental proof to its pres- response contribute to ghting off the cancer
ence came from the work of Shankaran et al. in from our body.
the last decade (Table 12.1, Fig. 12.2) [6].
However, despite the experimental evidence of 12.2.1.1 The Key Players in Anticancer
its presence in mice, it has been difcult to Immunity
demonstrate it in the clinical scenario. Still, the The key players responsible for launching an
data obtained from various cancer registries effective immune response against cancer include
wherein a higher cancer incidence especially of the immune cells and soluble molecules secreted
viral etiology has been observed in immuno- into the tumor milieu (Fig. 12.3). In case, the
suppressed transplant recipients suggests its tumor exhibits high immunogenicity, a specic
existence in human subjects as well. Currently, immune response occurs against it. However, if
a similar trend has been noticed in the setting of tumor immunogenicity is low, the nonspecic
acquired immunodeciency syndrome [13,14]. effector responses gain importance.
The proponents of this stage in cancer immu- The major cell types involved in an antitumor
nity state that many of the cell transformation immune response are adoptive T cells, which
events occurring in our body may be removed not only kill tumor cells directly with the help
TUMOR
SITE APC
Plasma cell
Complement
O -
les
2
ADCC
O - molecu
2 O2 , N
Macrophage
AIL
Phagocytosis
TR
2 H
Lytic
IFN-
- ,
IFN NK cell
HSPs
chaperoning
tumor NK NK
F
Cytotoxic
CS
antigens +
CD8+ CD8 T-cells
-
GM
Antibodies
F,
against TA
TN
Antigen
IL-2
processing &
IL-1 -,
IFN
presentation Tumor cell
CD4+ CD4+
CD4+ B B Lytic tumor
PC
CD4+ cells
LYMPH
NODE
Activated T Helper cell Antibody
Molecule
Tumor CD4+ T Activated Perforin

antigens HSP MHC I MHC II TCR IL-2R CD4 B cell Granzyme
Helper cell
Fig. 12.3 Diagram showing key players involved in anti- counteract the tumors. In addition, tumors may directly
tumor immune response. The tumor releases Ags which activate the cytotoxic cells including CD8+ and NK cells
are chaperoned by heat-shock proteins and taken up by the and phagocytic cells. While the former two can cause
APCs which process them and present to CD4+ T cells. direct tumor lysis primarily via perforin and granzymes,
The later being the central point of immune response acti- the later may engulf tumor cells and kill them by releasing
vate various other cells including NK cells, CD8+ cells, lytic molecules or may process and present tumor Ags to
macrophages, and B cells which act in various ways to CD4+ T cells
of TNF- but are also essential for the activation factor (GM-CSF). The activated macrophages
of other components of the immune machin- may phagocytize tumor cells and kill them by
ery. The CD8+ cytotoxic lymphocytes (CTLs) releasing toxic free radicals including O2 and
are able to directly recognize tumor cells which NO2 or by becoming antigen-presenting cells
express MHC I and can also be activated by (APCs) which present tumor antigens to CD4+ T
CD4+ T-helper cells. They may cause lysis of the cells such as dendritic cells (DCs). Natural killer
tumor cells via perforin- and granzyme-depen- (NK) cells also have the potential to directly rec-
dent mechanisms. The CD4+ T cells also secrete ognize and destroy tumor cells via tumor necrosis
factors to induce proliferation of B cells and to factor-related apoptosis-inducing ligand (TRAIL)
promote their differentiation to antibody (Ab)- and IFN--dependent mechanisms. Loss of MHC
secreting plasma cells. The later may contribute class I as commonly observed in tumors may
to antitumor immunity by complement-mediated be responsible for their increased susceptibility
lysis or by antibody-dependent cellular cytotoxity to NK-cell-mediated lysis. In addition, NK-cell
(ADCC). The CD4+ T-helper cells also activate activity may also be enhanced by IL-2 and IFN-
macrophages by secreting IFN-, TNF, IL-4, produced by the CD4+ T-helper cells. NKT and
and granulocyte-macrophage colony-stimulating T cells also recognize the danger signals released
from the tumors and become activated. The NKT immunity. In experiments on MCA-induced
cells especially the invariant or the type I NKT, tumors in mice, Koebel et al. demonstrated the
which are CD4 CD8 and mainly recognize the presence of inert lesions in healthy mice, which
lipid/glycolipid antigens (Ags) via CD1d mol- grew when subjected to immunological oppres-
ecule, have been recognized to protect against sion (Fig. 12.4) [23]. The study served to be an
certain cancers. The protective role is however important milestone in proving the existence of
supposed to be indirectly exerted via secretion of the equilibrium phase in cancers. Likewise, the
IFN- and subsequent activation of NK and CD8+ tumors have been observed to stay dormant for
T cells. The T cells which represent 15 % of decades after remission in human cancer patients,
peripheral blood T cells are also reported to inl- which is believed to be due to the fact that immune
trate and cause lysis of tumors, both in vitro and system keeps them in check. The immune system
in vivo [1620]. is believed to synergize with chemoradiotherapy
In various clinical studies on different cancers in treatment-induced remission which renders the
including colon, ovary, lung carcinomas, and tumors silent. However, they relapse promptly
melanoma, the tumor-inltrating lymphocytes after any kind of immune insult, thereby, further
(TILs) have been associated with increased proving the presence of immune dormancy. The
time to disease recurrence, an enhanced 5-year minimal residual disease commonly observed in
survival, and an overall good prognosis. Also, hematological malignancies and the emerging
in a study on metastatic colorectal cancer, TIL donor-derived malignancies in immunosup-
density at the invasive margin was linked to a pressed transplant recipients are considered two
better chemotherapeutic response. Similarly, examples of the equilibrium phase in humans.
increased inltration by CD3+ and CD8+ T Even though the immune system prevents mono-
cells, NK cells, and T cells has been corre- clonal gammopathy of unknown signicance
lated with improved outcomes in epithelial (MGUS) from progressing to myeloma, it fails to
ovarian cancers. Some of the above studies eliminate the MGUS cells [24,25].
have done quantitative assessment of the TILs Adoptive T cells, both CD4+ and CD8+, have
in tumors, thus impressed upon the need to have been observed to play a pivotal role in cancer
a scoring system for TILs in order to determine immune equilibrium. Immune-sufcient mice
the exact tumor behavior [21,22]. with inert tumors are shown to develop into full-
edged tumors only upon depletion of T cells/
IFN-/IL-12. However, the depletion of innate
12.2.2 The Equilibrium Phase: immune cells was not found to result in the devel-
The Most Controversial opment of tumors. Moreover, tumor cells were
and the Least found to be highly immunogenic during the equi-
Understood Phase librium phase, as they are unedited by the immune
system and become less immunogenic at the end
This phase represents an intermediate stage of of this phase [23,26,27].
immune response in cancer. During this phase, In addition, the mechanisms including cellular
the cancer and the immune system both coexist and angiogenic dormancy also complement the
without allowing each other to dominate. The immune system in maintaining cancer cells in the
immune system cannot eliminate the cancer dur- dormant state. In the former, the tumor cells hide
ing this phase; however, it does not allow it to themselves in specialized niches, become quies-
expand or metastasize. The cancer in turn is cent, and wait for the opportunity to regrow. In
sculpted by the immune system, thus leading to the later condition, expansion is not possible, due
the emergence of variants resistant to the immu- to the lack of adequate vascularization. When
nological attack [3]. faced with favorable conditions, tumor cells
Various studies in mice have pointed toward come out of their slumber and undergo a series of
the occurrence of the equilibrium phase in cancer genetic and epigenetic modications which
a Low dose MCA

25g subcutaneously
C57BL/6&
129/SvEv mice
Wild
200-230 days
Atypical Fibroblasts
Ki67 Ki67
Tunnel Tunnel
Progressive tumors Small stable tumors
Control T-cell and IFN- depleting
Removed Monoclonal antibody monoclonal antibodies
b
Anti -NK 1.1 /
-NKG2D /
-TRAIL
antibodies
No additional tumors Progressive tumors in 60%
No additional tumors
Fig. 12.4 Experiments conducted in mice by Koebel components of innate and adoptive immunity. The mice in
et al. demonstrating the presence of equilibrium phase in former two groups did not develop any additional tumors;
tumorigenesis. (a) Groups of wild-type C57BL/6 or 129/ however, those in the last group (T cell and IFN-
SvEv mice were injected with a single low dose of depleted) showed rapid tumor growth. This could only be
MCA. After monitoring for 200230 days, the mice with explained by cancer immune equilibrium in which the
rapidly growing sarcomas were set aside. (b) The remain- tumors were not removed, but restricted by the immune
ing mice displaying small stable masses at injection site process. However, on suppression of adoptive immunity
were injected with control Ab or mAbs depleting specic progressive tumor growth was observed
increase their immune resistance, eventually the escape phase has formed the basis for the
leading to the next phase of cancer immunity, development of various therapeutic agents with
known as immune escape. Studies are being con- the aim to stop the progress of the neoplastic pro-
ducted to identify the genetic and molecular sig- cess. Due to increasing genomic instability, can-
natures of dormant tumor cells which allow them cer cells acquire various characteristics enabling
to retain their dormant status or facilitate their them to ward off the immune process or to mod-
escape [23,2629]. ify it in such a way which is benecial to tumor
cells. Tumors utilize a number of strategies to
evade an effective immune response (Fig. 12.5).
12.2.3 Immune Escape: The Best The basis of an effective immune response
Studied Phase against any Ag is its recognition as a nonself and
its presentation to immune effector cells. Tumors
The escape phase represents the nal and most escape recognition by either presenting self Ags
extensively studied phase of the immunoediting to which the immune system is already tolerized
process. The unleashing of mechanisms underlying or by modulating their antigenicity. The later
Altered antigen presentation Defective T-cell response
Selection of antigen loss variants Apoptosis of T-cells via Fas/FasL
Altered antigen processing pathways T-cell anergy via engagement of
Deletion or mutations in MHC class I CTLA-4 and B7 family members
Co-stimulation IL-2 production
DC dysfunction, IL-12 expression of CD3 chain
Th2 phenotype
Accumulation of T-regulatory cells

Cancer
Immune
Evasion
Defects in innate immunity
Other mechanisms
NK cell defects
Altered cytokine milieu
levels of MICA
B-cell defects ? B-regulatory cells
NKG2D
Altered metabolism
NK cell number
Angiogenesis
Accumulation of M2 macrophages, MDSCs
Genetic and epigenetic changes
Defective NKT & T-cell function
Fig. 12.5 Mechanisms of immune evasion by the cancer
involves the shedding of tumor Ags into the cir- T-lymphocyte antigen-4 (CTLA-4) and PD-L1
culation from where they may be removed [30]. [34]. Anergic T cells are unable to produce cyto-
The next line of defense adopted by tumor cells is kines such as IL-2 and IFN-. Therefore, the
the modulation of APCs, rendering them incapa- autocrine and paracrine activation of CD4+ cells
ble of effectively presenting cancer Ags to and other immune cells including B cells, macro-
immune cells. The APCs like DCs are either phages, and CD8+ cells are blocked, leading to
deleted or functionally compromised in response further suppression of the immune cascade [35].
to the factors secreted by malignant cells [31]. Moreover, tumors also express Fas ligands on T
Tumor-induced co-inhibition of the second signal cells, leading to lymphocyte apoptosis [36]. Not
of the Ag presentation and consequent immuno- only do they suppress CD4+ and CD8+ cells, but
suppression has now been recognized in several also promote the suppressor T-cell phenotype
cancer types [32]. In addition, the tumors alter such as CD25+Foxp3+ T-regulatory cells. These
MHC molecules especially MHC class I and cells secrete IL-10, TGF-, and VEGF which
other components of Ag processing machinery in suppress the antitumor response and promote
the APCs, so as to further incapacitate the pre- tumoral angiogenesis (Table 12.2) [37]. Besides,
sentation of its Ags to the immune system [33]. tumors also inhibit innate immune response by
Besides, tumor cells plunge into an active battle induction of quantitative and qualitative defects
against the immune process by attacking its in NK cells, macrophages, and neutrophils. NK
adoptive and innate immune cells. Tumor cells cells have been found to exhibit decreased cyto-
subvert T cells and render them anergic through toxic potentiality due to the presence of tumor-
co-inhibitory molecules including cytotoxic secreted factors including TGF- in the tumor
Table 12.2 Mechanisms of immunosuppression induced was subsequently noticed that tumors may express
by T-regulatory cells and myeloid-derived suppressor cells
Ags which are quantitatively or qualitatively dif-
T-regulatory cells ferent from self Ags, thus rendering them sensitive
Secretion of immunosuppressive molecules like IL-10, to the immune attack. Quantitative differences
IL-35, and TGF
include signicantly increased expression of Ags,
Polarization of DCs toward tolerogenic phenotypes
which are less expressed in normal or benign con-
Direct cytolysis of effector T cells via granzyme B,
TRAIL, and galectin-1 ditions or reexpression of Ags only expressed
Metabolic changes like increased IDO in DCs and at a specic stage of embryonic development
increased conversion of ATP to adenosine promoting (Table 12.3). Moreover, the lineage-specic Ags
immunosuppression expressed normally in specic tissues may be
Stimulation of tumoral angiogenesis via VEGF expressed aberrantly in tumor cells. Qualitative
secretion
differences are produced due to mutational events
Myeloid-derived suppressor cells
occurring during carcinogenesis. Over the years,
Inhibition of effector T-cell proliferation and function
via L-arginine-dependent mechanisms several efforts have been made for the identica-
T-cell inhibition via production of ROS and TGF tion and mapping of the Ags expressed on tumor
Reduced T-cell homing via depletion of L-selectin cells; various nomenclatures have been used to
Promotion of Th2 and T-regulatory phenotypes via characterize them such as tumor-associated Ags
IL-10 secretion and tumor-specic Ags. Antigens capable of evok-
Inhibition of DC function via IL-10 ing a tumor-specic immune response have also
Promotion of angiogenesis via secretion of VEGF, been designated as tumor rejection Ags in some
basic broblast growth factor, HIF-1, etc.
textbooks, e.g., tyrosinase, MUC-1, Her-2/neu,
-catenin, caspase-8, etc. [44]. Previous studies on
microenvironment (TME) [38]. The later along tumor antigens (TAs) have mainly focused on the
with other cytokines (IL-4, IL-13, etc.) present in discovery of new Ags and their classication into
the tumor bed favors the accumulation of M2 two subclasses, a group which can evoke a protec-
macrophages, which also induce immunosup- tive immune response and another group serving
pression [39]. Recruitment of immature myeloid as potential therapeutic targets. However, the
cells like myeloid-derived suppressor cells advent of cancer immunoediting theory has
(MDSCs) further complements the tumor- changed our insight on TAs, as they are now con-
immunodecient environment by reducing T-cell sidered to be one of the prime targets of the above
and NK-cell activation and promoting neovascu- process. Currently, ongoing studies are attempting
larization via factors like VEGF [40]. to differentiate between the antigenicity of the
Other mechanisms such as anaerobic glycolysis, original or unedited tumors and those sculpted
hypoxia, and acidity of the TME along with the by the immune system [17,45,46]. Differences
existent defects in tryptophan metabolism induced between the immunogenicity of tumors derived
by increased expression of the enzyme indole- from carcinogen MCA (more immunogenic) and
amine 2,3-dioxygenase (IDO) further depress the those arising spontaneously (less immunogenic) in
antitumor immunity, thereby leading to cancer mice have been described by DuPage et al. [47].
progression and metastasis [4143]. They also showed that primary sarcomas are
edited by the immune system and, hence, become
less immunogenic in order to escape the T-cell
12.3 Tumor Antigens and Cancer response. In the same line, Matsushita et al.
Immunoediting obtained similar results in their study on tumor
exomes [48]. A recent study has revealed the pres-
Antigenicity of tumors has always been a matter of ence of antiinammatory antibodies to tumor-
discussion. In the past, it was believed that since associated Ags like NY-ESO-1, thereby suggesting
tumors are derived from self cells, the immune the importance of humoral immune system in can-
system is more receptive to their Ags. However, it cer immunoediting [49]. Novel genetic-based
Table 12.3 Examples of common categories of antigens present in tumors [44]

Characteristics
Antigen type Antigen class Antigen of antigens Tumor
Tumor- Oncofetal antigens CEA Expressed in fetal Colon cancer
associated AFP tissues, reexpressed in Germ cell tumors,
antigens tumors HCC
Differentiation and CD5 Normally in T cell but CLL
lineage-specic antigens aberrantly in B cells in
CLL
Melan A, tyrosinase Melanocyte lineage Melanoma
Gp 100 Prostate carcinoma
PSA
Cancer testes antigens MAGE 1 Expressed in germinal Melanoma
NY-ESO-1 tissues and reexpressed in
malignancies
Heat-shock proteins Gp 96 Fibrosarcoma,
HSP70 colon cancer
Gene amplication Her-2/neu Receptor tyrosine kinase Breast cancer
Ovarian cancer
Aberrant post MUC1 Under glycosylated Breast
translational modication mucin Pancreas
Tumor- Mutated oncogenes or Mutated p53 Point mutations Many tumors
specic proteins BCR-ABL Translocation 9;22 CML
antigens -Catenin Signal transduction Melanoma
pathway
Caspase 8 Apoptosis regulation Squamous cell
carcinoma
Oncoviral HPV 16, E6 and E7 Viral transforming gene Carcinoma cervix
proteins proteins products
CEA carcinoembryonic antigen, AFP alpha fetoprotein, Gp glycoprotein, PSA prostate-specic antigen, MAGE-1
melanoma-associated antigen 1, NY-ESO-1 New York-ESO-1, BCR-ABL breakpoint cluster region-Abelson, HPV
human papilloma virus
approaches including exome sequencing, in silico noediting process. During the elimination phase,
analysis, and CD8+ T-cell cloning are likely to fur- the milieu of the tumor comprises of factors
ther help in understanding the alterations in tumor which promote its eradication. Collaboration of
antigenicity occurring during different phases of factors including IFN- and lymphocytes has
cancer immunity [48]. been found to help in regulating the develop-
ment of tumors. In different studies, IFN- g - and
perforin-decient mice together with T-cell and
12.4 The Tumor NK-cell defects are found to exhibit a greater
Microenvironment During propensity for tumor development. Cytokines
Cancer Immunoediting like IL-2, IL-12, and IL-7 have been found to
promote antitumor immunity, suppress recruit-
The microenvironment surrounding the tumor ment of suppressor cells, and inhibit tumor
plays a critical role in determining cancer angiogenesis.
behavior. TME is composed of cells (tumor as During the equilibrium phase, TME assumes
well as immune), various factors secreted by the role of a niche, concealing relatively dormant
them, and the stroma. The TME is a dynamic cancer cells. The niche environment allows cancer
system switching from host protective to tumor cells to thrive without progression by maintaining
friendly during different phases of the immu- a balance between the cytostasis and cytolysis.
However, molecules which precisely maintain this peutic strategies to prevent, stop the progression,
balance during the immune equilibrium state or treat cancers. In addition, it has contributed to
remain to be dened. the development of new markers for the diagnosis
During the escape phase, tumor bed gets and prognostication of malignancies. Identication
packed with factors and cells which promote and manipulation of various molecules involved
immune suppression. Factors like IL-6, TGF-, in different phases of the immune response to
IL-8, and IL-10 help in generalized subversion cancer has emerged as a promising approach for
of an effective anticancer immune response. the development of novel immunotherapeutic
Growth factors like VEGF not only promote strategies for cancer treatment and eradication.
angiogenesis but also facilitate the recruitment Table 12.4 provides examples of the immuno-
of T-regulatory cells and MDSCs to the tumor therapeutic approaches directed toward the three
site. Besides, tumor cells induce downregulation phases of the immunoediting process.
of antitumor cytokines including IL-12 and Deciphering the nature of the cellular inltrate
IFN-. In addition, the abundant presence of and secretory molecules produced in response to
other factors within the TME including prosta- the transformation events and characterization of
glandin E2, reactive oxygen and nitrogen species the mechanisms involved in the elimination of
and phosphatidylserine, etc., aids cancer cells to tumor cells at early stages has led to the develop-
evade the immune response. Furthermore, the ment of novel cancer therapeutics. Moreover,
stroma including cancer-associated broblasts, quantitative as well as qualitative assessment of the
chemokines, matrix metalloproteinases, and immune cells present in TME may contribute to the
adhesion molecules also participates in cancers development of algorithms demonstrating tumors
conquest over antitumor immunity. response to chemoradiotherapy. In vivo or in vitro
Although the above few paragraphs have tried expansion of tumor-specic effector cells is being
to provide a simplied view of the events occur- applied as a strategy to boost up the antitumor
ring during various phases of the immunoediting immune response. Recognition of TAs which
process, there are several paradoxes involved. evoke an effective antitumor immune response has
One set of factors may play an immunostimula- served as the basis for the development of different
tory and antitumor role under particular condi- types of cancer vaccines. Monoclonal antibodies
tions, whereas they may exert an immune (mAbs) targeting diverse TAs have entered clinical
inhibitory and pro-tumor role under other cir- trials for several cancer types. Besides, TAs such as
cumstances. For example, IFN- which is a CEA have also been used as biomarkers for early
potent cytokine responsible for antitumor immu- detection and for determining tumor prognosis.
nity is now emerging as an important player in The concept of immunogenic chemotherapy which
cancer immune evasion. The pro-tumor effects of stimulates adaptive immunity is also gaining impe-
IFN- are believed to be related to an increase in tus in recent years.
T-regulatory cells and MDSCs and a decrease in The equilibrium phase has also emerged as a
neutrophilic inltrate in the TME [5053]. potential target to immunotherapists, as main-
taining cancer cells in the equilibrium phase
indicates prevention or delay in cancer progres-
12.5 Clinical Relevance sion and fatality. In cases treated with mAbs
of the Immunoediting which exert their effect via NK cells, an adop-
Process in Cancer tive T-cell response was also found to be evoked,
leading to the maintenance of tumors in equilib-
The introduction of immunoediting concept has rium phase [69]. Furthermore, development of
added a new insight to understanding of cancer sensitive techniques to seek out the occult tumor
immunity. A clear understanding of the mecha- cells in various organs may help in their specic
nisms underlying the three phases of cancer targeting, resulting in their complete eradica-
immunity is vital for designing the immunothera- tion. Identication and targeting of immune or
Table 12.4 Examples of therapeutic approaches targeting different phases of cancer immunoediting
Phases of immunoediting Approaches Outcome
Elimination In vivo or in vitro expansion of immune Sipuleucel T (autologous PBMCs, APCs,
effector cells and using them for therapy and recombinant fusion protein i.e.
DC-based approaches PA2024, PA, PAP fused to a GM-CSF),
Tumor antigen-based vaccines FDA approved for prostate cancer [54]
Tumor-specic monoclonal antibodies Trastuzumab (Her2/neu), rituximab
(CD20), cetuximab (EGFR) [5557]
Immunostimulatory cytokines IL-2, IL-7, IL-15 [5860]
Equilibrium Adoptive transfer of cancer-reactive Monitored for establishment
T cells of equilibrium phase [61]
Escape Anti-CTLA-4 Ipilimumab approved for melanoma [62]
Blockade of T-cell co-inhibition mAb against B7-H1 [63]
Depletion of T-regulatory cells Denileukin diftitox [64]
Lenalidomide [65]
Inhibition of MDSCs Sunitinib [66]
Inhibition of IDO 1-Methyl tryptophan [67]
Blockade of VEGF Bevacizumab [68]
PBMCs peripheral blood mononuclear cells, APCs antigen-presenting cells, DC cendritic cell, GM-CSF granulocyte
macrophage colony-stimulating factor, EGFR epidermal growth factor receptor, CTLA-4 cytotoxic T-lymphocyte anti-
gen-4, mAb monoclonal antibody, MDSCs myeloid-derived suppressor cells, IDO indoleamine-2, 3 dioxygenase, VEGF
vascular endothelial growth factor
nonimmune events shifting the balance from Further knowledge on the genetic and epigenetic
equilibrium to the elimination or to the escape features characterizing the three Es of cancer
phase may lead to tumor removal or at least pro- immunoediting are warranted for the develop-
gression restriction. ment of more precise cancer immunotherapeutic
As discussed in earlier sections, tumor cells approaches in the future.
apply a variety of tactics to combat with the host
immune system. The assessment of factors
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Apoptosis and Cancer
13
Contents 13.4.3 Proteasome Inhibitors .................................. 227

13.4.4 Inhibitor of Apoptosis Protein (IAP)
13.1 Introduction ................................................ 209 Antagonists .................................................. 229
13.2 Mechanisms of Apoptosis .......................... 211 13.5 Concluding Remarks ................................. 230
13.2.1 Extrinsic Apoptosis Pathway ....................... 212
13.2.2 Intrinsic Apoptosis Pathway ........................ 213 References ............................................................. 231
13.3 Apoptosis and Cancer ................................ 217

13.4 Apoptosis Signaling Pathways 13.1 Introduction
and Therapeutic Targets in Cancer.......... 220
13.4.1 TRAIL (TRAIL Ligands, The concept of life and death has been a topic of
Monoclonal Antibodies Against
TRAIL-R1 and TRAIL-R2) ......................... 220 interest among scientists, philosophers, and theo-
13.4.2 Bcl-2 Family Proteins (BH3 Mimetics logians. It was such an intriguing subject that an
and Bcl-2 Antisense).................................... 225 Immortality Project was established in mid-2012
to nd answers to human immortality. The highly
funded project, headed by a well-known philoso-
pher, employs empirical studies to address
M.L. Tan, PhD (*) research areas such as near-death experiences,
Advanced Medical and Dental Institute, Universiti
alleged out-of-body experiences, postmortem
Sains Malaysia, Persiaran Seksyen 4/9,
Bandar Putra Bertam, Kepala Batas, survival, and the inuence of beliefs about
Pulau Pinang 13200, Malaysia immortality on human behavior, attitudes, and
Malaysian Institute of Pharmaceuticals character. Scientically, life and death are essen-
& Nutraceuticals, Ministry of Science, Technology tial parts of a natural cycle of all multicellular
& Innovation (MOSTI), Block 5A, Halaman Bukit organisms. Cell division, death, shape modica-
Gambir, Minden, Pulau Pinang 11700, Malaysia
tion, and cell rearrangements form critical pro-
e-mail: tanml@usm.my; drtanmelan@yahoo.com
cesses on which tissues are shaped and organs are
H.K. Tan, BSc (Hons)
made [1]. The orchestration of these processes
Malaysian Institute of Pharmaceuticals
& Nutraceuticals, Ministry of Science, Technology depends on a genetic program operating on cell
& Innovation (MOSTI), Block 5A, Halaman Bukit behavior: for example, some signaling molecules
Gambir, Minden, Pulau Pinang 11700, Malaysia and growth factors promote cell divisions and
e-mail: hengkean@gmail.com
control tissue size, whereas other proteins control
T.S.T. Muhammad, PhD the orientation of cell divisions and cell rear-
Institute of Marine Biotechnology,
rangements. Control of tissue size is manifested
Universiti Malaysia Terengganu,
Kuala Terengganu 21030, Terengganu, Malaysia in the process of cell competition whereby faster
e-mail: sifzizul@umt.edu.my growing cells can out-compete slow growing

210 M.L. Tan et al.
Functional classification of cell

death
Other
Autophagic Mitotic
Apoptosis Necroptosis modalities of
cell death catastrophe
cell deaths
Intrinsic Extrinsic
apoptosis apoptosis
Mediated by death Mediated by

Caspase-dependent Caspase-independent
receptors dependence receptors
Fig. 13.1 Functional classication of cell death modalities as described by the Nomenclature Committee on Cell
Death (NCCD) [19]
cells. Competition also involves apoptotic elimi- An imbalance between cell growth and cell
nation of the slow growing cells and their engulf- death is implicated in a variety of human diseases
ment by fast growing cells [1, 2]. Hence, cell including cancer, autoimmune diseases, neurode-
death plays an important role in the development generative disorders, viral infections, and AIDS
and homeostasis of normal tissues [3, 4]. Cells [1115]. Cell death has a profound effect on cancer
produced in excess during the development growth and progression [1618]. Malfunction of
process eventually undergo cell death, thereby the cell death machinery, as a direct consequence
contribute to sculpturing of organs and tissues [5]. of mutations of the signaling molecules involved
Historically, cell death phenomenon was rst either directly or indirectly in the cell death path-
reported in 1842 by Carl Vogt [6, 7]. Subsequently, ways, has long been identied as an important
the term programmed cell death (PCD) was men- contributing factor in cancer. Continuous efforts
tioned by Lockshin and Williams in 1965 [8]. in deciphering the mechanisms and signaling
The phenomenon describes coordinated deaths of pathways of these cell deaths have also brought
certain larval muscles during transformation into forward a new paradigm of which cancer may
adult moths. Kerr and co-workers later described be efciently targeted. Novel and specic cancer
a series of similar morphological characteristics therapeutics and techniques directed at members
following the death of a variety of tissue sources, of the cell death signaling pathways have been
which was coined as apoptosis [9]. About the developed, and newer generation of drugs is cur-
same time, Horvitz and colleagues started a sys- rently being tested in clinical trials.
tematic search for genes controlling PCD in the Figure 13.1 illustrates the most recent cell
nematode worms, Caenorhabditis elegans. The death classications by the Nomenclature
discovery of cell death defective genes, such as Committee on Cell Death (NCCD). NCCD has
ced-3, ced-4, and ced-9, implicated that PCD is a suggested limiting the use of the term pro-
process with strict genetic program [10]. This grammed only for those physiological
was quickly followed by the identication of sub- instances of cell death, irrespective of the
strates and homologous genes in mammals and modality by which they are executed, and which
realization that mutations of some of these cell occur in the context of embryonic and postem-
death genes were contributing factors in various bryonic development and tissue homeostasis
cancers. The 2002 Nobel Prize in Physiology or [19]. On the other hand, the term regulated
Medicine was awarded jointly to Sydney Brenner, cell death should be used to indicate cases of
H. Robert Horvitz, and John E. Sulston for their cell death, be it programmed or not and whose
extensive work and discoveries on genetic regu- initiation and/or execution is mediated by a
lation of organ development and PCD. dedicated molecular machinery and can be
13 Apoptosis and Cancer 211
inhibited by targeted pharmacologic and/or 21]. Apoptotic bodies are quickly taken up by
genetic interventions [19]. Apoptosis and its nearby cells and degraded within their lysosomes,
possible roles in tumorigenesis and some of the usually with no associated inammation [9, 20].
novel antitumor strategies and therapeutics will It is important to note that despite the various
be discussed in this chapter. types of apoptosis characterized by their biochem-
ical features and signaling pathways, they share
similar morphological features. Biochemically,
13.2 Mechanisms of Apoptosis apoptosis is universally characterized by the
double-stranded cleavage at the linker regions
The term apoptosis was introduced by Kerr and between nucleosomes, resulting in the formation
co-workers in 1972, derived from a Greek term of multiple DNA fragments [21] and phosphati-
meaning dropping off of leaves or petals from dylserine externalization [22], and is accompanied
trees or owers [9]. Earlier methods to dene cell by a series of gene and protein expressions.
death rely much on morphological criteria and the Figure 13.2 illustrates the morphological charac-
use of microscopes [7]. The earliest recognized teristics of apoptosis and how it is compared with
morphological changes in apoptosis involve necroptosis and autophagic cell death.
compaction and segregation of nuclear chromatin The NCCD has formulated several rounds
and condensation of the cytoplasm [9, 20]. The of recommendations to propose guidelines
process is followed by the convolution of the and unify criteria on the use of cell death ter-
plasma membrane and cell blebbing in a orid minologies [19, 23]. According to the latest
manner, producing fragments of cells known as NCCD publication, apoptosis is functionally
apoptotic bodies. These fragments are membrane- classied into intrinsic or extrinsic apoptosis.
bounded and contain nuclear components [20, Intrinsic apoptosis is either caspase-dependent
Cell and mitochondrial swelling
Formation of
isolation
membrane
Normal cell
Expansion
Nuclear changes Cell shrinkage

and chromatin
condensation
Completion of
vesicle and fusion
with lysosome
Plasma membrane Cell blebbing and

Nuclear and cell
rupture formation of
fragmentation
apoptotic bodies
Formation of
autophagosomes
Spillage and generation of
Plasma membrane massive number of
of cytoplasmic
intact vacuoles
contents
Necroptosis Apoptosis Autophagic cell

death
Fig. 13.2 Morphological characteristics of cells undergoing apoptosis, autophagic cell death, and necroptosis
212 M.L. Tan et al.
or caspase-independent, whereas extrinsic apop- of netrin-1 by recruiting a signaling complex

tosis is categorized depending on source of trig- consisting of protein phosphatase 2A (PP2A) and
ger, as mediated either by death receptors or by death-associated protein kinase 1 (DAPK1) [31].
dependence receptors. In the presence of netrin-1, the PP2A complex is
repressed by the recruitment of cancerous inhibi-
tor of PP2A (CIP2A) into the UNC-5B/DAPK1
13.2.1 Extrinsic Apoptosis Pathway complex, of which DAPK1 is autophosphory-
lated and remained inactive. Conversely, netrin-1
Extrinsic apoptosis is essentially caspase- withdrawal is associated with a conformational
dependent and is induced by extracellular stress change in UNC-5B, resulting in the exposure of
signals which are mediated by specic transmem- the death domain, releasing of CIP2A, and the
brane receptors. In the extrinsic apoptosis induced recruitment of PP2A to the UNC-5B-DAPK1
by death receptors, the signaling pathway is medi- complex. PP2A-mediated dephosphorylation of
ated by receptors belonging to the tumor necrosis DAPK1 results in the activation of downstream
factor (TNF) receptor superfamily and is charac- apoptotic pathway. PP2A-like activity has been
terized by extracellular cysteine-rich domains linked to the formation of DISC, and is known to
(CRDs) and intracellular death domain (DD). inhibit B-cell lymphoma 2 (Bcl-2) phosphoryla-
Ligands such as TNF ligand, TNF ligand super- tion, leading to apoptotic cell death [32, 33]. In
family member 10 (TNFSF10), FAS ligand, and certain cell types, where the extrinsic apoptotic
tumor necrosis factor-related apoptosis-inducing pathway is triggered but lower levels of DISC
ligand (TRAIL) interact with their respective death followed by lower levels of active caspase-8 are
receptors [FAS/CD95, TNF- receptor 1 (TNFR1), formed, amplication of the death signal is pos-
or TRAIL receptor (TRAIL-R1 or TRAIL-R2)], sible through the cleavage of Bid by caspase-8,
recruit Fas-associating protein with a death domain which directly mediates Bak/Bax oligomeriza-
(FADD), and form the death-inducing signaling tion, and triggers the release of cytochrome (Cyt)
complex (DISC) [24, 25]. This complex recruits c [34, 35].
pro-caspase-8 and pro-caspase-10, leading to the Another signaling pathway mediated by
activation of the executioner caspase-3, caspase-6, dependence receptors are the DCC and the
and caspase-7 [26, 27]. Patched dependence receptor (Ptc). DCC encodes
On the other hand, extrinsic apoptotic signals an approximately 200 kDa type I membrane pro-
can be alternatively mediated by dependence tein, which displays homology with cell adhesion
receptors such as UNC-5 homolog family molecules in its extracellular domain, suggesting
receptors (UNC-5A, UNC-5B, UNC-5C, and that DCC may play a role in cell-cell or cell-
UNC-5D) and deleted in colorectal cancer (DCC) matrix interactions [36, 37]. DCC appears to
family receptors. These receptors are activated by drive apoptosis independent of both
netrins, a family of extracellular proteins that mitochondrial-dependent and death receptor/
direct cell and axon migration during embryo- caspase-8 pathways. DCC interacts and drives
genesis [28]. Netrins are members of the laminin the activation of caspase-3 through caspase-9
superfamily and contribute to the regulation of without requiring Cyt c or Apaf-1 [38]. Ptc, iden-
cell-cell adhesion and tissue organization [29]. tied as a tumor suppressor, induces apoptosis,
Netrin-1 has been recently identied to be an but is suppressed by its ligand, sonic hedgehog
anti-apoptotic survival factor in tumorigenesis (Shh) [39, 40]. Ptc interacts with the adapter pro-
[30]. DCC and UNC-5 homologs mediate cell tein DRAL/FHL2 in the absence of Shh and
death in the absence of netrin-1 and the binding recruits a protein complex that includes DRAL/
of the ligand to these receptors switches between FHL2, the CARD-containing domain protein
a pro-apoptotic signal and the promotion of sur- TUCAN, and apical caspase-9. Ptc triggers cas-
vival and motility [30]. UNC-5B (also known as pase-9 activation and enhances cell death via a
UNC-5H2) complex responds to the withdrawal caspase-9-dependent mechanism [41, 42].
The death receptor and dependence receptor rearrangements including blebbing during apop-
pathways converge at the activation of caspase-3, tosis [52, 53].
followed by cleavage and activation of down- FAK is a tyrosine kinase of which its phos-
stream caspases. Caspases or cysteine aspartic phorylation state and activity are linked to cell
acid-specic proteases are synthesized as inac- adhesion to the extracellular matrix through inte-
tive zymogens (or proenzymes) and are usually grin receptors. It has a direct inuence on the
cleaved to form active enzymes or undergo auto- cytoskeleton, structures of cell adhesion sites,
proteolysis in a cascade manner. Initiator caspases and membrane protrusions, leading to regulation
such as caspase-8, caspase-9, and caspase-10 of cell movement [54, 55]. Caspase-mediated
couple cell death stimuli to the downstream cleavage of FAK is known to contribute to the
effector caspases such as caspase-3, caspase-6, morphological changes in apoptosis. On the other
and caspase-7. The major proteolysis activity that hand, PAK, a serine-threonine kinase, regulates
takes place during apoptosis is carried out by morphological and cytoskeletal changes in a vari-
effector caspases. Caspase-3 appears to be the ety of cell types [56, 57]. Blocking PAK function
major executioner caspase during the demolition during Fas-induced apoptosis inhibits the mor-
phase of apoptosis [43, 44]. Caspase-3 cleaves a phological changes, but accelerates the phospha-
number of structural proteins such as fodrin, gel- tidylserine externalization in the membrane.
solin, rabaptin, nuclear lamin B, and vimentin Stable Jurkat cell lines that expressed a dominant-
[4446]. On the other hand, caspase-6 appears to negative PAK mutant are resistant to Fas-induced
merely cleave the nuclear lamin A during apopto- formation of apoptotic bodies and cleavage of
sis [44]. Caspase-3 also cleaves diverse regula- PAK [58].
tory proteins and enzymes, including focal PARP cleavage is believed to attenuate the
adhesion kinase (FAK), protein kinase C delta, cells ability to carry out DNA repair [45, 59].
retinoblastoma protein (Rb) (a protein involved Caspase-8 is also found to cleave PARP-2,
in cell survival), p21-activated kinase (PAK), U1 a member of the PARP family involved in DNA
small nuclear ribonucleoprotein (U1snRNP), repair, suggesting that caspase-8 is both an initia-
DNA fragmentation factor 45 (DFF45)/inhibitor tor and effector caspase [60]. Active caspase-3 or
of caspase-activated DNase (ICAD), receptor caspase-7 proteolytically cleaves DFF45, which
interacting protein (RIP), X-linked inhibitor of subsequently releases active DFF40, the inhibi-
apoptosis protein (X-IAP), signal transducer and tors associated endonuclease. It is responsible
activator of transcription-1 (STAT1), and topoi- for the degradation of chromosomes into nucleo-
somerase I [44, 45, 47]. Initially, poly (ADP- somal fragments, considered as the characteristic
ribose) polymerase (PARP) is reported to be an hallmark of apoptosis [61, 62]. Cleavage of both
exclusive substrate for caspase-7 [44], but a later structural and regulatory proteins is essential for
study proved that it is cleaved by both caspase-3 the apoptotic-associated chromatin condensa-
and caspase-7 [48]. tion, DNA fragmentation, nuclear collapse, and
Caspase-mediated cleavage of structural pro- morphological changes such as cell shrinkage
teins is essential for the apoptosis-associated and detachment, membrane blebbing, and forma-
morphological changes. For example, cleavage tion of apoptotic bodies. Figure 13.3 illustrates
of gelsolin in multiple cell types causes cells to the extrinsic apoptosis signaling pathway.
round up, detach from the plate, and undergo
nuclear fragmentation [49]. Inactivation of
rabaptin-5 causes fragmentation of endosomes 13.2.2 Intrinsic Apoptosis Pathway
during the execution phase of apoptosis [50].
Fodrin is a major component of the cortical cyto- Intrinsic apoptosis is known as either caspase-
skeleton of most eukaryotic cells; it has binding dependent or caspase-independent, and both sig-
sites for actin, calmodulin, and microtubules naling pathways are centrally mediated by the
[51]. Its proteolysis contributes to structural mitochondria. Intrinsic apoptosis can be triggered
214 M.L. Tan et al.
Soluble Monoclonal
recombinant antibodies against
TRAIL TRAIL-R1/R2
Dependence
Death receptors
receptors UNC-5B
Ptc and
DCC
PP2A DAPK1
DISC DRAL
TUCAN
MOMP Pro-caspase-9
Pro-caspase-8 tBid
Bid Cytochrome c
Apoptosome
NF-B
Caspase-8 Caspase-9
Gelsolin
Lamin B Pro-caspase-3
Fodrin
Rabaptin-5
DFF45/ICAD
IAPs
FAK and PAK Caspase-3 Smac/Diablo
U1snRNP mimetics
X-IAP
Topo 1
STAT1 Caspase-6
RIP
PARP
NF-B
Lamin A
Caspase-7
Proteasome
PARP inhibitors
Apoptosis
DFF45
ER
stress
Fig. 13.3 Extrinsic apoptosis signaling pathway and antitumor therapeutic targets
by DNA damage, -irradiation, oxidative stress, [78, 79]. MOMP causes generalized and irrevers-
cytosolic Ca2+ overload, serum deprivation, and ible inner mitochondrial transmembrane potential
many other intracellular stress conditions. Upon (m) dissipation. In the inner mitochondrial
stimulation, various molecules are released into membrane (IM) of a healthy cell, the frontier
the cytoplasm including Cyt c [27, 63], second between the intermembrane/intercristal space and
mitochondria-derived activator of caspases/direct the matrix is nearly impermeable to all ions,
IAP-binding protein with low pI (Smac/Diablo) including protons which help create the proton
[64, 65], apoptosis-inducing factor (AIF; pro- gradient required for oxidative phosphorylation
motes chromatin condensation) [66], endonucle- [70]. The charge imbalance that results from the
ase G (EndoG; facilitates chromatin degradation) generation of an electrochemical gradient across
[67, 68], and high-temperature requirement pro- the IM forms the basis of the m [70]. A loss of
tein A2 (HtrA2/Omi) [69]. Cyt c binds to and the m or long-lasting or permanent m dis-
activates Apaf-1 protein in the cytoplasm, induc- sipation can lead to cell death [80]. MOMP causes
ing the formation of apoptosome which subse- the release of toxic proteins from the mitochon-
quently recruits the initiator pro-caspase-9, dria to the cytosol as mentioned above. Pro-
yielding activated caspase-9 and nally mediat- apoptotic Bcl-2 proteins appear to cause the
ing the activation of caspase-3 and caspase-7 release of Cyt c, Smac/Diablo, and HtrA2/Omi
[35]. Loss of Cyt c from the mitochondria also but not EndoG and AIF [81]. On the other hand,
results in the inhibition of the respiratory chain. BH3-only protein Bid cleavage by caspase-8
The condition elicits and aggravates reactive serves to engage a mitochondrial amplication
oxygen species (ROS) overproduction and is loop during extrinsic apoptosis. Caspase-8 cleaves
thought to activate a feedforward circuit for the Bid, generating a truncated fragment known as
amplication of the apoptotic signal [70]. The truncated Bid (tBid) that can permeabilize the
function of Cyt c and its role in apoptosis are mitochondrion, resulting in MOMP [82].
widely reviewed and discussed elsewhere Inhibitors of apoptosis proteins (IAPs) play an
[7173]. important role in the regulation of apoptosis.
Bcl-2 family of proteins plays an important Eight human IAPs have been identied consist-
role in the regulation of mitochondrial-linked ing of X-IAP, IAP-like protein-2 (ILP-2), cIAP-
apoptosis [74]. Bcl-2 subfamilies such as Bax, 1, cIAP-2, melanoma inhibitor of apoptosis
Bak, and Bcl-2 homolog (BH)3-only subfamily protein (ML-IAP), neuronal apoptosis inhibitory
proteins (e.g., Bid) play a pro-apoptotic role, protein (NAIP), survivin, and apollon [83].
while Bcl-2 and Bcl-XL are functionally anti- Human IAP family members such as X-IAP,
apoptotic. Activated Bax and Bak form homo- cIAP-1, and cIAP-2 are potent caspase inhibitors
oligomer which creates pores on the mitochondrial [84, 85]. X-IAP, cIAP-1, and cIAP-2 block Cyt
membrane and releases toxic proteins from the c-induced activation of caspase-9, thus prevent-
mitochondria. Bcl-2 and Bcl-XL inhibit the action ing the activation of caspase-3, caspase-6, and
by blocking the activation of Bax and Bak and caspase-7. Furthermore, these IAPs bind to and
preventing the release of pro-apoptotic proteins inhibit the enzymatic activity of caspase-3 fol-
[75]. Nevertheless, the activation of Bax and Bak lowing its activation by caspase-8, thereby arrest-
can be restored with the presence of pro-apoptotic ing the proteolytic cascade initiated by the
BH3-only proteins. BH3-only proteins function initiator caspase [86]. X-IAP primarily inhibits
as antagonists of specic subsets of their caspase by disrupting the conformation of the
pro-survival relatives [76, 77]. The pore-forming active caspase and masking the substrate-binding
activities of Bax and Bak trigger a condition active site [83].
known as mitochondrial outer membrane permea- Smac/Diablo and HtrA2/Omi inhibit the anti-
bilization (MOMP). MOMP can also be triggered apoptotic function of several members of the IAP
by the opening of a multiprotein complex known family [87, 88]. Smac/Diablo and HtrA2/Omi are
as permeability transition pore complex (PTPC) two nuclear-encoded mitochondrial proteins
216 M.L. Tan et al.
functioning as IAP antagonists, identied in endogenous and recombinant AIF are found to
mammals [69, 8992]. After their release into the trigger peripheral chromatin condensation and
cytosol stimulated by apoptotic triggers, Smac/ large-scale DNA fragmentation in a caspase-
Diablo and HtrA2/Omi competitively bind to the independent manner [110, 111]. AIF is not known
BIR domains of IAPs via the IAP-binding motif, to possess nuclease activity; therefore, AIF is pos-
so that the BIR-bound caspases are released and tulated to directly interact with DNA and disrupt/
reactivated [9395]. Smac/Diablo and HtrA2/ collapse chromatin structure by displacing chro-
Omi manifest distinct physical characteristics matin-associated proteins and/or by recruiting
and biochemical activities, of which the active proteases and nucleases to form DNA-degrading
Smac/Diablo is a homodimer, whereas HtrA2/ complexes or degradosomes [104, 112].
Omi is a homotrimer [87, 96]. Despite Smac/ Another important signaling pathway affecting
Diablo, HtrA2/Omi is a mitochondrial serine pro- the regulation of apoptosis worth mention is the
tease [97, 98]. HtrA2/Omi has diverse roles, nuclear factor-kappa B (NF-B). NF-B is a
including maintenance of mitochondrial homeo- sequence-specic transcription factor known to
stasis and regulation of cellular apoptosis [99]. be involved in the inammatory and innate
A comprehensive proteome-wide analysis of immune responses. Under normal conditions,
Jurkat cell lysates leads to the identication of NF-B becomes activated only upon stimulation
potential HtrA2/Omi substrates, for example, the and subsequently upregulates the transcription of
cytoskeleton-associated proteins such as actin, - its target genes. NF-B is activated by many
and -tubulin, and vimentin, further suggest its divergent stimuli, including proinammatory
role in the caspase-independent pathway [100]. cytokines such as TNF-, TRAIL, interleukin-1
AIF and EndoG function in a caspase- (IL-1), epidermal growth factor (EGF), T- and
independent manner, by relocating to the B-cell mitogens, bacteria and lipopolysaccharides
nucleus, where they mediate large-scale DNA (LPS), viral proteins, double-stranded RNA,
fragmentation, independent of caspases [101, drugs, and a variety of physical and chemical
102]. Mammalian EndoG is a nuclear-encoded stresses [113]. However, in tumor cells, molecular
protein targeted to mitochondria and compart- alterations result in impaired regulation of NF-B
mentalized in the intermembrane space (IMS) and become constitutively activated in such cases,
and is known to possess DNase/RNase activity leading to deregulated expression of NF-B-
[103]. It is implicated in the mitochondrial DNA controlled genes [114]. Some genes targeted by
replication and is shown to be involved in apop- NF-B include cytokines/chemokines and their
totic DNA degradation [102]. In isolated non- modulators, immunoreceptors, transcription fac-
apoptotic nuclei, EndoG rst generates large tors, and regulators of apoptosis such as Bcl-XL,
fragments of DNA (>50 kb) and then cleaves at Fas, FasL, and IAPs [113].
inter- and intra-nucleosomal sites [104]. NF-B is also known to play a pro-apoptotic
Although EndoG apoptotic activity appears to role, in addition to its more common anti-
occur in the absence of caspase activation, the apoptotic role. Examples of its pro-apoptotic
pathway leading to EndoG-dependent DNA effects in cells include those found in B cells
damage remains controversial [105, 106]. [115], T cells [116, 117], and neuronal cells [118,
AIF was originally discovered as an IMS 119]. On the other hand, the anti-apoptotic effects
component capable of inducing chromatin con- of NF-B appeared to be cell-type specic and/or
densation and DNA loss in the nuclei isolated dependent on the inducing signal. Normally,
from healthy cells [104, 107]. AIF is a avopro- NF-B is transcriptionally inactive in the cyto-
tein which was rst proposed to act as a protease plasm of most cells as it is bound to its cytoplas-
or protease activator [108]; notably, its apopto- mic inhibitor IB. Upon stimulation with
genic activity is not affected by z-VAD-fmk proinammatory cytokines, such as TNF- or
[109]. Contribution of AIF to apoptosis depends IL-1, IB protein is phosphorylated, ubiquiti-
on the cell types and death triggers [104]. Both nated, and subsequently degraded by the
proteasome (the role of proteasome is further cancer development. Deregulation in the

discussed under proteasome inhibitors). This apoptosis pathway is one of the reasons why
process exposes the previously masked nuclear neoplastic cells gain extended lifespan, develop
localization signal of NF-B, allowing it to trans- genetic mutations capable of growth under stress
locate into the nucleus upon IB proteolysis and conditions, and undergo angiogenesis [12].
subsequently activate the expression of important Several key pathways controlling apoptosis are
target genes involved in cell growth, survival, and commonly altered in cancer [127]. Tumor resis-
adhesion [120, 121]. Activated NF-B leads to tance to apoptotic cell death is often a hallmark
the activation of A1/B-1, a member of the Bcl-2 of cancer and contributes to chemoresistance
family, which suppresses Cyt c release from the [12]. Alteration of many proteins involved in
mitochondria [122]. NF-B activation blocks both intrinsic and extrinsic signaling pathways
caspase-8 cleavage and Cyt c release, indicating has been described, and many more to be discov-
that NF-B suppresses the earliest signaling ered in near future. For example, overexpression
components of the caspase cascade. The IAP of certain anti-apoptotic proteins, such as Bcl-2,
family genes (cIAP-1 and cIAP-2) and TRAF Bcl-XL, Akt, NF-B, and IAP protein family, is
family genes (TRAF1 and TRAF2) are positively found in various human tumors [128].
regulated by NF-B with rapid kinetics following The apoptotic pathway of Fas, one of the TNF
TNF addition [123, 124]. Another member of the receptor family members, is frequently blocked
IAP family, X-IAP, has been shown to be acti- by several mechanisms in cancer, one of which is
vated by NF-B in endothelial cells [125, 126]. Fas gene mutation [129131]. Fas mutations
Thus, NF-B activation functions to suppress have been detected in several types of human
apoptosis at multiple levels. cancers with frequent allelic losses of chromo-
The Nomenclature Committee on Cell Death some 10q24 where the gene resides [130132].
(NCCD) suggests to dene intrinsic apoptosis Both TRAIL-R1 and TRAIL-R2 genes are mapped
as cell death mediated by MOMP and associated on chromosome 8p21-22 [133, 134]. Allelic
with generalized and irreversible m dissipa- losses of the chromosome 8p21-22 have been
tion, release of IMS proteins, and respiratory reported as a frequent event in several cancers,
chain inhibition [19]. On the other hand, differen- including non-Hodgkin lymphoma (NHL), lung
tiation between caspase-dependent and caspase- cancer, breast cancer, colon cancer, prostate can-
independent intrinsic apoptosis pathways is based cer, hepatocellular carcinoma, and head and neck
on the extent of cytoprotection as conferred by cancer [135141]. Mutations of TRAIL-R2 gene
inhibition of caspases. The caspase-independent have been reported in head and neck cancer [142]
mechanisms mediated by AIF, EndoG, or ATP and non-small cell lung cancer (NSCLC) [143].
depletion tend to prevail over caspase inhibition In addition, somatic mutations of TRAIL-R1 and
and kill cells in conditions that would have been TRAIL-R2 genes are found in NHL [144] and
rapidly executed by the caspase cascade [19]. breast cancer [145]. The number of pancreatic
However, the caspase-independent signaling tumor tissues with positive membrane staining
pathway is still vague, and the exact mechanisms for TRAIL-R1 and TRAIL-R2 is lower than non-
remain to be investigated. Figure 13.4 illustrates tumor tissues [146]. Loss of TRAIL-R2 expres-
the caspase-dependent and caspase-independent sion is associated with poorer prognosis in
intrinsic apoptosis pathway. patients [146]. A signicant association is also
observed between lower expression of TNF gene
and poor prognosis in childhood adrenocortical
13.3 Apoptosis and Cancer tumors [147].
On the other hand, PP2A inactivation in can-
Apoptosis is an essential developmental process cer occurs frequently through the upregulation of
to maintain tissue homeostasis. Therefore, defect CIP2A, a PR65 interactor and PP2A inhibitor
in apoptosis regulation plays an important role in [148]. PR65, a scaffold protein which interacts
218 M.L. Tan et al.
Proteasome
Bcl-2 inhibitors
BH3 mimetics
antisense
IB
NF-B Proteasome
Generation of ROS
Bcl-2 m
Bcl-XL Bax/Bak
Mitochondrial
ATP synthesis
m MOMP Mitochondrion PTPC arrest
G
do
En
Ap
af
-1
Cyto
chro
me AIF
c
Smac/Diablo HtrA2/Omi
DNA
fragmentation
Pro-caspase-9
Apoptosome
Cleavage of
cytoskeleton
Caspase-9 proteins
IAPs
Pro-caspase-3
NF-B
Apoptosis
Caspase-3
Caspase-independent
Smac/Diablo
intrinsic apoptosis
mimetics
Caspase-dependent
intrinsic apoptosis
Apoptosis
Fig. 13.4 Caspase-dependent and caspase-independent intrinsic apoptosis signaling pathway and antitumor targets
with the catalytic subunit of PP2A, appears to Studies have reported that polymorphic
play a key regulatory role in cancer. This scaffold variants of the caspase-8 gene are associated
protein is decreased or mutated in a large fraction with the risk of multiple cancers [168172]. For
of human cancers and has been recently linked to example, a six-nucleotide insertion-deletion
cancer development [149]. On the other hand, variant polymorphism (6 N ins/del) of caspase-8
Ptc is a tumor suppressor and mutations of Ptc promoter is linked to a signicant decreased risk
are associated with neoplasia, especially in basal of bladder and lung cancer in Chinese popula-
cell carcinoma and medulloblastoma [39, 40]. tions [171, 172]. Since cancer cells are highly
DCC expression is shown to be markedly reduced dependent on these genetic changes in the apop-
in more than 50 % of colorectal tumors. The loss totic pathways for survival, designing novel anti-
of DCC is not restricted to colon carcinoma, but cancer drugs that selectively kill cancer cells
has been observed in other tumor types, includ- while sparing normal cells seem appealing [173].
ing carcinoma of the stomach, pancreas, esopha- Survivin, a member of the IAP family, is unde-
gus, prostate, bladder, and breast, male germ tectable in terminally differentiated adult tissues,
tumors, neuroblastomas, gliomas, and some leu- but abundantly expressed in human cancers such
kemias [36, 150, 151]. as lung, colon, pancreas, prostate, and breast
Members of the Bcl-2 family of proteins as [167]. Increased survivin mRNA is associated
prominent regulators of apoptosis signaling are with decreased overall survival in colon cancer
often deregulated in many cancers, including patients [174]. Furthermore, increased levels of
lung carcinoma, lymphoma, and glioblastoma cIAPs in malignant cells are associated with a
[152156]. Aberrant expression of Bcl-2 is com- shorter relapse-free survival in patients with
mon in chronic lymphocytic leukemia (CLL) and prostate cancer [175]. Livin or ML-IAP, another
is associated with poor response to chemotherapy member of the IAP family of proteins, is found to
and decreased overall survival [157]. Bcl-2 gene be expressed in tumor cells [176, 177]. Thus, the
amplication is reported in diffuse large B-cell possibility of IAP inactivation through therapeu-
lymphomas (DLBCL) and overexpression of tic intervention is rather attractive and has gained
Bcl-2 protein has been associated with poor much interest over the years.
prognosis in some forms of NHL [158160]. Another important pathway linked to the
Myc/Bcl-2 co-expression in DLBCL is associated apoptotic cell death is the p53 pathway, which is
with aggressiveness, is more common in the often inactivated and deregulated in human can-
unfavorable activated B-cell-like subtypes, and cers [178, 179]. The p53 protein is a transcription
contributes to the overall inferior prognosis of factor with tumor suppressor activities. Its role in
patients with activated B cell-DLBCL [161]. tumor suppression relies partly on its ability to
Single-nucleotide polymorphisms in Bcl-2 are regulate the transcription of genes important in
found to have an association with survival in cell cycle arrest and in apoptosis. The p53 protein
advanced-stage NSCLC patients who received upregulates the expression of a number of genes
chemotherapy [162]. Furthermore, mutations in response to genotoxic stress, including the pro-
that inactivate the pro-apoptotic Bax gene have apoptotic Bax [180]. It is also found to inhibit the
been observed in solid tumors and hematologic expression of the Bcl-2 gene [181]. Studies have
malignancies [163, 164]. Higher Bcl-2 to Bax also shown that Bid is a p53-responsive chemo-
ratios has been associated with progression of sensitivity gene which may enhance the cell
CLL, shorter remission duration, and shorter sur- death response to chemotherapy [182]. The fact
vival [165, 166]. Therefore, cancer therapeutics that a majority of human cancers harbor muta-
that specically inhibit the anti-apoptotic pro- tions in the p53 gene suggests that such mutations
teins or activate the pro-apoptotic members of the would have contributed to the apoptosis-resistant
Bcl-2 family proteins are an attractive strategy to environment. However, the p53 network and the
reverse the intrinsic or acquired resistance of can- mechanism by which p53 determines the fate of
cer cells to apoptosis [167]. cells remain to be explored.
220 M.L. Tan et al.
13.4 Apoptosis Signaling chemotherapy (paclitaxel and carboplatin) and

Pathways and Therapeutic targeted anti-angiogenesis agent (bevacizumab)
Targets in Cancer in advanced NSCLC has led to a randomized
phase II study [192]. Despite the encouraging
13.4.1 TRAIL (TRAIL Ligands, phase Ib results, the addition of rhApo2L to pacli-
Monoclonal Antibodies taxel/carboplatin or paclitaxel/carboplatin/bevaci-
Against TRAIL-R1 zumab combination did not improve the outcome
and TRAIL-R2) and produced a higher incidence of treatment-
related adverse effects [194]. Similarly, the addi-
TRAIL (Apo2 ligand) induces cell death via the tion of rhApo2L to rituximab did not improve the
extrinsic pathway by recruiting and activating objective outcome in phase II NHL study despite
caspase-8 and caspase-10 to its R1 and R2 recep- its promising activity in phase Ib study [193, 195].
tors [183]. It activates the intrinsic pathway via Adverse effects commonly associated with
the TRAIL-caspase-8-tBid-Bax cascade, through rhApo2L include neutropenia and serum lipase
the cleavage of Bid, which promotes Bax and elevation [194, 195]. Phase I trials of rhApo2L in
Bak oligomerization, leading to Cyt c release and colorectal cancer are ongoing (Table 13.1).
activation of caspase-9 [184]. These processes Mapatumumab, a fully human agonistic mAb
collectively amplify the activities of the related targeting TRAIL-R1, either used alone or in
executioner caspases. TRAIL is a promising can- combination with other chemotherapy drugs in
cer therapeutic agent, known to induce apoptosis phase I or phase II trials, has yet to produce
in a wide variety of tumor cells while sparing impressive trial outcomes, as in most cases, few
normal cells [185, 186]. TRAIL activity is also patients ended with partial response or stable dis-
known to be independent of the p53 status, mak- ease [260263]. Despite its favorable safety pro-
ing it potentially effective against chemotherapy- le, mapatumumab demonstrated limited or no
resistant tumors [187]. Early clinical trials have clinical activity in phase I and II trials in advanced
been initiated in cancer patients, using soluble solid malignancies [196, 197], NHL [199],
recombinant TRAIL (rhApo2L, codeveloped by NSCLC [201], refractory colorectal cancer [200],
Genentech and Amgen) [188, 189] and monoclo- and advanced hepatocellular carcinoma [198].
nal antibodies (mAbs) (agonists) targeting Additional trials of mapatumumab in advanced
TRAIL-R1, such as mapatumumab [HGS-ERT1 hepatocellular carcinoma and advanced cervical
is developed by Human Genome Sciences cancer may provide additional data on the useful-
(HGS)], and anti-TRAIL-R2 agents, such as ness of this drug (Table 13.1).
lexatumumab (HGS-ETR2 is developed by Lexatumumab, apomab, and conatumumab
HGS), conatumumab (developed by Amgen), are agonistic human mAbs against TRAIL-R2.
and apomab (developed by Genentech) [190]. Generally, the percentage of patients who devel-
In an early phase I safety and pharmacokinetic oped partial response or stable disease in several
trial of rhApo2L used as a single agent in patients early phase I trials involving these novel drugs is
with advanced solid tumors and NHL, of 32 low, despite being well tolerated by patients. For
patients with post-baseline tumor assessment, 17 example, no objective activity of apomab was
(53 %) had stable disease and 13 (41 %) pro- demonstrated in a phase II study among patients
ceeded with disease progression. Only a single with NHL [206], despite some evidence of activ-
patient was reported to have a partial response to ity in phase I study in patients with advanced
the drug [188]. Phase I/Ib trials of rhApo2L in malignancies [204]. The effects of apomab in
advanced cancer [191], advanced NSCLC [192], phase II NSCLC trial coincide with rhApo2L,
and NHL [193] reported that this drug was well where addition of apomab to paclitaxel/carbopla-
tolerated by patients and no anti-rhApo2L Abs tin/bevacizumab combination did not improve
were detected. Promising outcome in phase Ib the efcacy, while increasing the rate of some
trial of rhApo2L in combination with cytotoxic adverse effects [194, 205].
Table 13.1 Current therapeutic targets in the apoptosis signaling pathway and clinical trial stages
Therapeutic Clinical trial stages (published
targets Current drugs reports)/type of cancer Combined with References
TRAIL-R1 rhApo2L Phase I: advanced cancer [191]
and TRAIL-R2 (dulanermin) Phase Ib: NHL Rituximab [193]
Phase Ib: advanced NSCLC Paclitaxel, carboplatin, and bevacizumab [192]
Phase II: NHL Rituximab [195]
Phase II: advanced NSCLC Paclitaxel, carboplatin, and bevacizumab [194]
13 Apoptosis and Cancer
Ongoing phase I: colorectal cancer Cetuximab and irinotecan or FOLFIRI http://clinicaltrials.gov/show/NCT00671372

regimen and bevacizumab
Ongoing phase I: colorectal cancer FOLFOX regimen and bevacizumab http://clinicaltrials.gov/show/NCT00873756
TRAIL-R1 Mapatumumab Phase I: advanced solid tumor Gemcitabine and cisplatin [196]
Phase I: advanced solid tumor Paclitaxel and carboplatin [197]
Phase Ib: advanced hepatocellular Sorafenib [198]
carcinoma
Phase Ib/II: NHL [199]
Phase II: colorectal cancer [200]
Phase II: advanced NSCLC Paclitaxel and carboplatin [201]
Ongoing phase I/II: advanced cervical Cisplatin and radiotherapy http://clinicaltrials.gov/show/NCT01088347
cancer
Ongoing phase II: advanced Sorafenib http://clinicaltrials.gov/show/NCT01258608
hepatocellular carcinoma
TRAIL-R2 Lexatumumab Phase I: advanced solid tumor [201]
[203]
Apomab [204]
Phase I: solid tumor
(drozitumab; [205]
Phase I: advanced cancer
PRO95780) [206]
Conatumumab Phase II: NSCLC Paclitaxel, carboplatin, and bevacizumab [207]
(AMG 655) Phase II: NHL Rituximab [208]
Phase I: advanced solid tumor [209]
Phase Ib: pancreatic cancer Gemcitabine [211]
Phase Ib: advanced NSCLC Paclitaxel and carboplatin [212]
Phase Ib: colorectal cancer mFOLFOX6 and bevacizumab [213]
Phase I/II: soft tissue sarcoma Doxorubicin [214]
221
(continued)
Table 13.1 (continued)
222

Phase Ib/II: colorectal cancer Panitumumab [215]
Phase II: pancreatic cancer Gemcitabine [216]
Phase II: colorectal cancer FOLFIRI
Phase II: advanced NSCLC Paclitaxel and carboplatin
Ongoing phase I/II: colorectal cancer mFOLFOX6 and bevacizumab http://clinicaltrials.gov/show/NCT00625651
Ongoing phase II: advanced solid AMG 479 http://clinicaltrials.gov/show/NCT01327612
tumors
Anti-apoptotic AT-101 Phase I: CLL [217]
Bcl-2 family Phase I/II: SCLC Topotecan [218]
members Phase II: SCLC [219]
Phase II: NSCLC Docetaxel [220]
Ongoing phase II: advanced laryngeal Docetaxel and cisplatin/carboplatin http://clinicaltrials.gov/show/NCT01633541
cancer
Ongoing phase II: SCCHN Docetaxel http://clinicaltrials.gov/show/NCT01285635
Obatoclax Phase I: leukemia and myelodysplasia [221]
mesylate Phase I: advanced CLL [222]
(GX15-070) Phase I: solid tumor Topotecan [223]
Phase I: advanced solid tumor or [224]
lymphoma
Phase II: SCLC Topotecan [225]
Phase II: Hodgkin lymphoma [226]
Ongoing phase I: CLL Fludarabine and rituximab http://clinicaltrials.gov/show/NCT00612612
Ongoing phase I: solid tumor, Vincristine sulfate, doxorubicin, and http://clinicaltrials.gov/show/NCT00933985
lymphoma, or leukemia dexrazoxane hydrochloride
Ongoing phase I/II: SCLC or Topotecan http://clinicaltrials.gov/show/NCT00521144
advanced solid tumor
ABT-263 Phase I: lymphoid tumor [227]
(navitoclax) Phase II: SCLC [228]
Ongoing phase I: solid tumor Erlotinib or irinotecan http://clinicaltrials.gov/show/NCT01009073
Ongoing phase I/IIa: CLL http://clinicaltrials.gov/show/NCT00481091
Ongoing phase II: lymphoid cancer http://clinicaltrials.gov/show/NCT00406809
M.L. Tan et al.
Bcl-2 mRNA Oblimersen Phase I: advanced melanoma Temozolomide and albumin-bound [229]
sodium paclitaxel
Phase II: CML Imatinib [230]
Phase II: SCLC Carboplatin and etoposide [231]
Phase III: advanced melanoma Dacarbazine [232]
Phase III: CLL Fludarabine and cyclophosphamide [233]
Phase III: CLL Fludarabine and cyclophosphamide [234]
Phase III: MM Dexamethasone [235]
13 Apoptosis and Cancer
Ongoing phase I: AML Cytarabine and daunorubicin hydrochloride http://clinicaltrials.gov/show/NCT00039117

Ongoing phase I: advanced melanoma Abraxane and Temodar http://clinicaltrials.gov/show/NCT00409383
Proteasome Bortezomib Approved by FDA for multiple [236]
myeloma (2005)
Approved by FDA for mantle cell [237]
lymphoma (2006)
Phase I: acute leukemia [238]
Phase I: advanced solid tumor Paclitaxel [239]
Phase I/II: MM Cyclophosphamide and prednisone [240]
Phase II: MM Dexamethasone [241]
Phase II: mantle cell lymphoma [242]
Phase II: Hodgkin lymphoma Gemcitabine [243]
Phase II: esophageal, gastric, and Paclitaxel and carboplatin [244]
gastroesophageal cancer
Phase II: mantle cell lymphoma [245]
Phase II: MM [246]
Phase III: MM Dexamethasone [247]
Ongoing phase I: prostate cancer Mitoxantrone http://clinicaltrials.gov/show/NCT00059631
Ongoing phase I: neuroblastoma Irinotecan http://clinicaltrials.gov/show/NCT00644696
Ongoing phase I: T-cell lymphoma Azacitidine http://clinicaltrials.gov/show/NCT01129180
Ongoing phase I: acute leukemia or Belinostat http://clinicaltrials.gov/show/NCT01075425
myelodysplastic syndrome
Ongoing phase I/II: mantle cell Rituximab and lenalidomide http://clinicaltrials.gov/show/NCT00633594
lymphoma
Ongoing phase I/II: Waldenstroms Everolimus and rituximab http://clinicaltrials.gov/show/NCT01125293
macroglobulinemia
(continued)
223
Table 13.1 (continued) 224

Ongoing phase II: plasma cell Liposome doxorubicin and dexamethasone http://clinicaltrials.gov/show/NCT01328236
leukemia
Ongoing phase III: MM Panobinostat and dexamethasone http://clinicaltrials.gov/show/NCT01023308
Ongoing phase III: MM Vorinostat http://clinicaltrials.gov/show/NCT00773747
Carlzomib Phase I: hematologic malignancies [248]
(PR-171) Ongoing phase I: AML or ALL http://clinicaltrials.gov/show/NCT01137747
Ongoing phase I/II: MM Immunomodulatory drugs http://clinicaltrials.gov/show/NCT01365559
Ongoing phase II: MM http://clinicaltrials.gov/show/NCT00884312
Ongoing phase III: MM Dexamethasone http://clinicaltrials.gov/show/NCT01568866
X-IAP mRNA AEG35156 Phase I: advanced cancer [249]
Phase I/II: AML Idarubicin and cytarabine [250]
Phase II: AML Idarubicin and cytarabine [251]
Pan-IAP LCL161 Phase I: advanced cancer [252]
Phase I: advanced solid tumor Paclitaxel [253]
Ongoing phase II: breast cancer Paclitaxel http://clinicaltrials.gov/show/NCT01617668
HGS1029 Phase I: advanced solid tumor [254]
TL32711 Phase I: advanced solid tumor and [255]
lymphoma
Ongoing phase I: advanced solid Gemcitabine http://clinicaltrials.gov/show/NCT01573780
tumor
Ongoing phase I/II: myelodysplastic Azacitidine http://clinicaltrials.gov/show/NCT01828346
syndrome
Ongoing phase I/II: AML, ALL, and http://clinicaltrials.gov/show/NCT01486784
myelodysplastic syndrome
Ongoing phase I/II: solid tumor http://clinicaltrials.gov/show/NCT01188499
Ongoing phase II: ovarian, fallopian http://clinicaltrials.gov/show/NCT01681368
tube, and peritoneal cancer
Survivin YM155 Phase I: advanced solid tumor or [256]
lymphoma
Phase II: NSCLC [258]
Phase II: melanoma [259]
Ongoing phase II: breast cancer Docetaxel http://clinicaltrials.gov/show/NCT01038804
M.L. Tan et al.
Ongoing phase II: NHL Rituximab http://clinicaltrials.gov/show/NCT01007292

As for conatumumab, a phase I study in posttranslational modications of Bcl-2-related

advanced solid tumors showed that this drug is proteins have been demonstrated to play important
generally well tolerated [207, 208]. Conatumumab roles in regulating cell survival and death.
in combination with gemcitabine shows evidence The Bcl-2 family is characterized by specic
of an improved 6-month survival rate and tolerable regions of homology termed Bcl-2 homology
toxicity in phase Ib and II metastatic pancreatic (BH1, BH2, BH3, and BH4) domains. Anti-
cancer trials [209, 214]. In metastatic colorectal apoptotic proteins have BH1BH4 domains (e.g.,
cancer, conatumumab improves progression-free Bcl-2 and Bcl-XL). Pro-apoptotic proteins have
survival (PFS) when combined with FOLFIRI either BH1BH3 domains (e.g., Bax and Bak) or
[215], but limited activity when combined with BH3-only domains (e.g., Bid, Bim, Puma, Bad,
modied FOLFOX6 and bevacizumab [211], and Noxa, Hrk, Bik) [77, 266, 267]. These domains
no activity when combined with panitumumab are critical to the function of these proteins, espe-
[213]. The effect of conatumumab in NSCLC is cially their impact on cell survival and cell death
similar as compared with rhApo2L [192, 194], of and their ability to interact with other family
which combination of this drug with paclitaxel members and regulatory proteins. The molecular
and carboplatin did not produce promising results surface of the multidomain anti-apoptotic Bcl-2
[210, 216]. Combination of conatumumab with protein contains a BH3-binding groove, which
other chemotherapy drugs also produces no evi- accommodates BH3 domain from pro-apoptotic
dence of activity in soft tissue sarcomas [212]. The Bcl-2 protein family members. The BH3-only
common adverse effects with this drug are neutro- proteins are known to function as antagonists of
penia and thrombocytopenia [214216]. Generally, anti-apoptotic Bcl-2 family proteins and act as
these early trials lacked data on the correlation tumor suppressors [77]. This forms the basis or
between patients TRAIL status and response to platform for subsequent drug discovery strategies
therapy. Preferential TRAIL sensitivity and pres- based on mimicking BH3 peptides with chemical
ence of TRAIL-R1 and TRAIL-R2 expression in compounds that bind in the same groove [268].
certain cancers are considered factors in patients The earlier observation that apoptosis deregu-
response. Therefore, rhApo2L and agonistic anti- lation in cancer cells primarily affects the
TRAIL-R therapies may be limited to patients upstream of the signaling pathways of Bax/Bak
with TRAIL-sensitive tumors. The efcacy of and mitochondria, leaving the downstream core
TRAIL targeting therapies may be improved if of the apoptotic machinery mostly intact, has led
diagnostic methods determining TRAIL sensitiv- to a therapeutic strategy of which manipulation
ity of clinically detectable human cancers are of the equilibrium between the pro- and anti-
available [190]. Trials are still ongoing, especially apoptotic Bcl-2 family members could possibly
those involving the combination of these agents restore apoptosis [128, 173]. Since pro-apoptotic
with current chemotherapy drugs. BH3 domains directly bind to the hydrophobic
grooves of pro-survival proteins with high afn-
ity, and are necessary and sufcient for initiation
13.4.2 Bcl-2 Family Proteins (BH3 of apoptosis, agents mimicking the BH3 domains
Mimetics and Bcl-2 Antisense) may provide some degree of selectivity against
cancer cells. This is mainly because cancer cells
Bcl-2 family proteins can regulate apoptosis both are postulated to be more sensitive to inhibition
positively and negatively. The Bcl-2 family mem- of pro-survival proteins compared with their nor-
bers consist of anti-apoptotic (Bcl-2, Bcl-XL, Bcl- mal counterparts [12]. Cancer cells often express
W, Bag-1, Mcl-1, and A1/B-1) as well as high levels of Bcl-2-like anti-apoptotic proteins
pro-apoptotic (Bad, Bax, Bak, Bcl-xs, Bid, Bik, and to evade the apoptotic fate imposed by aberrant
Hrk) molecules [264, 265]. The balance and inter- cell proliferation, activation of oncogenes, or
action between Bcl-2 gene family members and DNA damage [269]. Therefore, it is possible to
226 M.L. Tan et al.
design BH3 mimetics to target specic anti- demonstrated that the drug is well tolerated up to
apoptotic proteins that are overexpressed in a the highest dose. However, only a single patient
particular type of cancer for improved specicity with acute myeloid leukemia (AML) with mixed
[173]. Several chemicals mimicking BH3 pep- lineage leukemia t(9;11) rearrangement achieved
tides exclusively targeting the Bcl-2 anti- complete remission, which lasted 8 months, and
apoptotic proteins have since been described of 14 patients with myelodysplasia, only three
[268, 270, 271]. Another antitumor strategy is showed hematologic improvement [221]. In
direct inhibition of Bcl-2 mRNA, in the form of another phase I trial, where obatoclax was admin-
antisense. istered to patients with advanced CLL, activation
One of the earliest small-molecule BH3 of Bax and Bak was demonstrated in peripheral
mimetics or more accurately Bcl-2 and Bcl-XL blood mononuclear cells, and induction of apop-
inhibitor that went through several phase I/II tosis was related to overall obatoclax exposure,
clinical trials is gossypol, an orally available as monitored by the plasma concentration of oli-
compound derived from cottonseed extracts gonucleosomal DNA/histone complexes.
[272]. It binds to the BH3-binding grooves of Obatoclax is noted to have some biological activ-
Bcl-2, Bcl-XL, and Mcl-1 [273]. However, sev- ity and modest single-agent activity in heavily
eral past clinical trials have not indicated this pretreated patients with advanced CLL [222]. In
compound as an effective anticancer agent. Either advanced solid tumor and lymphoma, of 35
used alone or in combination, patients treated patients given obatoclax infusions, only one
with gossypol failed to show evidence of tumor patient with relapsed NHL achieved partial
regression or any therapeutic responses in several response of 2 months duration, and one patient
clinical trials [274276]. A derivative of R-(-)- had stable disease for 18 months [224]. In a phase
gossypol (AT-101) is found to be well tolerated in II study in patients with relapsed SCLC, obato-
a phase I trial involving CLL patients [217]. clax added to topotecan produced no difference
However, later studies showed that either AT-101 in response rates as compared to topotecan alone,
is not active in patients or the response rates are even though the drug was generally well toler-
too low that it did not meet the criteria for addi- ated [223, 225]. Obatoclax has also showed lim-
tional enrolment in further trials for small cell ited clinical activity in heavily pretreated patients
lung cancer (SCLC) [218, 219]. In NSCLC, with classic Hodgkin lymphoma (HL) [226].
patients did not meet the primary endpoint of Neurological symptoms are reported as the most
improved PFS when given a combination of common adverse effects in patients. Obatoclax
AT-101 and docetaxel [220]. Current trials to appears to have limited efcacy as a single agent
evaluate the potency of this drug in other forms or even in combination with some of the more
of cancer are still ongoing, for example, as a common anticancer drugs. Clinical trials using
combination therapy in squamous cell carcinoma obatoclax in combination with other chemother-
of the head and neck (SCCHN) and advanced apy drugs in solid tumor, leukemia, and SCLC
laryngeal cancer. A semisynthetic analog of gos- are currently ongoing (Table 13.1).
sypol with improved pharmacologic properties, Another BH3 mimetic, ABT-737, possesses
such as apogossypolone (ApoG2), was found to greater binding afnity to BH3-only proteins,
inhibit the growth of diffuse large cell lymphoma enhances the effect of death signal, and is syner-
cells in vitro and in vivo [277]. However, this gistic with cytotoxic agents and radiation [268].
compound has yet to proceed to clinical trials. To overcome the low solubility and oral bioavail-
GX15-070 (obatoclax mesylate) is an indole ability of ABT-737, the ABT-263 analog (navito-
derivative and a broad-spectrum inhibitor of pro- clax) was developed for clinical investigation.
survival Bcl-2 family proteins; it has been exten- Preclinical studies conrmed that navitoclax has
sively evaluated in clinical trials. A phase I high afnity for the anti-apoptotic Bcl-2 family
clinical trial of obatoclax mesylate in 44 patients proteins and kills cancer cells in a Bax/Bak-
with refractory leukemia and myelodysplasia has dependent manner [227]. In a phase II clinical
study, navitoclax exhibits limited single-agent differences in time to tumor progression or objec-
activity against advanced and recurrent SCLC tive response rate [235]. Interestingly, in a recent
[228]. Clinical trials of navitoclax as a single phase I study, the combination of oblimersen,
agent or as combination therapy in a variety of temozolomide, and albumin-bound paclitaxel was
cancers such as lymphoid, leukemia, and other well tolerated and demonstrated encouraging
solid tumors are ongoing. activity in patients with advanced melanoma, with
A nuclease-resistant phosphorothioate anti- objective response rate and disease control rate at
sense oligonucleotide targeting Bcl-2 mRNA 40.6 % and 75 %, respectively [229]. Some of the
(oblimersen sodium) has shown promising activ- common adverse effects associated with oblim-
ity for CLL and malignant melanoma in random- ersen sodium administration include fatigue,
ized phase III trials [232234]. It is an 18-mer transaminase elevation [279, 280], and hemato-
phosphorothioate antisense oligonucleotide logic disorders [231233]. There are a number of
designed to bind to the rst six codons of the trials listed in the NIH ClinicalTrials.gov website;
human Bcl-2 mRNA [278]. The use of oblim- some trials are terminated, and some are com-
ersen in combination with chemotherapy in a pleted, while the outcome of trials involving some
variety of cancers has shown diverse response other tumor types is unknown at this point of time.
rates with good tolerability. In the Oblimersen
Melanoma Study Group, the addition of oblim-
ersen to dacarbazine improved the multiple clini- 13.4.3 Proteasome Inhibitors
cal outcomes in patients with advanced melanoma
and increased overall patients survival [232]. In The proteasome is a multicatalytic enzyme com-
another phase III trial, the addition of oblimersen plex that degrades intracellular proteins by a tar-
to udarabine and cyclophosphamide signi- geted and controlled mechanism. The 26S
cantly increased the complete response/nodular proteasome, a large protein complex, composes
partial response rate in patients with relapsed or approximately 50 subunits that function as a
refractory CLL [233]. In the same study, a sig- highly specic molecular shredder by hydrolyz-
nicant 5-year survival benet was observed ing ubiquitinated proteins into small peptides
with oblimersen in combination with udarabine [281]. The 26S proteasome can be further divided
and cyclophosphamide. Among patients with into two sub-complexes, a central 20S proteolytic
udarabine-sensitive disease who had previously core particle (CP) that is capped at either end by
demonstrated maximum benet with the same one or two 19S regulatory particles (19S RP).
treatment, a 50 % reduction in the risk of death The 20S CP is the degradation unit and contains
was observed [234]. the active sites required to hydrolyze proteins
However, not all combination therapies pro- into peptides [281]. On the other hand, the 19S
duce desirable outcomes. In the Cancer and RP controls the degradation of ubiquitin-tagged
Leukemia Group B Study 10107 (CALGB), substrates by acting as a receptor for poly-
although the combination of oblimersen and ima- ubiquitinated proteins and facilitating their ATP-
tinib was safe and feasible, no clinical benets dependent translocation into the catalytic
were observed in imatinib-resistant chronic chamber of the 20S CP [281].
myeloid leukemia (CML) patients [230]. In a ran- The ubiquitin-proteasome pathway (UPP) is
domized phase II study of carboplatin and etopo- responsible for proteolytic degradation of the
side with or without oblimersen for extensive-stage majority of damaged and misfolded proteins
SCLC (CALGB 30103), the addition of oblim- within the eukaryotic cell. The UPP is essentially
ersen to a standard regimen did not improve any important for controlled degradation of key regu-
clinical outcome measure [231]. A randomized latory proteins involved in a wide variety of cel-
study of dexamethasone with or without oblim- lular functions such as apoptosis [282], cell cycle
ersen sodium in patients with advanced multiple control, proliferation [283], and transcriptional
myeloma (MM) demonstrated no signicant regulation [284]. However, overactivity of the
228 M.L. Tan et al.
UPP results in an accelerated turnover of proteins radiation, and reverses chemoresistance in a vari-
that regulate the cell cycle, leading to a deregu- ety of hematologic and solid malignancy models
lated mitosis, thereby supporting cancer growth in vitro and in vivo [292]. Bortezomib is a novel
[285]. A defect in the proteasome function is synthetic dipeptide boronic acid that reversibly
associated with the development of different dis- inhibits the chymotryptic-like activity and, to a
eases such as neurodegenerative disorders, car- lesser extent, the caspase-like activity of the 5-
diovascular and rheumatoid diseases, and and 1-subunits of the 20S CP [293].
cachexia, but not cancer, suggesting that cancer However, the role of NF-B as a key determi-
cells use the proteasome for their survival [286]. nant of bortezomib-induced cytotoxicity is rather
In humans, three deubiquitinases (DUBs) are controversial, as several studies have shown that
associated with the 19S RP. Two of these direct inhibition of NF-B signaling is insuf-
(UCHL5/Uch37 and USP14/Ubp6) are cysteine cient to induce apoptosis in bortezomib-sensitive
proteases and members of the ubiquitin cells [294296]. Recent studies also found that
C-terminal hydrolases (UCH) and ubiquitin- bortezomib exerts no inhibition of constitutive
specic proteases (USP) families, respectively. NF-B activity in MM or mantle cell lymphoma
The expression of the cysteine DUBs UCHL5 cells [297, 298]. Results of the genome-wide
and USP14 is also deregulated in cancer. siRNA screen performed by Chen and co-workers
Activities of UCHL5 (along with several other showed that bortezomib induces cell death by
DUBs) are found to be enhanced in tumor biop- interfering with ribosome function and DNA
sies of cervical carcinoma when compared to damage pathways and through deregulation of
adjacent normal tissues [287]. Myc signaling [299]. A separate screen by Zhu
The transcription factor NF-B is inactive in and co-workers demonstrates that knockdown of
the cytoplasm under normal conditions and is cyclin-dependent kinase 5 (CDK5), as well as a
activated when its binding partner, IB, is number of other genes, potentiated bortezomib-
degraded by the proteasome. Constitutive NF-B induced cytotoxicity in MM cells [300]. In addi-
activity has been observed in a variety of tumors tion, proteasome inhibitors are also potent
including MM; sustained activity of NF-B may inducers of endoplasmic reticulum (ER) stress
lead to aberrant expression of target genes pro- [295, 301]. Acute ER stress response caused by
moting tumor cell proliferation and survival proteasome inhibition results in apoptosis [301].
[288]. Bcl-2 is identied as a key target of NF-B In addition to ER stress, several reports indicate
in B-cell lymphoma [289]. NF-B, a centrally that proteasome inhibitors induce the rapid pro-
important transcription factor involved in duction of ROS, known to be involved in apop-
immune and inammatory cellular responses totic signaling [295, 302, 303].
affecting both cell growth and survival, appears Bortezomib (Velcade, Millennium
to be pivotally involved in the pathogenesis of Pharmaceuticals, Inc., Cambridge, MA and
aggressive lymphoid malignancies [290]. As a Johnson & Johnson Pharmaceutical Research
result, the inhibition of proteasome function and Development, L.L.C.) is the rst proteasome
serves as an important mechanism in anticancer inhibitor approved by the US Food and Drug
therapy. Proteasome inhibitors have recently Administration (FDA) in 2005 for the treatment
emerged as an interesting and potentially new of progressive MM in patients who have received
group of chemotherapeutic agents for various at least one prior therapy [236]. The drug is later
human cancers, including breast, prostate, and approved for the treatment of mantle cell lym-
lung carcinomas, that function in part by stabiliz- phoma, a lymphoid malignancy derived from
ing the IB protein and, nally, inhibiting mature B cells [237, 242, 245]. Bortezomib has
NF-B activation [121, 291]. Preclinical studies undergone a series of successful preclinical and
have shown that the proteasome inhibitor, bort- clinical studies. Phase I and II trial results pro-
ezomib, decreases proliferation, induces apopto- duced encouraging prospects. In a retrospective
sis, enhances the activity of chemotherapy and study [based on data from phase II (SUMMIT or
CREST) or phase III (APEX) registration stud- when bortezomib was used as a single agent in
ies] to clarify the utility of bortezomib as a repeat newly diagnosed patients, 52 % did not achieve a
therapy, bortezomib retreatment appeared to be partial response or a better outcome [246].
safe and effective in patients with relapsed MM Furthermore, the clinical response to bortezomib
[304]. In a separate phase I/II trial, weekly bort- in other hematologic malignancies and solid
ezomib plus oral cyclophosphamide and predni- tumors remains low [238, 306].
sone produced more than 50 % complete response Resistance to proteasome inhibitors has been
rate and an encouraging 1-year survival in examined in cell-based studies, and potential
relapsed/refractory patients with MM [240]. The clinical mechanisms of bortezomib resistance
regulatory approval of bortezomib was based on have been highlighted. Understanding the molec-
its efcacy and safety in a large, international, ular basis of resistance to proteasome inhibitors
multicenter phase III prospective study. This ran- in patients with myeloma and other malignancies
domized, open-label trial compared single-agent will aid in the development of therapeutic strate-
bortezomib with single-agent, high-dose dexa- gies to overcome bortezomib resistance. In an
methasone in patients with progressive MM after effort to overcome bortezomib resistance, novel
at least one prior therapy [236, 247]. Bortezomib proteasome inhibitors have been developed that
manifested signicant efcacy and safety, sup- act through mechanisms distinct from bortezo-
ported by an improved response rate, including mib. These newer proteasome inhibitors may also
achieving near-complete responses [236, 247]. possess side effect proles distinct from that of
Updated results of a multicenter phase II bortezomib. Second-generation proteasome
PINNACLE study of bortezomib in patients with inhibitors with novel properties, such as NPI-
relapsed or refractory mantle cell lymphoma 0052 and carlzomib, are currently evaluated in
indicate that single-agent bortezomib is associ- clinical trials and have shown evidence of anti-
ated with lengthy responses and notable survival myeloma activity. Carlzomib (previously
in these patients [245]. However, clinical trials known as PR-171) is a tetrapeptide epoxyketone-
using bortezomib in combination with other che- based, irreversible proteasome inhibitor, more
motherapy drugs in cancers such as HL [243]; potent and selective, and produces more sus-
advanced solid tumors such as breast, ovarian, tained inhibition of the proteasome [307, 308].
and prostate [239]; and metastatic gastroesopha- Although a recent phase I study of carlzomib
geal cancer [244] lacked favorable outcomes. revealed tolerability and some clinical activity in
It is clear that although bortezomib has potent patients with multiple hematologic malignancies,
anti-multiple myeloma activity, not all patients the response rates were rather low [248].
respond to bortezomib, and most responders ulti- Currently, various trials are ongoing for carlzo-
mately relapsed [305, 306]. To date, however, no mib, either as a single agent or in combination
marker has been identied and validated in a man- with other chemotherapy drugs for MM patients
ner that would allow clinical use and to distin- who have relapsed or are refractory to bortezomib-
guish patients likely to respond to bortezomib containing treatments. Other clinical studies are
treatment from those who would not [305]. The currently exploring the potential benet of this
most common adverse events are gastrointestinal drug in patients with relapsed AML or acute lym-
symptoms, fatigue, thrombocytopenia, and sen- phoblastic leukemia (ALL) (Table 13.1).
sory neuropathy, which comprised a major reason
of treatment discontinuation [241]. Despite the
clinical success of bortezomib in MM and mantle 13.4.4 Inhibitor of Apoptosis Protein
cell lymphoma, resistance to this drug remains a (IAP) Antagonists
clinically signicant problem. For example, in
studies of bortezomib in relapsed refractory During apoptosis, natural IAP antagonists such
patients [241, 306], almost all responding patients as Smac/Diablo and HtrA2/Omi translocate from
ultimately experienced disease progression. Even the mitochondria and inactivate IAPs to facilitate
230 M.L. Tan et al.
caspase activation and cell death. Smac/Diablo combination with paclitaxel in advanced solid
and HtrA2/Omi promote apoptosis by antagoniz- tumors is currently underway. Early report shows
ing the IAPs, such as X-IAP, cIAP-1, and cIAP-2, that this combination is well tolerated and dem-
which are often upregulated in many cancer cells onstrates preliminary antitumor activity in breast
[309]. X-IAP is a potent direct inhibitor of cas- cancer patients [253]. Two other small-molecule
pase-3, caspase-7, and caspase-9 [310]. Smac/ IAP antagonists, HGS1029 and TL32711, are
Diablo contains an IAP-binding motif which also reported to be well tolerated in phase I stud-
forms the basis for the design of the novel class ies and have produced some evidence of antitu-
of anticancer drugs named Smac mimetics [311]. mor activity as well as suppression of cIAP-1
Peptides that mimic Smac/Diablo functions are level [254, 255]. YM155, a small-molecule
capable of inducing death or increasing the apop- inhibitor of survivin, another human IAP, has
totic effect of chemotherapeutic agents [64, 309]. also demonstrated to be safe and to possess anti-
In a preclinical study, the synthesized Smac/ tumor activity in phase I studies [256, 257].
DIABLO-N7 peptides are found to increase the However, a phase II trial reported modest single-
apoptosis-inducing potential of chemotherapeu- agent activity of YM155 in NSCLC [258]. In
tic drugs (paclitaxel, doxorubicin, and tamoxi- patients with stage III or IV melanoma, prespeci-
fen) and irradiation; in addition, they sensitize ed primary endpoint was not achieved in a
TRAIL-resistant cells to undergo apoptosis phase II trial [259]. LY2181308, a survivin anti-
[312]. Smac mimetic such as AEG40730 triggers sense oligonucleotide, has been reported to be
the autoubiquitination of cIAP-1 and cIAP-2 and safe in the rst-in-human phase I study, although
targets them for proteasomal degradation. Loss further studies would be needed to assess its
of cIAPs leads to TNF-dependent cell death in activities [314]. The overall efcacy of IAP
some cell types [313]. antagonists remains uncertain at this point of
AEG35156, an X-IAP antisense oligonucle- time. Table 13.1 summarizes the various drugs
otide, is the rst IAP antagonist that has advanced targeting the apoptosis pathways and clinical
to human clinical trial. A phase I trial of trial stages based on published reports as well as
AEG35156 in advanced refractory cancer pro- ongoing trials listed in the NIH ClinicalTrials.
duced reduction in X-IAP mRNA level; however, gov website.
the suppression was not preserved. Nonetheless, The crosstalk between apoptosis, autophagy,
it is well tolerated in patients after intravenous and necroptosis signaling pathways and future
infusion and some clinical evidence of antitumor directions of cancer therapeutic targets will be
activities are observed [249]. However, in a ran- discussed in Chap. 14.
domized phase II trial of patients with primary
refractory AML, the addition of AEG35156 to
idarubicin and cytarabine did not improve the 13.5 Concluding Remarks
rate of remission as compared with the control
arm consisting of cytarabine and idarubicin alone Resistance to cell death induction has long been
[250, 251]. The mRNA level of X-IAP was not recognized as a hallmark of cancer. Therefore,
determined in this study, therefore, whether ef- increased understanding of the underlying molec-
cient knockdown of X-IAP mRNA was achieved ular events regulating different cell death mecha-
in this phase II trial remains unknown [251]. nisms such as apoptosis, necroptosis, and
A phase I report of another novel IAP antago- autophagy has provided new possibilities for tar-
nist, LCL161, indicated that this orally bioavail- geted interference of these pathways. Various
able agent is well tolerated in patients with phases of clinical trials have been conducted
advanced cancer. However, no objective which interfere with these pathways.
responses were observed, despite the fact that
LCL161 treatment results in target inhibition, as Acknowledgments The authors would like to acknowl-
shown by cIAP-1 degradation and cytokine edge the Ministry of Science, Technology and Innovation
induction [252]. Phase Ib study of LCL161 in Malaysia and Universiti Sains Malaysia.
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Autophagy and Necroptosis
in Cancer 14
Ahmed Ismail Hassan Moad,

14.1 Introduction .............................................. 243
The ubiquitin-proteasome system (UPS) and
14.2 Autophagy and Cancer ............................ 247
lysosomes are two primary intracellular protein
14.3 Autophagy Signaling Pathways degradation pathways recognized in eukaryotic
and Therapeutic Strategies
cells. Differences between these two major pro-
in Cancer................................................... 249
14.3.1 mTOR Signaling Pathway Inhibitors ......... 249 tein degradation systems depend on their func-
14.3.2 Pro-autophagics ......................................... 250 tional signicance and the type of substrates
14.3.3 Autophagy Inhibitors ................................. 251 taken in for degradation [1]. The UPS catalyzes
14.4 Mechanisms of Necroptosis ..................... 252 the rapid degradation of abnormal proteins and
14.5 Necroptosis and Possible
short-lived regulatory proteins, leading to a con-
Therapeutic Targets in Cancer ............... 260 trol of a diversity of essential cellular processes
[2]. In the lysosomal protein degradation path-
14.6 Crosstalk in Apoptosis, Autophagy,
and Necroptosis ........................................ 261 way, degradation of extracellular materials is
mediated by endocytosis, whereas degradation of
14.7 Future Directions ..................................... 263
intracellular long-lived cytoplasmic proteins and
14.8 Concluding Remarks ............................... 263 damaged organelles is mediated by three types of
References ............................................................... 264
M.L. Tan, PhD (*)

Advanced Medical and Dental Institute, Universiti
Sains Malaysia, Persiaran Seksyen 4/9,
Bandar Putra Bertam, Kepala Batas,
Pulau Pinang 13200, Malaysia
Malaysian Institute of Pharmaceuticals & Nutraceuticals,
Ministry of Science, Technology & Innovation (MOSTI), A.I.H. Moad, PhD
Block 5A, Halaman Bukit Gambir, Minden, Department of Medical Laboratories,
Pulau Pinang 11700, Malaysia College of Medicine & Health Sciences,
e-mail: tanml@usm.my; drtanmelan@yahoo.com Hodeidah University, Hodeidah, Yemen
e-mail: ahmd7891@yahoo.com
H.K. Tan, Bachelor of Science (Hons)
Malaysian Institute of Pharmaceuticals & Nutraceuticals, T.S.T. Muhammad, PhD
Ministry of Science, Technology & Innovation (MOSTI), Institute of Marine Biotechnology,
Block 5A, Halaman Bukit Gambir, Minden, Universiti Malaysia Terengganu,
Pulau Pinang 11700, Malaysia Kuala Terengganu 21030, Terengganu, Malaysia
e-mail: hengkean@gmail.com e-mail: sifzizul@umt.edu.my

244 M.L. Tan et al.
autophagy: macroautophagy, microautophagy, including Class III PI3K (the mammalian ortho-
and chaperone-mediated autophagy (CMA), log of vascular protein sorting 34; Vps34), p150
which are classied based on their transport of (the mammalian ortholog of Vps15), Beclin-1
cytoplasmic materials into the lysosome for deg- (the mammalian ortholog of Atg6, also called
radation [3, 4]. Vps30), and Atg14; (3) two ubiquitin-like conju-
Autophagy literally means self-digestion in gation systems, Atg12 and Atg8; and (4) Atg9
Greek [5]. Macroautophagy, usually referred to and its cycling system [14]. The unc-51-like
autophagy, is responsible for the turnover of kinases (ULKs; the mammalian orthologs of
unnecessary or dysfunctional organelles and pro- Atg1), which exist in a large complex with mam-
teins, such as damaged mitochondria [6]. These malian Atg13 (mAtg13), focal adhesion kinase
processes are important to maintain a well- family interacting protein of 200 kDa (FIP200;
controlled balance between anabolism and catab- the mammalian homolog of Atg17), and the
olism to facilitate normal cell growth and recently identied Atg101, plays a crucial role in
development. It is also a survival pathway, autophagy induction [1519]. Phosphorylation of
required during starvation or growth factor depri- Atg13 and FIP200 by ULK1 is an important step
vation as it provides an alternative energy source in the initiation of autophagy, although the exact
[7, 8]. Autophagy process provides catabolic role of phosphorylation in generating autophago-
intermediates for intracellular production of ATP somes is currently unclear.
when energy supplies are limited. It plays an The early stages of the phagophore membrane
essential role during starvation, cellular differen- nucleation are dependent on the Class III PI3K com-
tiation, cell death, cell survival, aging, and tumor plex which consists of the Class III PI3K protein,
prevention [4, 6, 9]. its regulatory protein kinase p150, and Beclin-1
Autophagy is a multistep process character- [20]. Beclin-1 is a 60 kDa tumor suppressor pro-
ized by induction, vesicle nucleation, extension, tein and is identied from a yeast two-hybrid
and completion of an isolation membrane to form screen as a Bcl-2 interacting protein [21]. Recent
an organelle called autophagosome [10]. Briey, studies have demonstrated that several binding
the autophagy process begins with the formation molecules positively regulate Beclin-1 activity
of a pre-autophagosomal structure known as iso- and autophagosome formation and maturation.
lation membrane or phagophore [11]. The isola- Ultraviolet (UV) radiation resistance-associated
tion membrane engulfs and elongates to form the gene (UVRAG), Atg14L, and autophagy/Beclin-1
autophagosome, surrounding the components regulator 1 (Ambra1) associate with Beclin-1 to
destined to be recycled. The autophagosome, activate autophagy [2226].
which is a double membrane-bounded structure, The next stage of phagophore membrane elon-
undergoes maturation and fuses with both endo- gation (expansion and closure of the autophago-
somal and lysosomal vesicles to form autolysosome) requires two ubiquitin-like systems [27].
some [1113]. The sequestered contents are The ubiquitin-like protein Atg12 conjugates with
subsequently degraded by lysosomal hydrolases Atg5 in an Atg7- and Atg10-dependent manner
and are recycled. Based on morphological fea- [1]. The Atg5-Atg12 complex interacts with
tures, the term autophagic cell death has been Atg16 to form a stable and large multimeric com-
described in instances of cell death that are plex called the Atg16L complex, which localizes
accompanied by massive cytoplasmic vacuoliza- on the outer surface of the extending autophago-
tion. The morphology characteristics of cells somal membrane [10]. This complex is important
undergoing autophagic cell death are depicted in in the stimulation and localization of the
Chap. 13 (Fig. 13.2). microtubule-associated protein 1 light chain 3
The core autophagy machinery composes of (LC3) conjugation reactions. LC3 is rst cleaved
four major functional groups: (1) the Atg1- by Atg4 to expose a C-terminal glycine residue
Atg13-Atg17 kinase complex; (2) the Class III required for subsequent activation and conjuga-
phosphoinositide-3-kinase (PI3K) complex I, tion reactions [28]. It is then conjugated to the
14 Autophagy and Necroptosis in Cancer 245
lipid phosphatidylethanolamine (PE), also via cell death to be used based on some biochemi-
Atg7 and E2-like Atg3, and is subsequently cal and functional considerations, before indicat-
recruited to both outer and inner surfaces of the ing that a cell death is mediated by autophagy.
autophagosomal membrane [27, 29]. Actually, Some of the considerations include making sure
two forms of LC3 are produced posttranslation- that the investigated cell death can be suppressed
ally in various cells; the unconjugated form by the inhibition of the autophagic pathway
(LC3-I) is in the cytosol, while the conjugated using chemicals and/or genetic means (e.g., gene
form (LC3-II) targets to the autophagosomal knockout or RNAi silencing of essential autoph-
membrane with the assistance of the Atg16L agy modulators such as AMBRA1, Atg5, Atg12,
complex [29, 30]. The Atg5-Atg12-Atg16 com- or Beclin-1) [37].
plex is recycled, while the LC3 complex stays on One of the most studied and important path-
the membrane until it is degraded by the lyso- ways involved in autophagy regulation is the
some [1]. In mammalian autophagy, LC3-II pro- PI3K-Akt-mTOR signaling pathway. The mam-
tein is used as an index of autophagosome malian target of rapamycin, commonly known
formation or as an autophagosomal marker [31]. as mTOR, is a serine/threonine kinase which
Atg16L complex is a ubiquitin-protein ligase belongs to the family of phosphatidylinositol-
(E3)-like enzyme that functions as a scaffold for 3-kinase-related kinases. It regulates translation
LC3-II lipidation by localizing to the source and cell growth by its ability to phosphorylate
membranes for autophagosome formation [30, both binding protein of eukaryotic translation
32]. The association of LC3-II to the autophago- initiation factor 4E (4E-BP1) and p70 ribosomal
some is crucial for membrane elongation of the S6 kinase (p70S6k). Upon stimulation by a vari-
autophagosome and the nal limitation of the ety of signals including cytokines, growth fac-
membrane to form the vacuoles [1]. These conju- tors, cellular stress such as heat shock, hypoxia,
gation systems are considered to be uniquely and oxidative stress, PI3K is recruited to the inner
important to the autophagosome formation and cell membrane via phosphorylated receptor tyro-
have been identied as possible drug targets in sine kinases and catalyzes the phosphorylation of
cancer [33]. On the other hand, Atg9 provides phosphatidylinositol-3,4-bisphosphate (PIP2) to
lipids to the isolation membrane by cycling phosphatidylinositol-3,4,5-triphosphate (PIP3).
between distinct subcellular compartments. The The recruitment of inactive Akt from the cytosol
cycling of Atg9 requires Atg1/ULK1 and the to the plasma membrane requires that the pleck-
kinase activity of Vps34 [34]. However, the role strin homology (PH) domain of Akt binds to PIP3
of Atg9 is currently not completely understood. synthesized at the plasma membrane by PI3K. Akt
The completed autophagosome mem- is then phosphorylated at Thr 308 by phosphati-
brane subsequently fuses with lysosome via dylinositol-dependent kinase 1 (PDK1) [38, 39].
the actions of the lysosomal proteins including PTEN phosphatase antagonizes PI3K-Akt signal-
the lysosomal-associated membrane protein 1 ing by converting PIP3 back to PIP2 [38].
(LAMP1), LAMP2, member RAS oncogene Upstream PI3K and Akt activation by growth
family (Rab7), and UVRAG [35]. The eventual factors leads to the activation of mTOR and sub-
autolysosome is a single-membrane-bound acidic sequently phosphorylation of downstream sub-
vesicle where the contents are digested and recy- strates. Phosphorylation of p70S6k promotes
cled by lysosomal hydrolases such as cathepsins ribosome biogenesis and increases the capacity
(CTS), and its nutrient and energy are recycled of the translational machinery for protein synthe-
[36]. These single-membrane autolysosomes, sis [40]. Phosphorylation of 4E-BP1 initiates the
lled with degraded cytoplasmic materials, can transcription of a subset of mRNAs important for
be easily observed using transmission electron cell growth and proliferation [4042]. The mTOR
microscopy (TEM) [10]. As a precautionary kinase is a key regulatory component that con-
note, the Nomenclature Committee on Cell Death trols the induction of autophagy [43]. Inhibition
(NCCD) recommends that the term autophagic of mTOR (by nutrient depletion, starvation, or
246 M.L. Tan et al.
rapamycin) leads to cell cycle arrest, inhibition of rapid inactivation of p70S6k and hypophosphor-
cell proliferation, immunosuppression, and ylation of the 4E-BP1 [59].
induction of autophagy. Increased levels of the Studies have shown that mTORC1 controls
mTOR kinase are found to inhibit the autophagy autophagy through the regulation of a protein
process, resulting in an increase in cell growth complex consisting of ULK1, mAtg13, and
and tumor development [13]. Rapamycin, a spe- FIP200 [16, 18, 60]. The ULK complex is directly
cic mTOR inhibitor, complexes with the cyto- controlled by mTOR, leading to maintenance of
solic receptor FK506-binding protein (FKBP12) the mAtg13 hyperphosphorylation state and sup-
and subsequently binds to a distinct region of pression of autophagy induction [61]. A recent
mTOR upstream of the catalytic domain [44]. It study has demonstrated that inhibition of mTOR
induces autophagy and inhibits the proliferation by rapamycin leads to dephosphorylation of
of a variety of cells [45]. ULK1, ULK2, and mAtg13 and activates ULKs
In eukaryotic cells, mTOR exists in two differ- to phosphorylate FIP200. These results suggested
ent complexes: mTORC1, a rapamycin-sensitive that the ULK-Atg13-FIP200 complexes are
complex dened by its interaction with the sup- direct targets of mTOR and important regulators
plementary protein Raptor (regulatory-associated of autophagy in response to mTOR signaling
protein of mTOR), and mTORC2, a rapamycin- [18]. One of the most important proteins involved
insensitive complex dened by its interaction in the regulation of mTORC1 activity is the
with Rictor (rapamycin-insensitive companion of tuberous sclerosis complex (TSC), which is a
mTOR) [4648]. mTORC1 and mTORC2 acces- heterodimer of two proteins, TSC1 (also known
sorial complexes consist of mTOR, mammalian as hamartin) and TSC2 (also known as tuberin)
lethal with SEC13 protein 8 (mLST8) (also [56]. TSC1 and TSC2 function as a GAP
known as GL), and DEP domain containing (GTPase-activating protein) that negatively regu-
mTOR-interacting protein (Deptor) [49]. mLST8 lates a small GTPase called Rheb (Ras homolog
binds to the kinase domain of mTOR and stabi- enriched in brain). TSC1 and TSC2 inhibit
lizes the interaction of Raptor with mTOR in a mTORC1 signaling by transforming Rheb into
rapamycin-sensitive pathway [50]. Raptor is the its inactive GDP-bound state [62, 63].
rst protein shown to bind directly to mTOR that In contrast to mTORC1, relatively little is
is required to mediate mTOR regulation of known about the regulatory pathway inuencing
p70S6k and 4E-BP1 activities [47, 51]. On the mTORC2 (mTOR-Rictor) [64]. mTORC2 con-
other hand, PRAS40 and Deptor play roles as sists of mTOR, mLST8, Rictor, Deptor, mamma-
distinct negative regulators of mTORC1 [52, 53]. lian stress-activated map kinase-interacting protein
In a rapamycin-sensitive mTOR signaling 1 (mSIN1; also known as MAPKAP1), and the
pathway, much of the knowledge about mTORC1 recently identied protein observed with Rictor
function comes from the use of rapamycin, a bac- (PROTOR) [49, 65]. Rictor is dened as a novel
terial macrolide antibiotic [54]. Upon entering mTOR-interacting protein which is Raptor-
the cell, rapamycin binds FKBP12, its intracel- independent [46, 66]. Unlike mTOR-Raptor, the
lular receptor, which subsequently binds to the mTOR-Rictor complex does not bind to FKBP12-
FKBP12-rapamycin-binding domain (FRB) of rapamycin and is insensitive to rapamycin treat-
mTOR, thus inhibiting the mTORC1 functions ment [46, 48]. Therefore, rapamycin treatment
[55, 56]. Rapamycin weakens the interaction does not represent a complete inhibition of mTOR
between mTOR and Raptor [57]. However, the function [67]. mTORC2 stimulates cell signaling
exact mechanism of how rapamycin and several through activation and phosphorylation of the pro-
rapamycin derivatives bind to FKBP12 to inhibit proliferative and pro-survival kinase Akt [68].
mTORC1 signaling is not completely understood Inhibitors of the mTOR kinase domain have been
[58]. Various conditions including starvation or developed to suppress the activity of both mTOR
lack of nutrients such as amino acids and/or glu- complexes (mTORC1 and mTORC2) [69, 70].
cose mimic rapamycin treatment, hence inhibit Figure 14.1 illustrates the simplied autophagy
mTOR function in cultured cells, as indicated by signaling pathways.
P
PIP2 PIP3
LKB1
PTEN
PI3K
P
Akt TSC1
Dual PI3K and AMPK
mTOR TSC2 P
P
inhibitors
P
P
mTOR mTOR Rheb
inhibitors
P
4E-BP1
ULK P
complex p70S6K
Cell growth and

proliferation
Autophagy
Tumor cells with Pro-

defective autophagy autophagics
response
Tumor cells with Autophagy

intact autophagy inhibitors
response
Fig. 14.1 Autophagy signaling pathway and antitumor therapeutic targets
14.2 Autophagy and Cancer hepatic, pancreatic, and breast carcinoma exhibit
low autophagic activity, as compared with nor-
The role of autophagy in cancer is rather per- mal cells from the same origin [25, 72].
plexing. It is widely postulated that the autopha- Autophagic capacity is known to increase during
gic pathway is deregulated in tumor cells. the premalignant stages of pancreatic carcino-
Several proteins and pathways related to autoph- genesis and then decreases during the transition
agy signaling are deregulated during cancer of pancreatic adenoma into adenocarcinoma,
development [25, 71]. Cell lines derived from suggesting that a decreased autophagic activity
248 M.L. Tan et al.
possibly contributing to the malignancy of pan- lar growth continues (typical characteristic of
creatic cancer [73, 74]. A decrease in autophagic tumor cells), there are some exceptional cases.
capacity is also observed during animal experi- For example, a study in human epidermoid lung
mental carcinogenesis, where cells from preneo- carcinoma cells revealed that the autophagic
plastic liver nodules or primary hepatocellular pathway in response to nutrient deprivation is not
carcinomas induced by chemical carcinogens downregulated when compared to their normal
showed a decreased autophagic capacity as com- counterparts [93]. Human colon cancer cells
pared to normal liver cells [74, 75]. In addition, which are able to survive for long period of time
Beclin-1 is found to be mono-allelically deleted in the absence of nutrients have a high rate of
in a high percentage of ovarian, breast, and pros- autophagy activity [94]. Studies in colorectal
tate cancers (based on the 17q21 and gene map- cancer cells revealed that these cancerous cells
ping studies). It has been demonstrated to have a harbor functional autophagic machinery to pro-
direct link between tumorigenesis and the dis- long cell survival during shortages of nutrients
ruption of autophagy [25]. PTEN deletions as [95]. A recent study by Fuji and co-workers has
well as the amplications of both Class III PI3K also showed that strong LC3 expression in the
and Akt are found in several cancers [76, 77]. peripheral area of pancreatic cancer tissue is cor-
The mTOR signaling pathway is constitu- related with poor outcome and short disease-free
tively activated in many tumor types. For exam- period [96]. Activated autophagy observed in
ple, the mTOR pathway is frequently found to be pancreatic cancer cells is thought to be a response
hyperactive in cancers such as breast cancer, sug- to factors in the cancer microenvironment, such
gesting that mTOR is an attractive target for can- as hypoxia and poor nutrient supply.
cer drug development and therapy [7880]. The Autophagy has been identied as the key
mTOR signaling network contains a number of mechanism of cell survival in estrogen receptor-
tumor suppressor genes which includes PTEN, positive (ER+) breast cancer cells undergoing
LKB1 (liver kinase B1), TSC1/2, and a number of treatment with 4-hydroxytamoxifen (4-OHT)
proto-oncogenes such as PI3K, Akt, and eIF4E [97]. Antiestrogen therapy is the standard treat-
genes [81]. Cancer-related changes in pathways ment for ER+ breast cancers which improves
at the downstream of mTOR such as p70S6k and overall survival and provides chemoprevention
eIF4E are reported in breast carcinoma [82, 83]. [98, 99]. Unfortunately, approximately half of the
In addition, malignant cell types undergo autoph- women treated with antiestrogen therapy either
agic cell death when responding to anticancer do not respond or their breast cancer ultimately
agents and traditional herbs, indicating the poten- acquires resistance during treatment [100, 101].
tial utility of autophagic cell death induction in Studies have shown that autophagy activity
cancer therapy [13, 84, 85]. Autophagic cell reduces the efcacy of chemotherapy and tamox-
death characterized by an increase in the number ifen therapy in ER+ breast cancer cells [97, 102,
of autophagic vacuoles in the cytoplasm, fol- 103], supporting the thesis that blocking autoph-
lowed by cell demise, has been observed in vari- agy signaling pathways may provide a new
ous diseases such as Alzheimers disease [86], mechanism of anticancer therapy for resistant
Huntingtons disease [8790], and Parkinsons tumors.
disease [91]. Therefore, manipulation of autoph- In another example, electron microscopy
agy may provide an attractive strategy to increase examination of autophagic vesicles in melanoma
the efcacy of cancer treatments, prevent cancer tumors from 12 patients enrolled in a Phase II
development, and limit tumor progression. clinical trial of temozolomide and sorafenib ther-
However, autophagy is divergent in nature in apy revealed that autophagic index (mean num-
both tumor suppression and tumor progression ber of autophagic vacuoles per cell) is signicantly
[92]. Although the argument supports that if cells higher in patients who derived little or no clinical
cannot activate autophagy, protein synthesis will benet from the combination of temozolomide
predominate over protein degradation, and cellu- and sorafenib treatment. Patients who had stable
disease or responded to therapy had low levels of metastatic adrenocortical carcinoma, the same
autophagy in their tumors. These ndings vali- combination therapy results in 40 % of patients
date the emerging preclinical evidence that achieving prolonged stable disease [109].
autophagy plays a critical role in resistance to However, Phase I and II clinical trials with temsi-
chemotherapy. Results of this study indicate that rolimus and sorafenib carried out in patients with
pretreatment levels of autophagy can predict metastatic melanoma did not produce sufcient
resistance to therapy. Patients with aggressive activity to justify further use [110, 111]. In Phase
melanoma are more likely to have higher levels II trial for metastatic colorectal cancer, temsiroli-
of autophagy in their tumor and therefore may mus has limited efcacy in chemotherapy-
respond to autophagy inhibition as a therapeutic resistant KRAS-mutant disease [112].
strategy [104]. Hence, the divergent nature of Everolimus is another rapamycin analog
autophagy has resulted in strategies for using which is already approved as an anticancer agent.
pro-autophagics or autophagy inhibitors depend- Everolimus (RAD001; rapamycin derivative
ing on the inherent nature of the cancer involved. 001) is a hydroxyethyl ether derivative of rapamy-
cin that has been developed for oral administra-
tion [113]. This drug was approved by FDA for
14.3 Autophagy Signaling use in a variety of cancers, including advanced
Pathways and Therapeutic RCC, advanced pancreatic neuroendocrine
Strategies in Cancer tumors, renal angiomyolipoma, and HER2-
negative breast cancer. Everolimus, a derivative
14.3.1 mTOR Signaling Pathway of rapamycin, is structurally similar to temsiroli-
Inhibitors mus and binds to an intracellular protein, FKBP-
12, forming a complex that inhibits the mTOR
Rapamycin, as the rst prototype of an mTOR kinase. A recent Phase II trial showed that evero-
inhibitor, has a strong immunosuppressive prop- limus demonstrates efcacy and acceptable toler-
erty but poor aqueous solubility. Therefore, its ability in patients with advanced endometrial
utilization at doses capable of exerting anticancer cancer [114]. A randomized Phase II study indi-
effects is rather limited [105]. Nevertheless, trials cates that combination therapy of everolimus
utilizing rapamycin as a single-agent or combina- with tamoxifen increases the clinical benet rate
tion therapy are still being carried out. In a recent (dened as the percentage of all patients with
Phase I study of rapamycin and sunitinib in complete or partial response or stable disease at
patients with advanced non-small-cell lung can- 6 months), time to progression (TTP), and overall
cer (NSCLC), combination of rapamycin and survival compared with tamoxifen in postmeno-
sunitinib is reported to be well tolerated and has pausal women with aromatase inhibitor-resistant
warranted further investigation in Phase II trials metastatic breast cancer [115]. In patients with
[106]. advanced NSCLC, Phase I study showed that
Various rapamycin analogs have since been combination therapy with everolimus and erlo-
developed. Temsirolimus (CCI-779) is the rst tinib provides acceptable tolerability and disease
mTOR inhibitor approved by the US FDA for control [116].
cancer treatment and is considered a rst-line Ridaforolimus (deforolimus or AP23573) has
treatment for patients with advanced renal cell been tested in Phase I and II clinical trials and has
carcinoma (RCC) with poor prognostic features shown promising results in several tumor types
[107]. A great deal of clinical trials was carried including sarcoma [105, 117]. In a Phase II clini-
out for this drug, mainly as combination therapy cal study of ridaforolimus in 216 patients with
with other chemotherapy drugs. Generally, clini- advanced bone and soft tissue sarcomas, the over-
cal activity is observed in patients with bone and all clinical benet response (CBR) was 28.8 %
soft tissue sarcoma given a combination of temsi- with a median progression-free survival of
rolimus and cixutumumab [108]; in patients with 15.3 weeks. Interestingly, the archival tumor
250 M.L. Tan et al.
protein markers analyzed were not correlated with 14.3.2 Pro-autophagics

CBR [118]. Ridaforolimus receives fast track and
orphan drug status from the US FDA, as well as Temozolomide is the rst pro-autophagic cyto-
orphan status from the European Medicines toxic drug used to overcome apoptosis resistance
Agency. The FDA is currently reviewing its regis- in cancer cells and was approved for use in glio-
tration for maintenance therapy in patients with blastoma multiforme (GBM) [125]. It demon-
sarcoma [119]. In another Phase II trial study on strates therapeutic benets in patients with
the efcacy and safety of single-agent ridaforoli- glioblastoma and has been evaluated for several
mus in patients with relapsed or refractory hema- types of apoptosis-resistant cancers [126].
tologic malignancies, results were unremarkable. Temozolomide is a prodrug, is a monofunctional
Of the 52 patients evaluated, partial responses alkylating agent, and is chemically related to
were noted in ve subjects, while hematologic dacarbazine. It is the 3-methyl derivative of the
improvement/stable disease was observed in less experimental anticancer drug, mitozolomide. The
than half of the patients [120]. ability of temozolomide in inducing autophagic
PI3K-Akt-mTOR pathway is often constitu- cell death is reported in various preclinical stud-
tively activated in human tumor cells and thus ies [127130]. In addition, temozolomide also
has been considered as a promising drug target. demonstrates pro-apoptotic activities in malig-
NVP-BEZ235 is a potent imidazo(4,5-c)quino- nant melanoma cells [131]. In a systematic
line derivative that inhibits PI3K and mTOR assessment of three randomized controlled trials
kinase activities by binding to the ATP-binding addressing whether temozolomide holds any
cleft of these enzymes and induces G1 arrest advantage over conventional therapy for high-
[121]. Preclinical studies have suggested that grade gliomas, it was shown that temozolomide
NVP-BEZ235 is a potent dual PI3K/mTOR is an effective therapy for GBM. The drug pro-
modulator with favorable pharmaceutical prop- longs survival, delays disease progression, and
erties. For example, it inhibits VEGF-induced has a low incidence of early adverse events [132].
HUVEC cell proliferation and survival in vitro Similar outcomes were observed in a Phase II
and VEGF-induced angiogenesis in vivo [122]. study involving erlotinib in combination with
The compound also inhibits microvessel perme- radiation therapy and temozolomide to treat
ability in BN472 mammary carcinoma grown GBM and gliosarcoma. Patients treated with the
orthotopically in syngeneic rats, suggesting that combination of erlotinib and temozolomide dur-
this compound is potentially anti-angiogenic ing and following radiotherapy have better sur-
[122]. Deregulated angiogenesis and high tumor vival than historical controls [133].
vasculature permeability are known VEGF- In a recent Phase II trial, patients with unre-
mediated characteristics of human tumors. In sectable or multifocal glioblastoma, an upfront
addition, NVP-BEZ235 is found to produce sig- regimen of temozolomide and bevacizumab was
nicant tumor growth inhibition in xenograft well tolerated and provided a signicant level of
models of pancreatic cancers and breast cancer disease stabilization [134]. In patients with recur-
cells [123, 124]. Phase I/II clinical trials of NVP- rent glioblastoma, either used as a single agent in
BEZ235 in patients with advanced solid malig- a dose-intense schedule or in combination with
nancies and breast cancer were completed, but other chemotherapeutic agents, temozolomide is
reports on the safety and efcacy of this drug proven to be well tolerated and safe [135137].
have yet to be published. Other ongoing trials In pediatric patients with recurrent solid tumors
either as a single-agent or combination therapy or brain tumors, low-dose temozolomide
with other chemotherapy drugs in breast cancer, improves tolerability and is convenient as outpa-
prostate cancer, leukemia, and other advanced tient therapy [138]. Temozolomide in combina-
solid tumors are listed in the NIH ClinicalTrials. tion with vorinostat is also well tolerated in
gov website. children with recurrent central nervous system
(CNS) malignancies with myelosuppression In a Phase I clinical study, ATO given con-
[139]. However, good therapeutic effects are not comitantly with radiation therapy in children
observed in patients with NSCLC. In a current with newly diagnosed anaplastic astrocytoma,
efcacy and safety study of temozolomide in a glioblastoma, or diffuse intrinsic pontine glioma
total of 31 pretreated patients with NSCLC, only was safe and well tolerated by patients through-
two patients achieved partial response, and three out the entire dose escalation [147]. ATO is also
had stable disease [140]. Moreover, the research- reported to be well tolerated when used in combi-
ers pointed out that prolonged low daily doses of nation with temozolomide and radiotherapy in
temozolomide produces minimal activity in malignant gliomas [148] or when used in combi-
patients with advanced NSCLC. Hence, more nation with bortezomib, high-dose melphalan,
Phase II and III studies to characterize the ef- and ascorbic acid in multiple myeloma (MM)
cacy of this drug in various cancers are denitely patients [149]. A Phase II study to evaluate the
warranted. efcacy and feasibility of a sequential treatment
Arsenic trioxide (ATO) has recently been consisting of induction and consolidation with
introduced as part of a regimen in the therapy ATO followed by autologous hematopoietic cell
and management of acute promyelocytic leuke- transplantation for relapsed APL revealed that
mia (APL) [141]. It is now considered to be the ATO demonstrates outstanding efcacy. Of the
most biologically active single drug in APL by 23 patients who underwent autologous hemato-
a panel of international leukemia experts for the poietic cell transplantation with PML-RAR-
European LeukemiaNet. The combination of negative PBSC graft, posttransplant relapse
ATO and all-trans retinoic acid (ATRA) holds the occurred only in three patients, and there was no
promise to replace conventional approaches for transplant-related mortality. The 5-year event-
most, if not all, patients in the very near future free and overall survival rates were 65 % and
[142]. ATO is known to induce both autophagy 77 %, respectively [150]. Phase I/II/III clinical
and apoptosis depending on cell types; there- trials using ATO mostly as combination therapy
fore, its role as an autophagy inducer remains with other chemotherapy drugs are currently
largely uncertain. In some preclinical trials, ongoing for CML and APL.
ATO has induced the autophagic pathway in
ovarian carcinoma cells and has synergized with
everolimus to induce the cytotoxicity of ovar- 14.3.3 Autophagy Inhibitors
ian cancer cells. The enhanced cytotoxicity is
accompanied by the upregulation of Atg5-Atg12 The knowledge that autophagy plays a role as a
conjugate and LC3-II, a hallmark of autophagy cell survival pathway in response to therapeutic
[143]. In another recent study, ATO induced and cellular stresses in the tumor microenviron-
the autophagic degradation of the BCR-ABL1 ment (which is highly acidic and hypoxic) implies
oncoprotein, known to cause chronic myeloid that autophagy may work in favor of cancer cells.
leukemia (CML) and Ph+ acute lymphoblastic Therefore, inhibition of protective autophagy may
leukemia (ALL) [144]. However, in other stud- break the resistance mechanism for survival of the
ies, in the presence or absence of ionizing radia- harsh tumor microenvironment and lead to cell
tion and in specic low concentrations, ATO death [151]. Since autophagy activities are known
induced apoptosis in MTLn3 cells, known to be to differ according to stages of cancer, modulation
highly malignant and resistant to both radio- and of autophagy is postulated to enhance the efcacy
chemotherapy [145]. Interestingly, in human of anticancer therapy. In a preclinical study,
glioma cells, ATO induces both autophagy and effects of imatinib, with or without different types
apoptosis in vitro and in vivo, mediated by the of autophagy inhibitors, on human malignant
inhibition of PI3K/Akt and activation of MAPK glioma cells were carried out [152]. It is demon-
signaling pathway [146]. strated that suppression of imatinib-induced
252 M.L. Tan et al.
autophagy by 3-methyladenine (3-MA) or siRNA of cytoplasmic content [160, 161]. DNA in

against Atg5 (which inhibits autophagy at an necrotic cells is usually degraded randomly, giv-
early stage) attenuates the imatinib-induced cyto- ing rise to a smear of DNA [162]. The cellular
toxicity. On the other hand, inhibition of autoph- content leaks into the extracellular environment
agy at a late stage by balomycin A1 or RTA 203 and is usually associated with inammation [163,
enhanced imatinib-induced cytotoxicity through 164]. Release of cytokines and other factors from
the induction of apoptosis [152]. Thus, the authors the necrotic cells and the secretion of pro-inam-
have even suggested that therapeutic efciency of matory cytokines from activated macrophages
imatinib for malignant glioma may be augmented triggered by necrotic cells are thought to be
by inhibition of autophagy at a late stage, which responsible for the inammatory response [165,
could help sensitize tumor cells to anticancer 166]. Interestingly, in the past decade, studies
therapy [152]. have revealed necrosis as a form of regulated cell
The current autophagy inhibitors used in trials death, executed through a mechanism termed
for human cancer are chloroquine (CQ) and necroptosis or programmed necrosis [167, 168].
hydroxychloroquine. Both drugs are widely used Necroptosis can be stimulated via a class of death
as antimalarials and have recently received atten- receptors including TNFR1, TNFR2, TRAIL-R,
tion as potential chemosensitizers in treating and Fas. Upon binding to their agonists, these
tumors when used in combination with cytotoxic death receptors can induce cells toward either
chemotherapeutic agents [153155]. CQ inhibits survival or death. Depending on the circum-
lysosomal acidication and therefore prevents stances, the induction of cell death may be either
autophagy by blocking autophagosome fusion apoptosis or necroptosis. However, the exact
and degradation [154, 156, 157]. A number of mechanisms that dictate the cellular decision to
clinical trials are now revealing the promising undergo apoptosis or necroptosis remained
role of CQ, an autophagy inhibitor, as a novel largely unknown.
antitumor drug. For example, adding chloroquine TNF- can be massively generated during
to conventional treatment for GBM improves hyperinammatory shock, accumulated upon
midterm survival of patients [158]. In a Phase I infection or produced primarily by macrophages.
study involving patients with advanced NSCLC, It induces apoptosis in many cells, while trigger-
hydroxychloroquine, with or without erlotinib, ing necrosis in some [161, 169]. In necroptosis,
was safe and well tolerated, although the overall TNF- binds to the extracellular portion of the
response rate was as low as 5 % [159]. Other tri- death receptors and triggers downstream signal-
als on metastatic breast cancer, pancreatic cancer, ing pathway by forming complex I with proteins
RCC, NSCLC, and MM are currently ongoing. containing a death domain, such as TNF-receptor-
Table 14.1 summarizes the various drugs target- associated death domain (TRADD), receptor-
ing the autophagy pathways and clinical trial interacting protein kinase 1 (RIP1), and several
stages based on published reports as well as E3 ubiquitin ligases, such as TNF-receptor-
ongoing trials listed in the NIH ClinicalTrials. associated factor 2/5 (TRAF2/5), cIAP-1, and
gov website. cIAP-2. Ubiquitination of these proteins is
important for the regulation of the activity of
complex I and impacts the outcome of the cell
14.4 Mechanisms of Necroptosis survival [170]. The ubiquitination and phosphor-
ylation states of RIP1 determine whether it func-
Necrosis is initially known as a passive and tions as a pro-survival molecule or a kinase
uncontrolled death process usually caused by promoting cell death. RIP1 is a member of the
physical or chemical insult. An irreversible drop RIP family exhibiting a homologous N-terminal
in intracellular ATP and energy insufciency lead kinase domain and has recently emerged as an
to the morphological characteristics of organelle essential mediator of cellular stress and cell
swelling, plasma membrane rupture, and spillage death. RIP1 is polyubiquitinated by TRAF2/5,
Table 14.1 Current therapeutic targets in the autophagy signaling pathways and clinical trial stages
Therapeutic Clinical trial stages (published reports)/
Pathway targets Current drugs type of cancer Combined with References
mTOR mTOR Rapamycin Phase I: advanced NSCLC Sunitinib [106]
signaling (sirolimus) Ongoing Phase I: NSCLC BIBW 2992 http://clinicaltrials.gov/show/NCT00993499
pathway Ongoing Phase I: solid tumor http://clinicaltrials.gov/show/NCT01331135
Ongoing Phase II: hepatocellular http://clinicaltrials.gov/show/NCT01374750
carcinoma
Temsirolimus Approved by FDA for advanced RCC The US Food and Drug Administrationa
(CCI-779) (2007)
Phase I: melanoma Sorafenib [111]
Phase I: advanced adrenocortical Cixutumumab [109]
carcinoma
Phase II: melanoma Sorafenib [110]
14 Autophagy and Necroptosis in Cancer
Phase II: bone and soft tissue sarcoma Cixutumumab [108]

Phase II: colorectal cancer Irinotecan [112]
Ongoing Phase I: advanced cancer Vemurafenib http://clinicaltrials.gov/show/NCT01596140
Ongoing Phase I: solid tumor Erlotinib http://clinicaltrials.gov/show/NCT00770263
Ongoing Phase I: advanced cancer Bevacizumab and valproic acid http://clinicaltrials.gov/show/NCT01552434
Ongoing Phase I: Hodgkin lymphoma http://clinicaltrials.gov/show/NCT00838955
Ongoing Phase II: diffuse large B-cell Rituximab, cytarabine, cisplatin, http://clinicaltrials.gov/show/NCT01653067
lymphoma and dexamethasone
Ongoing Phase II: head and neck cancer http://clinicaltrials.gov/show/NCT01172769
Everolimus Approved by FDA for advanced RCC The US Food and Drug Administrationb
(RAD001) (2009), advanced pancreatic
neuroendocrine tumor (2011), renal
angiomyolipoma (2012), and hormone
receptor-positive, HER2-negative breast
cancer (2012)
Phase I: advanced NSCLC Erlotinib [116]
Phase II: advanced breast cancer Tamoxifen [115]
Phase II: advanced endometrial cancer [114]
Ongoing Phase II: advanced breast cancer Exemestane http://clinicaltrials.gov/show/NCT01783444
Ridaforolimus Phase II: hematologic malignancies [120]
(deforolimus; Phase II: advanced bone and soft tissue [118]
AP23573) sarcoma
253
(continued)
254
Therapeutic Clinical trial stages (published reports)/

Pathway targets Current drugs type of cancer Combined with References
PI3K/mTOR NVP-BEZ235 Ongoing Phase I: advanced solid tumor, Everolimus http://clinicaltrials.gov/show/NCT01482156
(BEZ235) breast cancer, or renal cell carcinoma
Ongoing Phase I: advanced solid tumor http://clinicaltrials.gov/show/NCT01343498
Ongoing Phase I: acute leukemia http://clinicaltrials.gov/show/NCT01756118
Ongoing Phase I: breast cancer Capecitabine http://clinicaltrials.gov/show/NCT01300962
Ongoing Phase I: advanced solid tumor http://clinicaltrials.gov/show/NCT01195376
Ongoing Phase Ib: castration-resistant Abiraterone acetate http://clinicaltrials.gov/show/NCT01634061
prostate cancer
Ongoing Phase I/II: castration-resistant Abiraterone acetate and http://clinicaltrials.gov/show/NCT01717898
prostate cancer prednisone
Ongoing Phase I/II: advanced solid tumor Everolimus http://clinicaltrials.gov/show/NCT01508104
Ongoing Phase Ib/II: breast cancer Paclitaxel http://clinicaltrials.gov/show/NCT01495247
Ongoing Phase II: advanced pancreatic Everolimus http://clinicaltrials.gov/show/NCT01628913
neuroendocrine tumor
Ongoing Phase II: perivascular epithelioid http://clinicaltrials.gov/show/NCT01690871
cell tumor
Ongoing Phase II: advanced pancreatic http://clinicaltrials.gov/show/NCT01658436
neuroendocrine tumor
Pro- Temozolomide Approved by FDA for GBM (2005) Radiotherapy [125]
autophagics Phase I: glioblastoma Tipifarnib and radiotherapy [137]
Phase I: primary brain or spinal cord Vorinostat [139]
tumor
Phase I: solid tumor or brain tumor Bevacizumab, vincristine, and [138]
irinotecan
Phase I/Ib: glioblastoma Lonafarnib [136]
Phase II: advanced NSCLC [140]
Phase II: GBM or gliosarcoma Erlotinib and radiotherapy [133]
Phase II: glioblastoma Bevacizumab [134]
Phase II: glioblastoma [135]
Ongoing Phase I: glioblastoma BKM120 http://clinicaltrials.gov/show/NCT01473901
Ongoing Phase I: acute leukemia Veliparib http://clinicaltrials.gov/show/NCT01139970
Ongoing Phase I/II: melanoma Doxycycline and ipilimumab http://clinicaltrials.gov/show/NCT01590082
Ongoing Phase I/II: melanoma Decitabine and panobinostat http://clinicaltrials.gov/show/NCT00925132
M.L. Tan et al.
Ongoing Phase II: hepatocellular Veliparib http://clinicaltrials.gov/show/NCT01205828
carcinoma
Ongoing Phase II: neuroblastoma Bevacizumab and irinotecan http://clinicaltrials.gov/show/NCT01114555
Ongoing Phase II: breast cancer Veliparib http://clinicaltrials.gov/show/NCT01506609
Ongoing Phase II: small cell lung cancer Veliparib http://clinicaltrials.gov/show/NCT01638546
Ongoing Phase III: glioblastoma Lomustine http://clinicaltrials.gov/show/NCT01149109
Ongoing Phase III: high-grade glioma Interferon- http://clinicaltrials.gov/show/NCT01765088
Arsenic trioxide Phase I: malignant glioma Temozolomide and radiotherapy [148]
Phase I: inltrating astrocytomas of Radiotherapy [147]
childhood
Phase II: multiple myeloma Bortezomib, melphalan, and [149]
ascorbic acid
Phase II: APL [150]
14 Autophagy and Necroptosis in Cancer
Ongoing Phase I: CML Tyrosine kinase inhibitors http://clinicaltrials.gov/show/NCT01397734

Ongoing Phase II: small cell lung cancer http://clinicaltrials.gov/show/NCT01470248
Ongoing Phase II: APL Tretinoin http://clinicaltrials.gov/show/NCT01404949
Ongoing Phase II: APL Gemtuzumab ozogamicin and http://clinicaltrials.gov/show/NCT01409161
ATRA
Ongoing Phase III: APL ATRA http://clinicaltrials.gov/show/NCT00378365
Ongoing Phase IV: APL http://clinicaltrials.gov/show/NCT00504764
Autophagy Chloroquine Ongoing Phase I: pancreatic cancer Gemcitabine http://clinicaltrials.gov/show/NCT01777477
inhibitors Ongoing Phase I/II: ductal carcinoma in http://clinicaltrials.gov/show/NCT01023477
situ
Ongoing Phase II: multiple myeloma Bortezomib and http://clinicaltrials.gov/show/NCT01438177
cyclophosphamide
Ongoing Phase II: breast cancer Taxane or taxane-like drugs http://clinicaltrials.gov/show/NCT01446016
Hydroxychloroquine Phase I: advanced NSCLC Erlotinib [159]
Ongoing Phase I/II: RCC Everolimus http://clinicaltrials.gov/show/NCT01510119
Ongoing Phase I/II: NSCLC Getinib http://clinicaltrials.gov/show/NCT00809237
Ongoing Phase I/II: pancreatic cancer Gemcitabine http://clinicaltrials.gov/show/NCT01506973
Ongoing Phase I/II: colorectal cancer FOLFOX and bevacizumab http://clinicaltrials.gov/show/NCT01206530
a
http://www.fda.gov/AboutFDA/CentersOfces/OfceofMedicalProductsandTobacco/CDER/ucm129247.htm
b
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm317385.htm
255
256 M.L. Tan et al.
cIAP-1, and cIAP-2 at the 63rd position of lysine Cleavage of RIP3 by caspase-8 induces caspase-
(K63) [171, 172]. K63-linked ubiquitination dependent apoptosis [181]. However, if the apop-
of RIP1 by cIAP-1/cIAP-2 is known to inhibit totic machinery is decient or when the apoptosis
TNF--induced apoptosis [173, 174]. pathway is blocked by pan-caspase inhibitors
Complex I is crucial for activating the NF-B such as Z-VAD-FMK, or caspase-specic inhibi-
and mitogen-activated protein kinase (MAPK) tors such as cytokine response modier A (CrmA)
pathways. cIAPs direct the formation of polyu- or in cells decient in FADD or caspase-8, trig-
biquitin chains on RIP1, allowing it to interact gering TNFR1 results in necrosis [182186]. This
with TGF (transforming growth factor)-- process involves the formation of complex IIb,
activated kinase 1/TAK-1-binding protein 2/3 consisting of mainly RIP1 and RIP3. It appears
(TAK1/TAB2/3) complex. TAK1 activates the that FLICE-inhibitory protein (FLIP), together
IB kinases (IKK) complex and, in turn, phos- with caspase-8, is recruited to FADD and the for-
phorylates IB. When IB is degraded by the mation of this complex is dominant for inhibiting
proteasome, it allows NF-B to translocate to the apoptosis [187]. Cellular FLICE-inhibitory pro-
nucleus and activate its target genes in a pro-sur- tein (c-FLIP) is known as a crucial inhibitor of
vival manner. Inhibitors of NF-B are known to death receptor-mediated apoptosis by interfering
facilitate TNF--induced necrotic cell death, sug- with caspase-8 activation at the death-inducing
gesting that NF-B suppresses the necrotic cell signaling complex (DISC) signaling [188]. Due
death pathway [175]. On the other hand, deubiq- to its structural similarity to caspase-8 and cas-
uitination of RIP1 could inhibit NF-B pathway, pase-10, c-FLIP can bind to FADD and inhibit
leading to cell death pathways. Cylindromatosis complete caspase-8 processing and activation
(CYLD) blocks the activation of NF-B by cleav- [189]. However, the involvement of FADD, cas-
ing K63-linked polyubiquitin chains, and its deu- pase-8, and FLIP in complex IIb remains unclear.
biquitinating activity on RIP1 facilitates the RIP3 has an N-terminal kinase domain and
direct interaction of RIP1 with caspase-8 and ini- a C-terminus lacking a death domain or CARD
tiation of cell death [176, 177]. Knockdown of motif. RIP3 binds RIP1 through this unique
CYLD inhibits TNF--induced necroptosis, C-terminal segment to inhibit RIP1- and TNF-
which indicates that the deubiquitination of RIP1 receptor-1-mediated NF-B activation [190].
is an important step in TNF--induced necropto- However, the interaction between RIP1 and RIP3
sis [178]. CYLD also interacts directly with via the RIP homotypic interaction motif (RHIM)
TRAF2, an adaptor molecule involved in signal- domain is required for necroptosis [191]. RHIMs
ing of the TNF/nerve growth factor family recep- of RIP1 and RIP3 mediate the assembly of het-
tors. TRAF2, an E3 ligase, has been demonstrated erodimeric functional amyloid signaling com-
to be essential for TNF--induced necroptosis, as plex which is ultrastable [192]. Mutations in the
TRAF2/cells are resistant to TNF--induced RHIMs of RIP1 and RIP3 which render them
necroptosis [175]. defective in interactions compromise kinase acti-
When RIP1 is deubiquitilitized by CYLD, vation and necroptosis in vivo, indicating the cru-
RIP1 can dissociate from complex I and is released cial role of RHIM in necroptosis [192]. Mutations
into the cytoplasm, forming complex II with of RHIMs in RIP1 or RIP3 block the formation
FADD, RIP3, and caspase-8. If the conditions are of necrosomes and protect cells from necroptosis
apoptotic competent, TNF- stimulation induces [190]. RIP3 acts upstream to phosphorylate RIP1,
the sequential protein complexes, complex I and which in turn mediates downstream RIP3 phos-
complex IIa, leading to the activation of NF-B phorylation. Phosphorylation of RIP3 is essential
and apoptosis, respectively [168, 179]. However, in necroptosis, but the exact mechanism remains
proteolytical cleavage of RIP1 by caspase-8 dur- unclear [191]. Both RIP3 and the kinase activity
ing TNF-induced apoptosis abolishes NF-B of RIP1 are essential for stable formation of the
activation and enhances pro-apoptotic signaling RIP1-RIP3 pro-necrotic complex, which criti-
through the TRADD-FADD interaction [180]. cally controls downstream ROS production [191].
RIP3 is essential in necroptosis induced by vari- substrate. GLUD1 is a mitochondrial matrix

ous stimuli, and RIP3 knockdown leads to a enzyme that converts Glu to -ketoglutarate.
notable inhibition of necroptosis [191, 193]. Cells GLUL and GLUD1 are essential for the use of
with low levels of RIP3 expression are resistant amino acid Glu or Gln as substrates for adenos-
to necroptosis, but transfection of these cells with ine triphosphate (ATP) production by means of
the RIP3 gene enables them to undergo necropto- oxidative phosphorylation [197]. Taken together,
sis when the apoptotic pathway is blocked, clearly these enzymes increase substrates for oxidative
highlighting RIP3 as an essential mediator in phosphorylation, which is a major source of ROS
TNF--induced necroptosis [194]. In addition to in the cell. RIP3-decient cells have reduced
TNF-, IFN- also induces an NF-B-dependent ROS production downstream of TNF- signal-
transcriptional response that is cytoprotective. ing [191]. Zhang and co-workers postulated that
However, in mammalian cells decient in NF-B RIP3 activation of all these enzymes results in
signaling, IFN- promotes mitochondrial ROS an increased energy metabolism and subsequent
accumulation, loss of mitochondrial membrane ROS production [197].
potential, and necroptosis [195]. The necroptosis Nicotinamide adenine dinucleotide phosphate
signaling pathway is illustrated in Fig. 14.2. oxidases (NADPH) are a family of enzymes spe-
Necroptosis shows identical subcellular events cically important in ROS production and have
with necrosis and secondary necrosis. The cellu- been implicated in TNF--induced necroptosis.
lar disintegration phases are characterized by For example, TNF treatment induces the forma-
lysosomal membrane permeabilization, mito- tion of a signaling complex containing TRADD,
chondrial hyperpolarization, oxidative burst, and RIP1, Nox1, and the small GTPase Rac1. RIP1 is
eventually plasma membrane permeabilization; shown to be essential for Nox1 recruitment, and
however, the kinetics and timing may be different activation of Nox1 is implicated in ROS produc-
[196]. A number of events have been implicated tion [198]. Other NADPH oxidases, such as Nox1,
and proposed to contribute to the downstream Nox2, Nox3, and Nox4, are also shown to be
events in necroptosis. One important downstream upregulated in the presence of TNF- [198200].
event is the production of ROS which acts as an In addition, riboavin kinase (RFK), a TNFR1-
executioner of necroptosis [168]. The ROS is binding protein, functionally couples TNFR1 to
implicated to play an important role in necropto- NADPH oxidase. RFK binds to both the TNFR1
sis; in addition, RIP, TRAF2, and FADD are cru- death domain and p22phox, the common subunit
cial in mediating ROS accumulation in of NADPH oxidase isoforms and triggers TNF-
TNF-induced necroptotic cell death [175]. This induced ROS production [201]. Both RFK and
was based on the observation that in TNF-induced the NADPH oxidases are found to be crucial for
necroptosis, the cellular ROS level was signi- downstream ROS production [198, 201].
cantly elevated in wild type, but not in RIP(/), ROS are thought to act by oxidizing MAP
TRAF2(/), and FADD(/) cells [175]. kinase phosphatases (MKPs) whose normal func-
Interestingly, RIP3 has been reported to interact tion is to downregulate the c-Jun N-terminal
with several metabolic enzymes including glyco- kinase (JNK) signaling pathway [202]. Sustained
gen phosphorylase (PYGL), glutamate-ammonia JNK activation is required for Cyt c release and
ligase (GLUL), and glutamate dehydrogenase 1 caspase-3 cleavage in apoptosis as well as necrop-
(GLUD1) [197]. PYGL plays a key role in using tosis [202]. In necroptosis, JNK is activated in the
reserved glycogen as an energy source and cata- downstream of RIP1 and TRAF2 [203, 204]. ROS
lyzes the rate-limiting step in the degradation of and JNK appear to form a loop to enhance necrop-
glycogen by releasing glucose-1-phosphate. On tosis as JNK also affects the mitochondrial func-
the other hand, GLUL is a cytosolic enzyme cata- tion and produces ROS [205]. JNK activation is
lyzing the condensation of glutamate (Glu) and required for mitochondrial depolarization, AIF
ammonia to form glutamine (Gln). Gln transfers translocation, and subsequent cell death in PARP-
into the mitochondria to function as an energy 1-hyperactivated cells [204].
258 M.L. Tan et al.
IFN TNF
Complex I
TRADD
TRAF RIP1 CYLD
B2/3
-TA cIAP-1/2
K1
TA
IKK +Ub
+Ub
NF-B
activation
Complex IIb Complex IIa

Caspase-8-FLIP Caspase-8
RIP3 RIP3
RIP1
RIP1 RIP1
FADD FADD
Z-VAD-FMK,
CrmA,
c-FLIP
Ca2+
Calpains
MPT
Apoptosis
Mitochondrial LMP
ATP synthesis
arrest Generation of
ROS
AIF
DAMPs and inflammation
DNA Necroptosis
fragmentation
Fig. 14.2 Necroptosis signaling pathways
Activation of phospholipase A2 (PLA2) and arachidonate-containing phospholipids and facil-

lipoxygenase pathways are also known to con- itates the release of arachidonic acid. Arachidonic
tribute to the TNF--induced necrotic death acid is the main substrate for lipoxygenase, which
[206]. Cytosolic PLA2 (cPLA2), a subfamily of further catalyzes the conversion of the fatty acids
PLA2, is an intracellular enzyme that hydrolyzes into hydroperoxides [163]. Overexpression of
cPLA2 sensitizes TNF--resistant cells to TNF- inducers of MPTP opening [218220]. The
-induced necrosis, emphasizing the role of induction of MPT, which increases mitochon-
cPLA2 in necroptosis [207, 208]. Lipid peroxida- drial membrane permeability, causes the mito-
tion leading to disruption of organelle and plasma chondria to become further depolarized, resulting
membranes are key features of necrosis. in the abolishment of m as well as allowing
The mitochondrion has been implicated down- the release of ROS and other molecules such as
stream of RIP1. Mitochondrial synthesis of ATP AIF and necrotic danger-associated molecular
requires ADP transport from cytosol into mito- patterns (DAMPs) into the cytosol.
chondria by the inner mitochondrial membrane AIF is a FAD-dependent oxidoreductase that
ADP/ATP carrier adenine nucleotide translocase has a vital role in oxidative phosphorylation [221].
(ANT) [209]. ADP/ATP exchange depends on After a caspase-independent cell death insult,
transition between two conformational states of AIF is cleaved by calpains and/or cathepsins to
ANT. In the cytosolic state (c-state), the hydro- yield truncated AIF (tAIF), the pro-apoptotic AIF
philic loop of the ANT nucleotide-binding site form (~57 kDa) [222, 223]. The tAIF relocates
faces the cytosol, while in the matrix state from the mitochondria to the cytosol and nucleus,
(m-state), this binding site faces the matrix [209, where it associates with histone H2AX in the
210]. The interaction of ANT with cyclophilin D nucleus, through its C-terminal proline-rich-
(CYPD) and voltage-dependent anion channel binding domain (PBD, residues 543559). This
(VDAC) is important in regulating the mitochon- interaction generates an active DNA-degrading
drial permeability transition pore (MPTP); in complex with cyclophilin A, leading to chro-
addition, CYPD is an important regulator of matin condensation and DNA fragmentation, as
MPTP [211]. Z-VAD-FMK is found to block the observed in necroptotic cells [224].
ability of ANT to transport cytoplasmic ADP, Interestingly, necroptosis induced by high
thereby inducing a massive ATP depletion in the doses of the alkylating DNA-damaging agent
mitochondria [212]. The inhibition of ADP/ATP N-methyl-N-nitro-N-nitrosoguanidine (MNNG)
exchange coincides with the loss of interaction is found to be regulated by the kinase RIP1 and
between ANT and CYPD as well as with the executed by the activation of PARP-1, Ca2+-
inability of ANT to adopt the cytosolic confor- dependent calpain Cys proteases, and the pro-
mational state, which prevents Cyt c release and apoptotic Bcl-2 member Bax [225]. MNNG
subsequently necroptosis [212]. treatment induces a PARP-1 hyperactivity that
The release of cytosolic Ca2+ and overactiva- leads to calpain activation. Calpains generate
tion of calpains are also thought to play important tBid, which redistributes from the cytosol to
roles in necroptosis. Yamashima and co-workers mitochondria, where it regulates Bax activation.
postulated that excessive Ca2+ overload leads to Once activated, Bax provokes mitochondrial
calpain-mediated lysosomal disruption with damage and tAIF mitochondrial release. The
releases of cathepsins B and L [213]. Cathepsin tAIF relocalizes to the nucleus, associates with
B is shown to be involved in caspase-independent H2AX and cyclophilin A, and subsequently
cell death induced by death receptor ligands induces chromatinolysis [226]. PARP-1 is a
[213]. Lysosomal membrane permeability (LMP) nuclear enzyme activated by DNA strand breaks
is associated with activation of PLA2, causing the and plays a key role in repairing DNA damage.
production of ROS [214]. LMP is also known to PARP-2, the closest homolog to PARP-1, has
activate mitochondrial permeability transition been identied as one of the essential regulators
(MPT), leading to cell death. The opening of of necroptosis by a genome-wide siRNA screen
MPTP accounts for the MPT resulting in disrup- study [178]. PARP activation is also found to
tion of the inner mitochondrial transmembrane play a critical role in glutamate-induced necrop-
potential (m) during cell death [215217]. tosis [227]. The mechanisms of PARP-induced
Oxidative stress can serve as a facultative inducer mitochondria dysfunction in necroptosis remain
of MPTP opening; moreover, ROS are potent to be explored.
260 M.L. Tan et al.
Necroptotic cells spill their contents which mutated CYLD(C/S) [catalytically inactive form
contain DAMPs. DAMPs can trigger inamma- of CYLD that mimics the identied mutations of
tion by activating pattern recognition receptors CYLD in human tumors] exhibit a faster growth,
(PRRs), including Toll-like receptors (TLRs), are poorly differentiated, have robust angiogenesis
nucleotide-binding oligomerization domain activity, and are aggressive tumors [237]. Both
(NOD)-like receptors, and retinoic acid-inducible cIAP-1 and cIAP-2 may promote cancer cell sur-
gene I (RIG-1)-like receptors [228]. DAMPs are vival by functioning as E3 ubiquitin ligases that
intracellular molecules that have inammation- maintain constitutive ubiquitination of the RIP1
inducing capacities when released from cells, adaptor protein [174]. cIAP-1 and cIAP-2 directly
resulting in the activation of macrophages and ubiquitinate RIP1 and induce constitutive RIP1
subsequently the inammation processes [229]. ubiquitination in cancer cells which then associ-
ates with the pro-survival kinase TAK1 and sup-
presses apoptosis [174]. Immunohistochemical
14.5 Necroptosis and Possible analysis of cIAP-1 and cIAP-2 in archival bladder
Therapeutic Targets in Cancer specimens revealed that both cIAP-1 and cIAP-2
expression are signicantly increased in bladder
Necroptosis is found to occur during the early cancer compared with normal bladder urothelium.
phases of T-cell clonal expansion, indicating that Nuclear cIAP-1 expression is strongly correlated
this mode of cell death may be involved in the to bladder cancer stage, tumor grade, and tumor
regulation of the immune system [230]. In addi- recurrence suggesting the possibility of using
tion, virus-infected cells, which are resistant to cIAP-1 as a marker in bladder cancer prognosis
apoptosis, are found to be highly sensitive to [238]. Furthermore, X-IAP, cIAP-1, and cIAP-2
necroptosis, indicating that it may serve as an are found to be highly expressed in chronic lym-
alternative mechanism of cell death other than phocytic leukemia samples [239242]. Thus, the
apoptosis [231]. In acute lymphoblastic leukemia IAPs should be an attractive antitumor strategy. In
(ALL), necroptosis cell death can be induced to addition, RIP1 polyubiquitination by TRAF2/
overcome the glucocorticoid resistance of ALL TRAF5 at the position of K63 inhibits TNF--
cells. Resistance to the initial phase of chemo- induced apoptosis [171, 172] and TRAF2 knock-
therapy, in particular poor response to glucocorti- down by siRNA radiosensitizes cancer cells via a
coids, is a strong predictor of adverse outcome reduced NF-B activation, suggesting that TRAF2
for childhood ALL. In a clinical study using pri- may also be an attractive target for anticancer
mary leukemia cells from patients with very activity [243].
high-risk disease, obatoclax mesylate, a Bcl-2 Necroptosis in cancer cells is induced by vari-
antagonist, and rapamycin increased RIP1 activ- ous approaches including administration of
ity and restored the response to dexamethasone alkylating DNA-damaging agents [244] and the
by inducing a type of cell death morphologically application of photodynamic therapy through
consistent with necroptosis [232]. In addition, which photosensitizing compounds accumulated
necroptosis appears to cause cancer cell death as in tumor cells generate ROS following excitation
a response to several anticancer treatment strate- with light from various spectra [245, 246].
gies, clearly indicating the role of this cell death Necroptosis induction is speculated to be useful
in cancer [233236]. in cancers which are resistant to the apoptotic
Defects in necroptosis or variations of effects of chemotherapy. One preclinical study
necroptosis-related genes may contribute to the on shikonin, a naturally occurring naphthoqui-
pathological process of human malignancies, none, has demonstrated that induction of necrop-
based on several observations and studies. tosis by the compound is able to overcome
Deubiquitination of RIP1 by CYLD is important resistance to cancer drugs mediated by
for the formation of complex II, leading to either P-glycoprotein, Bcl-2, and Bcl-XL in cancer cell
apoptosis or necroptosis. Tumors carrying the lines [236]. All these results indicate that
necroptosis could be a potent therapeutic strategy in common with apoptosis. Starvation and oxida-
for the treatment of cancer. The further explora- tive stress can trigger both apoptosis and autoph-
tion of the necroptosis signaling pathway will be agy. Bcl-2 proteins function to inhibit both
important to identify strategies and novel antitu- apoptosis and autophagy, providing another clue
mor drugs which can be brought forward to the to the interplay between both processes. Beclin-1,
human clinical trials. the essential autophagy protein and haplo-
insufcient tumor suppressor, interacts with sev-
eral cofactors such as Ambra1, Bif-1, and
14.6 Crosstalk in Apoptosis, UVRAG to activate the lipid kinase Class III
Autophagy, and Necroptosis PI3K and induce autophagy [255]. In normal
conditions, Beclin-1 is bound to and inhibited by
Functional relationships between apoptosis and Bcl-2 or the Bcl-2 homolog Bcl-XL, well-charac-
autophagy are gaining much interest, as both cell terized apoptosis regulators, which involve an
deaths are not mutually exclusive. Perturbations interaction between the BH3 domain in Beclin-1
in the apoptotic machinery, such as caspase and the BH3-binding groove of Bcl-2/Bcl-XL.
inhibition, have been reported to induce both BH3-only proteins can competitively disrupt the
autophagic cell death and necroptosis [247, 248]. interaction between Beclin-1 and Bcl-2/Bcl-XL
Inhibition of autophagy in cancer cells results to induce autophagy. Nutrient starvation can
in an accelerated cell death that manifests the stimulate the dissociation of Beclin-1 from its
hallmarks of apoptosis including chromatin con- inhibitors, either by activating BH3-only proteins
densation, MOMP, and activation of caspases (such as Bad) or by posttranslational modica-
[249]. In some cases, mixed phenotypes of both tions of Bcl-2 (such as phosphorylation) that may
autophagy and apoptosis are detected in response reduce its afnity for Beclin-1 and BH3-only
to common stimuli [156, 249]. Studies in a vari- proteins [255]. Anti-apoptotic Bcl-2 family mem-
ety of experimental systems indicate that autoph- bers participate in the inhibition of autophagy,
agy cell death is likely to be cell type dependent. whereas the pro-apoptotic BH3-only proteins
Autophagy can delay the onset of apoptosis, participate in the induction of autophagy.
following starvation, DNA damage, and hemo- A recent nding suggests a link between
dynamic stress [13]. For example, 1-day fasting autophagy and the extrinsic apoptotic pathway
causes liver autophagy in rats, but when starva- mediated by p62/SQSTM1. Autophagy is
tion is prolonged for a few days, hepatocytes recently known to be responsible in selective
succumb to apoptosis [250]. Similarly, hemato- degradation of polyubiquitinated proteins via
poietic cell lines withdrawn from growth factor sequestosome-1 (SQSTM1), which encodes for
rst activate autophagy and eventually apoptosis p62 protein. p62 interacts with LC3 via its LC3
[7]. Studies have also demonstrated that certain interacting region (LIR). Recent studies indicate
compounds have the ability to trigger both apop- that p62 is recruited to damaged mitochondria
tosis and autophagy cell deaths simultaneously via binding to ubiquitinated outer mitochondrial
in cancer cells [251, 252]. Blocking of one path- membrane proteins, suggesting that p62 may
way will trigger the activation of another [253]. serve as an autophagy receptor for ubiquitinated
Researchers have also hypothesized that there proteins and damaged mitochondria [256258].
are factors (either external or internal) that may In addition to its role in autophagy, p62 medi-
affect the preferential shunting into either bio- ates a cells decision to undergo apoptosis or
chemical cascades that will ultimately result in survival through its organization of signaling
either apoptosis or autophagic cell death [254]. complexes in the cytoplasm [257, 259, 260].
Crosstalks between autophagy and apoptosis Upon cytokine stimulation, p62 activates the
exist at multiple levels because both pathways NF-B pathway, which subsequently induces
share mediators and pathway regulators. Several the pro-survival genes, such as anti-apoptotic
signals and pathways involved in autophagy are and cell proliferation genes and induces the
262 M.L. Tan et al.
expression of inammatory genes such as cyto- cleavage of RIP1 [180]. Cleavage of RIP1 by
kines, chemokines, and adhesion molecules active caspase-8 constitutes a negative feedback
[260263]. However, p62 is also found to acti- loop to limit autophagic induction. In T cells,
vate caspase-8 in the extrinsic apoptosis path- autophagy is induced in response to energetic
way, resulting in the initiation of apoptosis and demands, resulting in formation of a DISC-like
cell death [259]. complex including Atg512/Atg16L, FADD,
The expression of Ptc induces apoptosis, but caspase-8, and RIP1. In addition, blocking of
this activity is suppressed by its ligand, sonic necroptosis by necroptosis inhibitor necrostatin-1
hedgehog (Shh). Interestingly, hedgehog inhibi- (Nec-1) was sufcient to rescue the hyperactive
tion is found to induce autophagy through upreg- autophagy and restored the cell cycle prole and
ulation of Bnip3 and is also found to increase survival capacity of actively dividing FADDdd
apoptosis in hepatocellular carcinoma cells at the (cells expressing a dominantly interfering form
same time [264]. In a very recent study, apoptosis of FADD) and caspase-8/ T cells. When autoph-
suppressed by the knocking down of PP2A can agy is inhibited with 3-MA, Nec-1 reduces LC3
be reversed by the administration of 3-MA, a processing, suggesting that RIP1-dependent
known autophagy inhibitor. The elevated accu- necroptotic signaling, or perhaps necroptosis
mulation of LC3-II and the decline of the autoph- itself, promotes autophagy. Since autophagy is
agy substrate p62 are also observed in directly induced by RIP1 activity, RIP1 may
PP2Ac-small interfering RNA transfected cells. inuence autophagic signaling either directly or
However, overexpression of PP2Ac suppresses perhaps indirectly as a response to necroptotic
the accumulation of LC3-II and restores p62 stress [267, 269]. While autophagy is necessary
[265]. Interestingly, 3-MA increases cell death for rapid T-cell proliferation, studies suggest that
induced by diamindichloridoplatin (DDP), which FADD and caspase-8 form a feedback loop to
suggests the protective function of autophagy in limit autophagy and prevent this salvage pathway
DDP-induced cell death [265]. from inducing RIP1-dependent necroptosis.
The relationship between autophagy and Thus, the linkage of FADD and caspase-8 to
necroptosis is said to be complex, at least at this autophagic signaling intermediates is essential
point of time. There is increasing evidence sug- for rapid T-cell clonal expansion and may serve
gesting that necroptosis is associated with to promote caspase-dependent apoptosis under
autophagy [232], is suppressed by autophagy hyperautophagic conditions, thereby averting
[266], or is not associated with autophagy at all necroptosis and inammation in vivo [267].
[230]. For example, in ALL cells, reversal of glu- However, other reports tend to demonstrate
cocorticoid resistance occurred through rapid that inhibition of autophagy promotes necropto-
activation of autophagy-dependent necroptosis, sis in various human cancer cells. For example,
and the effect was associated with dissociation of TNF- signicantly induces necroptosis and
the autophagy inducer Beclin-1 from the anti- autophagy in murine brosarcoma L929 cells.
apoptotic Bcl-2 family member Mcl-1, as well as Nec-1 completely blocks TNF--induced necrop-
a marked decrease in mTOR activity. Combination tosis and autophagy, but inhibition of autophagy
of rapamycin with the glucocorticoid dexametha- with 3-MA or Beclin-1 siRNA promotes necrop-
sone triggered autophagy-dependent cell death, tosis, indicating that autophagy acts as a negative
with characteristic features of necroptosis [232]. regulator of TNF--induced necroptosis [270]. In
In addition, necroptosis signaling appears to other studies, T-cell receptor-induced necroptosis
activate autophagy process as a cleanup mecha- is found to be death receptor and autophagy inde-
nism for cell death. Experiments using proliferat- pendent, indicating the existence of an alternate
ing T cells have shown that caspase-8-decient T RIP1-dependent necroptotic pathway down-
cells exhibit RIP1-dependent necroptosis [267, stream of T-cell receptor signaling [230]. The
268]. On the other hand, caspase-8 is known to molecular link between necroptosis and autoph-
inhibit autophagy [269], probably through direct agy remains elusive.
14.7 Future Directions kinase. In some other cases, utilizing both autoph-
agy and apoptosis inducers may present a deadly
There is increasing evidence that the three major strategy against highly resistant tumors. Devising
cell deaths, i.e., apoptosis, necroptosis, and personalized pharmacotherapeutic strategy based
autophagic cell death, share overlapping molecu- on the autophagy status of the tumors has become
lar pathways and can occur in parallel under simi- an attractive option and offers signicant potential
lar conditions. Fundamental knowledge in to be translated into the clinic.
apoptosis, necroptosis, and autophagy has also So far, targeted drugs like oblimersen, bortezo-
generated a great deal of insight into the patho- mib, and mTOR inhibitors such as everolimus and
genesis of cancer and has provided important con- ridaforolimus have shown to be useful in some
siderations in strategizing cancer pharmacotherapy. clinical trials. These novel classes of drugs appear
Much effort and investment has been devoted to to work synergistically in combination with other
experimental drugs modulating autophagy or chemotherapeutics and have also showed specic
apoptosis, and scientists are beginning to look at activities against certain cancers. Since these drugs
necroptosis in a different light. A number of drugs are specically targeted against certain molecules
have proven to be promising during preclinical or receptors in the pathway, further unveiling of
studies and experimental anticancer therapies, but the tumors characteristics such as receptor or pro-
these drugs appear to be effective in one type of tein status may be critical in assessing patients
cancer and not the other. The percentage of response and clinical trial success. Furthermore, a
patients who totally responded or partially number of known genes that play a role in these
responded to these treatments, either as single- cell death pathways are either activated or inacti-
agent or in combination therapies, is relatively vated in several cancers. This will certainly affect
low, even though the outcome of these trials sug- not only the promotion and progression of cancer
gests some potential. These unforeseen effects are but also their response to treatment. Therefore, to
probably due to the specic-targeted nature of the optimize and personalize treatment strategies, the
therapy, in addition to the interconnected relation- genetic prole of the tumors is important. This
ships between these cell death pathways. The con- may provide information on the optimal point in
tradictory role of autophagy and the status of the pathway to be targeted and can be identied as
autophagy in the human tumors concerned remain prognostic markers. At the same time, the devel-
speculative and further complicate the response to opment of both robust tissue markers and relevant
conventional anticancer treatment. techniques that can be used in the clinical context
Thus, modulating apoptosis, necroptosis, and needs to occur along with novel treatments, which
autophagy by various means may be an important will be another challenge.
strategy to ght against the disease. Cancers,
which are resistant to the apoptotic effects of cer-
tain chemotherapy drugs, may be sensitive to 14.8 Concluding Remarks
drugs that evoke necroptosis or autophagic cell
deaths. An intact autophagy pathway has a role in Although recent studies have incorporated some
promoting carcinogenesis as well as suppressing predictive biomarkers by examining tumor status,
it. It also has a role in the development of resis- the utility of such practice remains non-conclusive.
tance to treatment. Therefore, if autophagy For example, the expression of peptidyl
response and activity are normal in tumors, com- O-glycosyltransferase GaLNT14 has been pro-
bining standard chemotherapy drugs with autoph- posed to be a potential marker of dulanermin or
agy inhibitors may sensitize tumor cells to Apo2L/TRAIL activity in NSCLC as high
anticancer agents. Cancer cells which present GaLNT14 mRNA and protein expression in
defects in the autophagy pathway may be man- tumor cell lines are associated with Apo2L/
aged by replacement of autophagy-inducing sig- TRAIL sensitivity [271]. An increase in
nals, e.g., pro-autophagics, or by inhibiting mTOR progression-free survival and overall survival was
264 M.L. Tan et al.
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Prognostic Value of Innate
and Adaptive Immunity in Cancers 15
Fabio Grizzi, Giuseppe Di Caro, Federica Marchesi,
and Luigi Laghi

15.1 Introduction ................................................ 275
It is now accepted that human carcinogenesis is a
15.2 Immune Inltration as a Major Player
dynamic process depending on multiple variables
of the Tumor Microenvironment .............. 276
and is regulated at multiple spatial and temporal
15.3 Cellular Players of the Innate scales [14]. According to the theory of multistep
Immunity in Cancer................................... 277
15.3.1 Tumor-Associated Macrophages (TAM) ..... 277 carcinogenesis, cancer cells accumulate a num-
15.3.2 Tumor-Associated Neutrophils (TAN)......... 278 ber of molecular changes to eventually become
15.4 Cellular Players of the Adaptive
fully malignant. The reductionist view of can-
Immunity in Cancer................................... 278 cer expressed in myriads of molecular biology-
based investigations stated that all the information
15.5 Prognostic Value of Innate
and Adaptive Cells of the Immune necessary for a cell to transform itself into a neo-
System in Cancer ....................................... 279 plastic cell can be attributed to changes at the
genomic level [5]. This certainty is based on
the fact that the genome carries all of the infor-
References ................................................................. 281
mation related to any cell process and that any
cellular transformation is due to a specic
genomic change [6]. Today, cancer is recognized
as a highly heterogeneous disease: more than 100
F. Grizzi, PhD (*) G. Di Caro, PhD L. Laghi, MD distinct types of human cancer have been
Laboratory of Molecular Gastroenterology,
described, and various tumor subtypes can be
Via Manzoni 56, Rozzano, Milan 20089, Italy found within specic organs. In addition, tumors
e-mail: fabio.grizzi@humanitasresearch.it; have somatic mutations and epigenetic changes,
giuseppe.dicaro19@gmail.com; many of which are specic to the individual neo-
luigi.laghi@humanitas.it
plasm [7]. It is now recognized that this genetic
F. Marchesi, PhD and phenotypical variability primarily determines
Department of Immunology and Inammation,
the self-progressive growth, invasiveness, and
Via Manzoni 56, Rozzano, Milan 20089, Italy metastatic potential of neoplastic disease and its
Department of Biotechnologies
response or resistance to therapy. It seems that
and Translational Medicine, the multilevel complexity of cancer explains the
Humanitas Clinical and Research Center, clinical diversity of histologically similar neopla-
University of Milan, Via Manzoni 56, sia [8, 9]. In simple mathematical terms, carcino-
Rozzano, Milan 20089, Italy
e-mail: federica.marchesi@humanitasresearch.it
genesis is a nonlinear process, and the behavior

276 F. Grizzi et al.
of which does not follow clearly predictable and induced molecules. However, only recent murine
repeatable pathways. In linear systems, the models have unraveled the role of
behavior of a system changes linearly in response the immune system in cancer progression, a
to an environmental factor. In contrast, the behav- process termed cancer immunoediting [17].
ior of nonlinear complex systems may be per- Immunoediting is a dynamic process composed
ceived as surprising and unpredictable. Periods of three phases: rst, the elimination of tumor
of inactivity may be punctuated by sudden cells by immunosurveillance; then an equilibrium
change, apparent patterns of behavior may disap- phase, during which the tumor is subjected to
pear, and new patterns may unexpectedly emerge immune-mediated latency, and the immune sys-
[2]. Moreover, nonlinear systems do not react tem is in balance with the tumor; and the last
proportionally to the magnitude of their inputs phase, during which tumor cells escape immune
and depend on their initial conditions, i.e., small restraints and co-opt the immune system to pro-
changes in the initial conditions may generate mote malignancy. Tumor cells employ diverse
signicantly different end points. These charac- mechanisms to escape from immunosurveillance,
teristics are commonly highlighted by the fre- as well as to manipulate the immune system and
quency with which differences in progression or their microenvironment in order to facilitate the
therapeutic response are seen in the same tumor development of a malignant phenotype. These
type and by the fact that cancer morphology does include mechanisms that promote escape, such as
not always reveal a similar underlying biology the downregulation of TAA and the decrease in
[10]. It is now ascertained that tumors grow in a expression/secretion of proinammatory cyto-
complex network of epithelial, mesenchymal, kines, as well as mechanisms that induce immune
inammatory, and immune cells, as well as vas- suppression, such as the production of immuno-
cular and lymphatic vessels [1113]. Neoplastic suppressive cytokines, metabolites, and immune
cells take advantage from their surrounding checkpoint molecules. Immunoediting enables
microenvironment, as they are supplied by nutri- tumor cells to evade immune system detection,
ents supplied by the blood stream and growth disseminate from the initial niche, survive in the
factors produced by inammatory and stromal circulation, and settle at new metastatic sites.
cells, in addition to ghting for space to expand Histopathological analyses of solid tumors
and escape the immune attack [14]. When tumor reveal that they are inltrated by cells of
cells metastasize to distant organs, the same the innate and adaptive immunity [1820].
crosstalk is established at the new site. Therefore, Macrophages represent a signicant portion
these complex interactions determine the overall of the tumor mass, where they are commonly
tumor aggressiveness and the clinical outcome. termed tumor-associated macrophages (TAMs)
[21]. These cells are generated from blood
monocytes [22], which differentiate into two
15.2 Immune Inltration as a Major distinct macrophage types, identied as M1 (or
Player of the Tumor classically activated) and M2 (or alternatively
Microenvironment activated). M1- and M2-polarized macrophages
are endowed with opposite functional roles in
Among the various factors inuencing tumor terms of tumor suppression and immune stimu-
establishment, growth, local invasion, and metas- lation, M1 cells enhance immune responses, and
tasis, the impact of immunity has been debated restrain tumor progression through eliciting the
for a long time [15]. While inammation is Toll-Like Receptor (TLR) pathway, whereas
known to contribute to cancer progression [16], M2 macrophages switch-off the immune sys-
the immune system is programmed to recognize tem and promote tumor development. Mast
tumors from their inception. Immunosurveillance cells, myeloid-derived suppressor cells (i.e., the
against the tumor is stimulated by the presence of most abundant type of hematopoietic cells in the
tumor-associated antigens (TAA) and by stress- immune system) [23] and neutrophils [24] have
15 Prognostic Value of Innate and Adaptive Immunity in Cancers 277
also been reported to invade the intra-tumoral from clear; B cells have recently been appre-
space. Dendritic cells (DCs) are found in dif- ciated as paracrine mediators of solid tumor
ferent locations within a tumor, most immature development [39]. However, their capability
Langerhans cell-type DCs home in the tumor to enhance T cell activation might have a posi-
nests, and are tightly linked to malignant cells, tive impact on the organization of the antitumor
whereas both immature interstitial DCs and immune response [40]. Here, the roles played by
plasmacytoid DCs are located in the stroma innate and adaptive immune system in the local
[25]. Mature DCs concentrate in lymphoid islets progression and metastasis of human cancers
adjacent to the tumor nests and some draining of unrelated histologic origin are discussed; in
lymph nodes. NK cells are usually found in the addition their prognostic roles understood and
stroma of most tumors [26, 27] but can also be exploited to date are pointed out.
found in close contact with tumor cells in renal
cell carcinoma. The distribution of lymphocytes
may be differently orchestrated depending on 15.3 Cellular Players of the Innate
the tumoral architecture [28]. T lymphocytes Immunity in Cancer
are mainly located in the core, often referred
to as the center of the tumor, its invasive mar- Rudolf Virchow (18211902) observed inltrat-
gin and in adjacent lymphoid islets. Among T ing leukocytes in tumors for the rst time and
lymphocytes, most have a memory phenotype, proposed the inammatory microenvironment as
with nave cells being found mostly in adjacent a primary site of cancer occurrence [41]. Later,
lymphoid aggregates [29]. Some CD8+ T lym- epidemiological and experimental studies have
phocytes contact malignant cells, whereas oth- associated chronic infections to about 1520 %
ers are dispersed in the stromal compartment. of tumors [42, 43] and linked inammation to
Forkhead/winged helix transcription factor tumorigenesis by modulation of a variety of com-
(FoxP3)+ T lymphocytes, T lymphocytes helper plex processes, including the increased cell pro-
17 (Th17), T follicular helper (TFH) cells, and liferation, rate of mutagenesis, angiogenesis, and
B lymphocytes concentrate in the stromal tissue inhibition of apoptosis. Therefore, inammation
and in lymphoid islets. A similar organization has been acknowledged as a critical element in
is found in metastatic sites, as in the primary cancer occurrence and has been included as a
tumors; however, their organization may vary new hallmark of cancer [16].
among tumors and between patients. Signicant
correlations between the level of immune cell
inltration in tumors and their clinical outcome 15.3.1 Tumor-Associated
have been investigated in several cancers of Macrophages (TAM)
unrelated histological origin [3033]. A strong
lymphocytic inltration is found to be associated A number of studies appraised tumor-associated
with good clinical outcome in different tumor macrophages (TAM) as crucial mediators of the
types and subtypes, including melanoma, head connection between inammation and cancer
and neck, breast, bladder, ovarian, colorectal, occurrence [44, 45]. TAMs secrete a plethora of
renal, prostatic, and lung cancer [3335, 30, 36, cytokines and chemokine, which are the soluble
31, 37, 38]. The role of other T lymphocyte inl- mediators of inammation and are mainly respon-
trates has also yielded apparently contradictory sible to mediate such processes [46]. It is widely
results. It is reported that Th17 cell inltration accepted that in the majority of cancers TAMs
is associated with poor prognosis in colorectal, have a pro-tumoral effect [47]. However, these
lung, and hepatocellular carcinoma, whereas it cells are intrinsically plastic in their functions,
is considered as a predictor of better survival in and they were shown to acquire antagonistic
some esophageal and gastric cancers. The effect properties ranging from immunosuppressive to
of intra-tumoral B lymphocytes in cancer is far immune-stimulatory properties in the complexity
of tumor microenvironment. While the antitumor [59]. Notably, TAMs exert their pro-tumor func-
role of TAM has been previously linked to the tions both directly, by acting on tumor cells, and
orchestration of T lymphocyte antitumor immune indirectly, by orchestrating suppression of the
response, recent ndings have shown that tumor adaptive immune response. Macrophages, when
immunosurveillance can be rmly directed by adequately activated, have the capability to both
TAMs when educated by specic treatments, directly kill tumor cells [60, 61], a property medi-
in a T cell independent fashion [48]. The func- ated by contact-dependent [62] as well as inde-
tional plasticity of macrophages is regulated by pendent mechanisms [48], and to orchestrate an
environmental stimuli, thus their immune prole antitumor adaptive immune response, through
results in the identication of two distinct polar- the activation of cytotoxic lymphocytes.
ized functions, schematically simplied as M1/
M2 classication. Macrophages are recruited at
peripheral sites by locally secreted chemotactic 15.3.2 Tumor-Associated
factors and cytokines, including inammatory Neutrophils (TAN)
chemokines and growth factors [i.e., vascular
endothelial growth factor (VEGF), platelet- Although TAMs are the most prevalent innate
derived growth factor (PDGF), and macrophage cellular components of the tumor microenviron-
colony-stimulating factor (M-CSF)] [49]. These ment, the role of tumor-associated neutrophils
cytokines can also promote macrophage sur- (TANs) on tumor progression has been reconsid-
vival and polarization. Although mobilization ered [63, 64]. Accordingly, TANs have been
of the circulating pool of monocytes is the main recognized as a source of cytokines and chemo-
mechanism of macrophage recruitment, local kine, as well as anti-inammatory mediators in
proliferation can contribute to macrophage accu- different settings, thus likely to mediate a dual
mulation at the tumor site [50]. In the tumor effect on tumor progression depending on their
context, both tumor and stromal cells secrete a polarization state, i.e., N1 and N2 [65, 66]. TAMs
variety of chemoattractants for blood-circulating and TANs functional polarization and prognostic
monocytes, including CCL-2, originally discov- value reect the intrinsic plasticity as it varies
ered as a tumor-derived chemotactic factor [51]. along with the tumor type, location in the tumor
Molecular proling analyses of both human and tissue (i.e., necrotic and hypoxic areas), and the
murine TAMs have evidenced a prole closer to tumor stage. Studies have demonstrated specic
that of M2 macrophages [52, 53], whose remod- examples of tumor-mediated signals (such as
eling, immunosuppressive activities, and produc- transforming growth factor-, TGF-) that induce
tion of trophic factors for tumor and stromal cells the formation of a pro-tumorigenic N2 phenotype
functionally correlate to important pro-tumor capable of supporting tumor growth and suppress-
activities [54], including proteolytic activity [55], ing the antitumor immune response. However,
remodeling of the extracellular matrix [56], and there are evidences showing that TAN can also
induction of angiogenesis [57]. Liu et al. have have an anti-tumorigenic N1 phenotype [67].
shown that M2-polarized TAMs increased bro-
blastic morphology, upregulated mesenchymal
markers (i.e., vimentin and Snail) at the mRNA 15.4 Cellular Players
and protein levels, and increased proliferation, of the Adaptive Immunity
migration, and metalloproteinase MMP2 and in Cancer
MMP9 proteolytic activity in pancreatic cancer
cells [58]. In addition, it has been shown that the It has been accepted that immune cells inltrate
MMP-9 inhibitor is associated with decreased the tumor stroma and are essential players of the
survival in breast cancer [59]. Leier et al. identi- tumor microenvironment. Cells of the adaptive
ed MMP-9 as a potent player in modulating the immune system are mainly represented by CD8+
innate immune response into antitumor activities cytotoxic T lymphocytes (CTLs) and CD4+
T-helper lymphocytes. The main function of where tissues harboring target Ags are inltrated
CD4+ T lymphocytes is to sustain activation of by cellular effectors of the adaptive immune sys-
other cells, including macrophages, B cells, and tem, which are organized anatomically and func-
CTLs, by the release of several cytokines, such as tionally as in secondary lymphoid organs, with
interleukin-2 (IL-2), tumor necrosis factor alpha recruitment of B cells and T cells, follicular den-
(TNF-), and interferon gamma (INF-). dritic cells with germinal centers, and specialized
Identication and specic elimination of tumor vessels suited to mediate trafc of immune cells
cells are mediated by CTLs CD8+ T cells [68, 69], [75, 76]. Those structures are named tertiary
which produce perforin and granzyme B [70]. lymphoid tissue (TLT) and might be involved in
Antigen (Ag) recognition by lymphocytes after the organization of the immune response. Few
the rst encounter is kept at a higher activation studies have reported the presence of TLTs in
level compared to baseline. Activated T lympho- cancer [77, 78]. Moreover, the concept of ectopic
cytes have a long life, are more reactive to stimu- lymphoid structures within solid tumors has only
lation than nave T lymphocytes, and are recently become appreciated, and it is still unclear
detectable by specic surface molecules, sug- whether these structures retain functional
gesting that their presence in the context of solid immune activities to mediate recruitment and
tumors has important implications. Accordingly, activation of TILs.
antigen-experienced CTLs phenotypically switch
CD45 isoform from CD45RA to CD45RO when
activated [71]. 15.5 Prognostic Value of Innate
T lymphocyte activation is also modulated by and Adaptive Cells
a subpopulation of T lymphocytes indicated as of the Immune System
Tregs, which suppress immune responses [72]. in Cancer
The transcription factor FOXP3 is a specic Treg
cell marker [73, 72]. Treg lymphocytes include The stromal compartment of solid tumors is inl-
different subpopulations, although the most trated by immune and inammatory cells express-
investigated are CD4+ CD25+ [72, 74]. However, ing a wide array of specic markers and exerting
these markers are not completely specic for critical effects on tumor outcome depending on
Tregs as CD25 and FOXP3 might also be their specic subset, density, spatial location
expressed by activated CTLs [71]. Moreover, it is [79], and the staging of tumor at diagnosis [80
not clear whether regulatory cells are capable to 82]. It is widely accepted that in preclinical stud-
suppress T lymphocytes with tumor antigen ies cellular mediators of the innate immunity
specicity. The identication and targeting of favor tumor progression [16, 54, 83]. Accordingly,
Tregs selectively suppressing tumor-specic T the quantication of the number of CD68+ TAMs
cells would avoid unwanted depletion of regula- was linked to a poor prognosis in pancreatic can-
tory cells involved in peripheral immune regula- cer and Hodgkin lymphoma [84, 85]. In the case
tion and generation of autoimmunity. Tregs may of pancreatic cancer, expression of M1 markers
exert different functions according to the tumor of macrophage polarization was associated with
contexture, i.e., they might block antitumor better prognosis, while M2 markers were linked
immunity or decrease chronic pro-tumor inam- to worst prognosis [85]. In lung cancer,
mation [71]. IL10+-CD68+ TAMs were associated with worst
In the clinical setting of some human cancers, prognosis in patients with late-stage disease at
lymphocytic reaction can comprise different diagnosis [86], while in a subsequent study a
components beside dispersed tumor inltrating high ratio of M1/M2 macrophages was a feature
lymphocytes (TILs) and include discrete lym- of patients with good outcome [87]. Thus,
phoid aggregates, resembling lymph-node-like according to the simplied view of macrophage
structures. These aggregates are similar to those polarization provided by Mantovani et al., in
observed in chronic inammatory conditions, clinical studies macrophages inltrate tumor nest
as a heterogeneous population, which seem to be concomitantly located at the tumor invasive

retain different functional and molecular proper- margin and intra-tumoral region in each CRCs
ties that may vary according to the instructions specimen [95]. By these means, these immune
provided by the tumor milieu. On the contrary, a features identied a benchmarking population
meaningful correlation between high number of with a dismal prognosis and devoid of TILs rep-
TAMs and better prognosis has been described in resenting only 6.5 % of the CRCs (stages IIII)
colorectal cancer [62, 88]; in addition, this cor- [95]. This strategy fostered statistical analysis,
relation held true regardless of TAM polarization but might not provide proper clinical prognostic
in another study [89]. Discrepancies among clini- relevance when addressing surveillance strate-
cal studies on prognostic abilities of innate gies and allocation to chemotherapy in the over-
immune cells underline the importance of the all population of CRC. The biological relevance
tumor type when trying to determine the inu- of tumor lymph node inltration in the context of
ence of TAMs on tumor progression. Further TILs prognostic abilities was previously shown
clinical data are warranted to determine whether in ovarian cancer in a study suggesting a negative
the effect of TAM differs along tumor progres- interaction of nodal status with antitumor immu-
sion, as well as in response to chemotherapy nity [81]. In CRC, the density of activated CD8+
treatments in a clinical relevant scenario. Several TILs decreased in patients with metastatic lymph
retrospective clinical studies on colorectal, mela- nodes and advanced tumor staging, suggesting
noma, ovarian, breast, and non-small-cell lung that immune escape might occur along CRC
tumors have generally underlined tumor inltra- disease progression [96]. Accordingly, in a
tion of the adaptive immune cells as a prognostic different study, the expression of eumesodermin,
indicator of good prognosis [9092, 79, 93, 77, a transcription factor critically involved in the
94]. Variability with respect to prognostic poten- production of perforin, was inversely associated
tial of the markers employed relies on the specic with tumor lymph node involvement [97]. In
population of T lymphocytes and the type of melanoma, these observations were supported by
tumor settings investigated. In this view, colorec- the fact that a primary tumor devoid of TILs was
tal cancer represents a paradigm since its milieu shown to predict sentinel lymph node metastasis.
is highly permeated by adaptive immune cells These studies underline that the plasticity of TILs
with potential antitumor abilities. A seminal with regard to their recruitment and antitumor
paper by Galon et al. claimed that concomitant activity seems to differ along the clinical progres-
local inltration of CD3+ lymphocytes at the sion of different solid cancers [82]. Therefore,
tumor invasive margin and in the intra-tumoral future design of clinical trials aimed to employ
location was a better predictor of survival com- TILs as diagnostic tools or novel immunothera-
pared to the tumor-node-metastasis (TNM) stag- peutic strategies should take these considerations
ing system [79]. However, TNM is still the gold into account. Recruitment of Treg cells into the
standard predictor of CRC patient prognosis, tumor milieu is another mechanism of tumor
while TILs have not been employed in clinical immune evasion. In ovarian cancer, recruitment
practice to date. A subsequent study by Laghi of Tregs decreased specic antitumor TILs and
et al. raised doubts on previous claim and showed was associated with a worst prognosis [98]. In
that while CD3+ T-inltrating lymphocytes hepatocellular, renal cell, and breast carcinomas,
(TILs) were not independent from TNM staging the number of CD4+CD25+Foxp3+ cells was
in predicting patients prognosis, TILs were a associated with worst patients outcome [99101],
strong prognostic factor only among lymph- although not independently by other histopatho-
node-negative but not among lymph-node- logical features in the case of breast cancer.
positive CRCs [80]. Later Mlecnik et al. showed Counterintuitively, different CRC studies showed
that an immune score was re-proposed, although that a high density of Foxp3+ cells was
represented by partly overlapping subpopulations independently associated with better prognosis
of TILs (i.e., CD8+ and CD45RO+), which had to [102104]. This discrepancy might be explained
by hypothesizing that Foxp3+ cells instead of remains largely, if not entirely, to be addressed in
inhibiting antitumor immunity decrease chronic prospective studies [111, 112]. In parallel, under-
pro-tumor inammation. However, the biological standing the mechanisms of efcient immune
basis explaining differing roles of Treg cells in reactions, the place where they are initiated, the
tumor progression with respect to the tumor type cellular and molecular mediators involved, and
is still unknown. New experimental models prop- their impact at different stages of the disease
erly simulating tumor development will be help- should provide new tools and goals for more
ful in better understanding Tregs activity in effective and less toxic targeted therapies.
tumor.
Acknowledgments This work was supported by Italian
Association for Cancer Research (AIRC) Italy (grant
number MFAG-11677 to FM) and the Italian Ministry of
15.6 Concluding Remarks University and Research, FIRB grant (RBAP11H2R9).
Solid tumors contain a heterogeneous mixture of

malignant and nonmalignant cells within an
extracellular matrix supported by an irregular
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Epigenetics and microRNAs
in Cancer 16
Petra M. Wise, Kishore B. Challagundla,
and Muller Fabbri

16.1 Introduction ................................................ 285
MicroRNAs (miRNAs) are small noncoding
16.2 MiRNAs Regulate Effectors
RNAs (ncRNAs) which regulate gene expression
of the Epigenetic Machinery ..................... 286
by directly binding mostly, but not exclusively,
16.3 MiRNAs Are Epigenetically Regulated to the 3-untranslated region (3-UTR) of target
in Several Types of Human Cancers ........ 289
mRNAs [1]. In 1993, Victor Ambros rst identi-
16.4 Concluding Remarks ................................. 291 ed a small ncRNA, called lin-4, able to regulate
References ................................................................. 292 the expression of a gene called lin-14 involved
in the development of C. elegans [2]. In 2001,
Lagos-Quintana M. et al. showed for the rst
P.M. Wise, PhD K.B. Challagundla, PhD time that many of these small ncRNAs (in the
Department of Pediatric Hematology/Oncology,
Childrens Center for Cancer and Blood Diseases,
meantime called microRNAs) are present not
Norris Comprehensive Cancer Center, only in invertebrates but also in vertebrates [3]. In
University of Southern, California, 2002, Croces group provided the rst evidence
Childrens Hospital Los Angeles, of miRNA involvement in cancer by showing
Los Angeles, CA, USA
that a specic cluster of miRNAs (namely, the
Department of Pediatric Hematology/Oncology, miR-15a/16-1 cluster) is located in the frequently
Childrens Hospital Los Angeles,
4650 Sunset Blvd MS #57, Los Angeles,
deleted chromosomal region 13q14 in chronic
CA 90027, USA lymphocytic leukemia (CLL) [4]. In 2005 Frank
e-mail: pwise@chla.usc.edu; Slack supported this molecular evidence of
kchallagundla@chla.usc.edu miRNA involvement by demonstrating that let-7
M. Fabbri, MD, PhD (*) directly targets the RAS oncogene in lung cancer
Department of Pediatric Hematology/Oncology, [5]. In the same year, Cimmino et al. found that
Childrens Center for Cancer and Blood Diseases,
Norris Comprehensive Cancer Center,
the miR-15a/16-1 cluster directly targets the anti-
University of Southern, California, apoptotic BCL2 gene in human CLL [6]. From
Childrens Hospital Los Angeles, this time on, we assist at a plethora of studies
Los Angeles, CA, USA identifying dysregulation of miRNAs in almost
Department of Pediatric Hematology/Oncology and all types of human cancers and unraveling their
Molecular Microbiology and Immunology, contribution to human carcinogenesis by identi-
Childrens Hospital Los Angeles,
4650 Sunset Blvd MS #57, Los Angeles,
fying which genes are modulated by the dysregu-
CA 90027, USA lated miRNAs. Overall, these studies clearly state
e-mail: mfabbri@chla.usc.edu that aberrancies of the miRNome (dened as the

286 P.M. Wise et al.
full spectrum of miRNAs in a specic genome) DNA promoter methylation and chromatin histone
contribute to human cancer development modications which are catalyzed by specic
and can be therapeutically targeted to restore enzymes, overall indicated as effectors of the epi-
miRNA expression to normal [7]. Moreover, it genetic machinery. However, if we consider the
has become clearer that miRNA involvement above denition, also miRNA gene regulation sensu
goes beyond cancer, since they are involved in stricto represents a component of epigenetics.
a variety of biological processes, spanning from Interestingly, it has been discovered that there is a
development, differentiation, apoptosis, and pro- two-way correlation between miRNAs and other
liferation to senescence and metabolism [813]. epigenetic mechanisms: miRNAs can regulate the
MiRNAs are genes, like any other protein expression of effectors of the epigenetic machinery
coding gene (PCG), transcribed by RNA poly- and miRNA genes undergo the same epigenetic
merase II into a capped and polyadenylated regulatory mechanisms of any other PCG. These
precursor, called pri-miRNA [14, 15]. A dou- two main aspects of miRNome-epigenome cross-
ble-stranded RNA-specic ribonuclease called regulation and their implications in human carcino-
Drosha, in conjunction with its binding partner genesis will be the main focus of this chapter.
DGCR8 (DiGeorge syndrome critical region
gene 8, or Pasha), cleaves the pri-miRNA into
a hairpin-shaped RNA precursor (pre-miRNA), 16.2 MiRNAs Regulate Effectors
about 70100 nucleotides (nt) long [16]. of the Epigenetic Machinery
Transferred to the cytoplasm by Exportin 5, the
pre-miRNA is cleaved into an 1824 nt duplex In 2007, Fabbri et al. provided the rst evidence
by a ribonucleoproteic complex, composed of that miRNAs can affect the expression of epige-
a ribonuclease III (Dicer), and TRBP (HIV-1 netically regulated PCG in cancer by directly tar-
transactivating response RNA binding protein). geting key effectors of the epigenetic machinery,
Finally, the duplex interacts with a large protein such as DNA methyltransferases (DNMTs) [20].
complex called RISC (RNA-induced silenc- The miR-29 family (composed of miR-29a, miR-
ing complex), which includes proteins of the 29b, and miR-29c) can directly silence the expres-
Argonaute family (Ago1-4 in humans), which sion of de novo DNMT3A and DNMT3B in
drives one strand of the duplex (the so-called non-small cell lung cancer (NSCLC), leading to a
mature miRNA) mainly, but not exclusively, to global hypomethylation status of cancer cells and
the 3-UTR of the target mRNAs. Overall, miR- re-expression of tumor suppressor genes (TSGs)
NAs exert its effect by modulating the expres- such as FHIT and WWOX, whose expression is
sion of the target mRNAs either by mRNA silenced in NSCLC by promoter hypermethyl-
cleavage or by translational repression. In 2007, ation. As a result of the re-expression of these
Vasudevan et al. discovered that miRNAs can TSGs, NSCLC cells undergo apoptosis both
also increase the expression of target mRNAs in vitro and in an in vivo xenograft model [20].
[17]. Each miRNA can target several different Subsequently, Garzon et al. showed that in addi-
transcripts. For instance, it has been demon- tion to directly targeting de novo DNMTs, miR-
strated that a cluster of two miRNAs (namely, 29b is also capable of targeting the maintenance
miR-15a and miR-16) can affect the expression DNMT1, even though in an indirect way: by
of about 14 % of the human genome in a leuke- directly silencing Sp1, a transactivator of DNMT1
mic cell line [18]. In addition, the same mRNA [21]. These combined effects of miR-29s on all
can be targeted by several miRNAs [19]. three major DNMTs highlight their relevance for
Epigenetics is dened as all heritable changes in epigenetic processes and explain the profound
gene expression not associated with concomitant effects of their restoration on the global methyla-
alterations in the DNA sequence. In a traditional tion status of cells. MiRNAs such as the miR-29
sense, gene epigenetic regulation usually includes family, able to directly target effectors of the
16 Epigenetics and microRNAs in Cancer 287
epigenetic machinery, have been called epi-miR- They showed that the expression of miR-342 is
NAs. In mouse embryonic stem (ES) cells, two inversely correlated to DNMT1 levels in colorectal
independent groups have shown that members of cancer (CRC) tissues and cell lines, and that this
miR-290 cluster directly target RBL2, an inhibitor miRNA targets DNMT1, leading to reactivation
of DNMT3 genes [22, 23]. ES Dicer null cells are of epigenetically silenced TSGs such as ADAM23,
characterized by no expression of the miR-290 Hint1, RASSF1A, and RECKS. Functionally, res-
cluster, overexpression of RBL2, and disruption of toration of miR-342 resulted in a reduction of
de novo methylation pathway, leading to increased DNMT1 expression, reduced cell proliferation,
telomere recombination and aberrant telomere and invasiveness in CRC cells and inhibition of
elongation. Restoration of the miRNA cluster tumor growth and lung metastasis formation in
reverted this phenotype [23, 22]. Interestingly, the nude mice [28]. In 2010, viral epi-miRNAs have
regulatory effect of miR-290 cluster on de novo been shown to control the epigenetic machinery of
DNMTs was not observed in human embryonic host cells through DNMTs [29]. MiR-K12-4-5p,
kidney 293 cells following Dicer knockdown, sug- a Kaposi sarcoma-associated herpesvirus (KSHV)
gesting that miR-290 targeting effect on DNMT3s miRNA, was found to regulate the expression of
might be cell- and/or species-specic [22]. DNMT1, 3A, and 3B indirectly, by targeting the
Another important family of epi-miRNAs is expression of Rbl2, a known repressor of DNMT1,
the miR-148a/b-152 family. In 2008, Duursma 3A, and 3B transcription. Ectopic expression of
et al. showed that miR-148a and miR-148b can miR-K12-4-5p reduces Rbl2 protein expression
indeed bind to the coding region (not the 3-UTR) and increases DNMT1, 3A, and 3B mRNA levels
of DNMT3b mRNA, affecting the expression of in 293 cells, thus affecting the overall epigenetic
this gene [24]. This seminal study also concluded reprogramming of the host cell [29].
that by binding to this unusual site, miR-148 fam- Epi-miRNAs are also involved in regulating
ily might be responsible for the several different the expression of histone deacetylases (HDACs)
splice variants of DNMT3b [24]. A role for the and Polycomb Repressive Complex (PRC) genes.
miR-148a/b-152 family was further conrmed in For instance, HDAC4 is a direct target of both
cholangiocarcinoma, where it was shown that these miR-1 and miR-140 [30, 31], while miR-449a
miRNAs, in addition to miR-301, can directly tar- binds to the 3-UTR region of HDAC1 [32].
get DNMT1, and their expression is silenced by HDAC1 is upregulated in several kind of cancers,
IL-6, which is involved in cholangio-cancerogen- and miR-449a re-expression in prostate cancer
esis [25]. This paper provided the rst evidence of cells induces cell-cycle arrest, apoptosis, and a
a correlation between epi-miRNAs, inammation, senescent-like phenotype by reducing the levels
and cancer. In 2010, Das et al. showed that all- of HDAC1 [32]. Recently, Jeon et al. showed that
trans-retinoic acid (ATRA)-treated neuroblastoma miR-449a,b regulate HDAC1 expression by
cells undergo downregulation of MYCN, hence directly targeting its 3UTR transcript, indicating
leading to overexpression of MYCN repressed that this might be one of the reasons for the low
miRNAs such as miR-152, miR-26a/b, and miR- miR-449a, b expression and the high expression
125a/b [26]. They also showed that these miRNAs of HDAC1 in lung cancer [33]. MiR-140 has also
are epi-miRNAs in this model, since they downreg- been shown to be involved in chemoresistance
ulate DNMT1 and DNMT3B expression, leading mechanisms by targeting HDAC4 [34]. Inhibition
to re-expression of epigenetically silenced NOS1, of endogenous miR-140 by locked nucleic acid
which promotes neural cell differentiation. Also, (LNA)-modied anti-miRNAs partially sensi-
the expression of miR-152 was normally down- tized resistant colon cancer stemlike cells to
regulated with concurrent increase of DNMT1 5-FU treatment by increasing HDAC4 levels,
expression in HBV-induced HCCs [27]. More leading to a G1 and G2 phase arrest [34]. Low
recently, Wang et al. identied miR-342 as another expression of miR-9 along with high expression
epi-miRNA involved in colon carcinogenesis [28]. levels of HDACs (HDAC4 and 5) were discovered
in Waldenstrom macroglobulinemia (WM) [35]. myogenin expression, and promotes muscle dif-
Mir-9 targets HDAC4 and HDAC5 in WM cells. ferentiation [43]. EZH2 is also highly expressed
Overexpression of miR-9 causes downregulation in nasopharyngeal carcinoma (NPC) patients
of HDAC4, 5, leading to an upregulation of acet- and correlates with a higher risk of relapse [44].
ylated-histone-H3 and acetylated-histone-H4. MiR-26a, miR-98, and miR-101, whose expres-
This provides evidence that the loss of miR-9 sion is consistently downregulated in human
might be responsible for upregulation of HDAC4 NPC specimens when compared to normal naso-
and HDAC5 in WM cells, contributing to the pharyngeal epithelial tissue samples, have been
pathogenesis of WM disease [35]. shown to directly target EZH2 [44], suggesting a
EZH2 is the catalytic subunit of the Polycomb prognostic role for these three miRNAs in
Repressive Complex 2 (PRC2) and is responsi- NPC. Recently, there has been an extensive
ble for heterochromatin formation by trimethyl- series of studies unraveling the central role of
ating histone H3 lysine 27 (H3K27me3), leading miR-101 in the regulation of EZH2, in several
to the silencing of several TSGs. Varambally types of cancer. In hepatoma tissues, it was
et al. showed that in prostate cancer cell lines shown that miR-101 and miR-29c are downregu-
and primary tumors, the expression of miR-101 lated, but their expression can be restored (lead-
decreases during cancer progression, inversely ing to reduced levels of EZH2, EED, and
correlating with an increase of EZH2. These H3K27me3 proteins) after treatment with TPA
ndings are suggestive of a role as epi-miRNA (12-O-tetradecanoylphorbol 13-acetate), which
for miR-101, a hypothesis which was tested and is dependent on protein kinase C (PKC) and
conrmed by showing that miR-101 directly tar- ERK pathways in HepG2 cells [45]. Also,
gets EZH2 both in prostate and in bladder cancer Smiths et al. have established a pro-angiogenic
models [36, 37]. Moreover, miR-101-mediated effect of miRNA-101 working together with
suppression of EZH2 inhibits cancer cell prolif- EZH2 and VEGF during the process of angio-
eration and colony formation, revealing a TSG genesis [46]. The group analyzed the expression
role for miR-101, mediated by its modulatory of miR-101 in endothelial cells derived from
effects on cancer epigenome [37]. The inverse glioma patients and found it to be low. VEGF
correlation between miR-101 and EZH2 was also downregulates the expression of miR-101 result-
observed in glioblastoma [38], gastric cancer ing in increased protein expression of EZH2 and
[39], and NSCLC [40]. In prostate cancer it has induces the elongation of endothelial cells lead-
been shown that miR-101 can be inhibited by ing to a pro-angiogenic response. Transfection
androgen receptor and HIF-1/HIF-1 [41]. with pre-miR-101, or EZH2 siRNA, or treat-
Ectopic expression of miR-26a targets EZH2 in ments with DZNep, a small inhibitor of EZH2
Burkitts lymphoma, leading to reduced cell pro- methyltransferase activity, reverses this process
liferation, increased percentage of cells in G1- in HBMVECs controls, providing a network
phase, and increased apoptosis in Raji and between VEGF/miR-101/EZH2 proteins toward
Namalwa cells [42]. Intriguingly, the authors pro-angiogenic response in endothelial cells
also found that c-Myc negatively regulates miR- [46]. A summary of the described epi-miRNAs
26a, therefore maintaining high EZH2 expres- is provided.
sion levels in cells and signicantly contributing Overall, these studies indicate that epi-
to c-Myc-induced tumorigenesis [42]. In 2009, miRNAs can modulate several key effectors of
Juan et al. analyzed a regulatory double-negative the epigenetic machinery, which indirectly affects
feedback loop between miR-214 and EZH2 in the expression of epigenetically regulated genes.
controlling PcG-dependent gene expression dur- Considering that inactivation of TSGs by epigen-
ing differentiation [43]. PcG proteins suppress etic mechanisms represents one of the main strat-
the transcription of miR-214 in undifferentiated egies adopted by cancer cells to promote their
skeletal muscle cells (SMC). Ectopic expression oncogenic phenotype, it is of the utmost impor-
of miR-214 directly targets EZH2, increases tance to completely dissect these mechanisms,
since they could provide new molecular targets phorylation status of CDK6-downstream effector
for anticancer treatments. Rb protein [48]. Prospers work has identied a
signature of 13 miRNAs embedded in CpG
islands, with high heterochromatic markers (such
16.3 MiRNAs Are Epigenetically as high levels of K9H3me2 and/or low levels of
Regulated in Several Types K4H3me3) in acute lymphoblastic leukemia
of Human Cancers (ALL) patients [49, 50]. Among these, miR-124a
was methylated in 59 % of ALLs, and its pro-
As previously anticipated, the relationship moter hypermethylation was associated with
between miRNome and epigenome is bidirec- higher relapse rate and mortality rate vs. non-
tional. Not only do miRNAs regulate the expres- hypermethylated cases; hence, miR-124a pro-
sion of effectors of the epigenetic machinery, but moter methylation status was an independent
they also undergo the same epigenetic regulation prognostic factor for disease-free and overall sur-
of any other PCG. vival [50]. Finally, supporting Lujambios results,
By treating bladder cancer cell lines with both also in ALL the impact of miR-124a in the
a DNA demethylating agent (5-aza-2- CDK6-Rb pathway was conrmed by showing
deoxycytidine, 5-AZA) and an HDAC inhibitor that miR-124a directly silences CDK6 [50].
(4-phenylbutyric acid), Saito et al. found that Hypermethylation of miR-124a promoter is also
about 5 % of all human miRNAs increased their involved in the formation of epigenetic eld
expression levels [47]. MiR-127 was the most defect which is a gastric cancer predisposing con-
upregulated after this treatment, and its re- dition characterized by accumulation of abnor-
expression led to direct targeting and downregu- mal DNA methylation in normal-appearing
lation of the oncogene BCL-6, inducing a tumor gastric mucosa, mostly induced by H. pylori
suppressor function. MiR-127 is part of a cluster infection [51]. These ndings also suggest that
which includes miR-136, miR-431, miR-432, miR-124a promoter hypermethylation is an early
and miR-433 and is embedded in a CpG island event in gastric carcinogenesis. MiR-107, another
region; however, miR-127 is the only member of epigenetically controlled miRNA, targets
the cluster whose expression increases upon CDK6 in pancreatic cancer as well and impacts
treatment with the two epigenetic drugs [47]. this oncogenic pathway [52]. In HCT-116 cells,
Moreover, when each drug was used alone, no decient for DNMT1 and DNMT3B, Bruckner
variation in miR-127 expression was observed et al. showed increased expression of let-7a-3, an
[47], suggesting that both DNA methylation and miRNA normally silenced by promoter hyper-
histone modications affect the epigenetic regu- methylation in the wild-type cell line [53]. In
lation of miR-127. This seminal work shows that lung adenocarcinoma, primary tumors let-7a-3
indeed miRNAs undergo epigenetic regulation, promoter was found hypomethylated with respect
that it is a complex epigenetic regulation (involv- to the normal counterpart [53], whereas hyper-
ing both methylation and histone modications), methylation of let-7a-3 promoter was described
and that there are differences among miRNAs in epithelial ovarian cancer, paralleled the low
which even belong to the same cluster. Lujambio expression of insulin-like growth factor-II expres-
et al. created a double knockout (DKO) for sion, and was associated with a good prognosis
DNMT1 and DNMT3B in the CRC cell line [54]. Therefore, DNA methylation could act as a
HCT-116 and compared miRNA expression pro- protective mechanism by silencing miRNA with
le of DKO and wild-type cells. About 6 % ana- oncogenic function. Also, the miRNA-200 fam-
lyzed miRNAs were re-expressed in the DKO ily participates in the maintenance of an epithe-
cells [48]. Among them, miR-124a (embedded in lial phenotype, and loss of its expression can
a CpG island heavily methylated in this cell line) result in epithelial to mesenchymal transition
was re-expressed, reducing the levels of its direct (EMT). Furthermore, the loss of expression of
target gene CDK6 and impacting on the phos- miR-200 family members is associated with an
aggressive cancer phenotype. Vrba et al. found activated due to the loss of BRCA1 [61]. Mazar
that hypermethylation of the miR-200c/141 CpG et al. studied which miRNAs were re-expressed
island is closely linked to their inappropriate upon treatment of a melanoma cell line with
silencing in cancer cells, and the epigenetic regu- demethylating agents [62]. Among the 15 re-
lation of this cluster appears evolutionarily con- expressed miRNAs, miR-375 and miR-34b were
served, since similar results were obtained in also involved in melanoma progression [62]. Liu
mouse [55]. Interestingly, no variation in miRNA et al. [63] found that miR-182 was signicantly
expression was observed in lung cancer cells upregulated in human melanoma cells after com-
treated with either demethylating agents or bined treatment with 5-AZA and trichostatin
HDAC inhibitors or their combination [56]. A. Genome sequence analysis revealed the pres-
Another miRNA which is under epigenetic con- ence of a prominent CpG island 810 kb upstream
trol is miR-1. In hepatocarcinoma, miR-1 is fre- of miR-182, but methylation analysis showed
quently silenced by promoter hypermethylation that this genomic region was exclusively methyl-
[57]. However, in DNMT1 null HCT-116 cells ated in melanoma cells, not in normal human
(but not in DNMT3B null cells), hypomethyl- melanocytes. Since miR-182 has been shown to
ation and re-expression of miR-1-1 were observed harbor oncogenic properties, this nding raises a
[57], revealing a key role for the maintenance possible concern for melanoma patients treated
DNMT in the regulation of this miRNA. Han with epigenetic drugs [63]. MiR-31 maps at
et al. observed that neither 5-AZA nor DNMT1 9p21, a genomic region frequently deleted in
deletion alone can recapitulate miRNA expres- solid cancers including melanoma. Asangani
sion prole of DKO DNMT1/DNMT3B HCT- et al. [64] found recurrent downregulation of
116 cells [58]. Also, Lehmann et al. found that in miR-31 in melanoma primary tumors and was
breast cancer cell lines, 5-AZA re-activates miR- associated with genomic loss or epigenetic
9-1 (hypermethylated in up to 86 % of primary silencing by DNA methylation and EZH2-
tumors), but not miR-124a-3, miR-148, miR- mediated histone methylation. Moreover, miR-31
152, or miR-663 (hypermethylated as well) [59]. overexpression resulted in downregulation of
Previously, Meng et al. observed that in malig- EZH2 and a derepression of its target gene rap-
nant, but not in normal cholangiocytes, 5-AZA 1GAP. The increased expression of EZH2 was
induces re-expression of miR-370 [60]. Overall, associated with melanoma progression and poor
these results indicate that the epigenetic control overall survival [64].
of miRNAs is both cancer specic and miRNA Nickel (Ni) compounds are well described
specic. More recently, Chang and Sharan human carcinogens. Recently an important regu-
reported that BRCA1 recruits the HDAC2 com- latory double-negative feedback loop has been
plex to the miR-155 promoter, which is conse- discovered between miR-152 and DNMT1 in
quently silenced epigenetically through the nickel sulde (NiS)-transformed human bronchial
deacetylation of H2A and H3 histones [61]. The epithelial (16HBE) cells [65]. Expression of miR-
study also showed the upregulation of miR- 152 was specically downregulated by promoter
155 in BRCA1-decient or BRCA1-mutant hypermethylation, whereas ectopic expression of
human tumors. The knockdown of miR-155 in a miR-152 resulted in a remarkable reduction of
BRCA1 mutant tumor cell line attenuates in vivo DNMT1 expression in transformed cells.
tumor growth. However, a knockdown of BRCA1 Interestingly, treatment with 5-AZA or knock
results in a two- to threefold increase in miR-155 down of DNMT1 reversed this process. Further,
levels in vitro. In contrast, a 50 to 150-fold inhibition of miR-152 expression in 16HBE cells
increase in miR-155 in human breast cancer cell was found to increase DNMT1 expression and
lines or tumor samples was observed, suggesting DNA methylation. Moreover, ectopic expression
that this increase may not be caused only by of miR-152 caused a signicant decrease of cell
BRCA1 loss; other transcription factors may acti- growth, whereas inhibition of miR-152 reversed
vate the miR-155 promoter after it is epigenetically this process in 16HBE cells, suggesting the
existence of an important functional negative MiR-34b/c cluster is also epigenetically regu-

feedback loop between miR-152 and DNMT1, lated in CRC (promoter hypermethylation in
likely to play an important role in NiS-induced 90 % of primary CRC samples vs normal colon
lung carcinogenesis [65]. The relationship mucosa) [69], whereas epigenetic silencing of
between miRNA and cognate host gene epigene- miR-9 and miR-148a (together with miR-152,
tic regulation was addressed by Grady et al. by miR-124a, and miR-663) was described also in
studying miR-342, located in an intron of the EVL breast cancer [59].
(Ena/Vasp-like) gene [66]. EVL promoter hyper- Finally, Fazi et al. showed that transcription
methylation occurs in 86 % of colorectal cancers factors can recruit epigenetic effectors at miRNA
and is already present in 67 % of adenomas, sug- promoter regions and contribute to the regulation
gesting that it is an early event in colon carcino- of their expression. The AML1/ETO fusion onco-
genesis. The combined treatment with 5-AZA and protein is the aberrant product of t(8;21) translo-
the HDAC inhibitor trichostatin A restores the cation in acute myeloid leukemia (AML) and can
synchronized expression of EVL and miR-342. bind to the pre-miR-223 region. The oncoprotein
The EGFL7 gene, frequently downregulated in recruits epigenetic effectors (i.e., DNMTs,
several cancer cell lines and in primary bladder HDAC1, and MeCP2), leading to aberrant hyper-
and prostate tumors, hosts miR-126 in one of its methylation of the CpG in close proximity to the
introns. While the mature miR-126 can be AML1/ETO binding site and H3-H4 deacety-
encoded by three different transcripts of the cog- lation of the same chromatin region [70]. In SkBr3
nate host gene, each of them with its own pro- breast cancer cell line, Scott et al. were able to
moter, miR-126 is concomitantly upregulated demonstrate that 27 miRNA expression levels are
with one of EGFL7 transcripts which has a CpG rapidly modied (5 up- and 22 downregulated) by
island promoter, when cancer cell lines are treated a treatment with the HDAC inhibitor LAQ824
with inhibitors of DNA methylation and histone [71], indicating that some miRNAs are mainly
deacetylation, indicating that silencing of intronic silenced by histone modications. In A549 lung
miRNAs in cancer may occur by means of epi- cancer cell line, the HDAC inhibitor SAHA
genetic changes of cognate host genes [67]. In deregulates 64 miRNA (>2 fold change) targeting
summary, miRNAs are encoded by either ncRNA genes involved in angiogenesis, apoptosis, chro-
genes with their own promoters or by noncoding matin modication, cell proliferation, and differ-
sequences in introns of PCGs. In the latter case, entiation [72]. A list of the discussed epigenetically
miRNA expression is usually driven by the same regulated miRNAs is provided.
promoters of the corresponding PCGs. In summary, these studies convincingly sup-
The role of miRNA epigenetic modications port an epigenetic regulation of miRNAs, and the
in the metastatic process has also been investi- fact that cancer cells adopt epigenetic mecha-
gated by several groups. Lujambio et al. treated nisms to silence/re-express key miRNAs modu-
three lymph-node metastatic cell lines with lating relevant PCGs for the development of their
5-AZA and identied 3 miRNAs which showed oncogenic phenotype. The metastatic process
cancer-specic CpG island hypermethylation: also seems to be driven, at least in part, by the
miR-148a, miR-34b/c, and miR-9 [68]. The rein- selected epigenetic regulation of miRNAs, in
troduction of miR-148a and miR-34b/c in cancer addition to the well-known epigenetic regulation
cells with epigenetic inactivation inhibited cell of relevant PCGs.
motility and their metastatic potential in xeno-
graft models and was associated with downregu-
lation of miRNA oncogenic target genes, such as 16.4 Concluding Remarks
c-MYC, E2F3, CDK6, and TGIF2 [68]. Finally,
promoter hypermethylation of these three miR- The series of studies listed in this chapter should
NAs was signicantly associated with metastasis have convinced the readers that a tight connec-
formation also in human malignancies [68]. tion relates miRNAs and epigenetics, and this
relationship harbors signicant implications in 2. Lee RC, Feinbaum RL, Ambros V. The C. elegans het-
erochronic gene lin-4 encodes small RNAs with anti-
the development and spreading of malignancies.
sense complementarity to lin-14. Cell. 1993;75(5):
Aberrancies of the miRNome can effectively be 84354.
reversed by overexpressing miRNAs that are 3. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl
downregulated in cancer and/or by silencing T. Identication of novel genes coding for small
expressed RNAs. Science. 2001;294(5543):8538.
miRNAs overexpressed by cancer cells.
4. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S,
Synthetically generated miRNA-mimic mole- Noch E, et al. Frequent deletions and down-regulation
cules can be effectively delivered to cancer cells. of micro- RNA genes miR15 and miR16 at 13q14 in
Conversely, miRNAs can be administered as chronic lymphocytic leukemia. Proc Natl Acad Sci U
S A. 2002;99(24):155249.
anti-miRNA molecules in case the silencing of a
5. Johnson SM, Grosshans H, Shingara J, Byrom M,
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Immunogenetics of Cancer
17
Armin Hirbod-Mobarakeh, Ali Akbar Amirzargar,
Behrouz Nikbin, Mohammad Hossein Nicknam,
Anton Kutikhin, and Nima Rezaei
Contents 17.4 Immunogenetics ..................................... 298

17.4.1 Background .............................................. 298
17.1 Introduction ............................................ 295 17.4.2 Immunogenetic Tools............................... 298
17.2 Cancers: Why Are There 17.5 Immunogenetics: A Champion
Different Faces?...................................... 296 in Fighting the Losing Battle
17.3 Immune Polymorphism ......................... 296 Against Cancer ....................................... 303
17.6 Human Leukocyte Antigen ................... 304
17.6.1 Background .............................................. 304
17.6.2 Genes Behind HLA .................................. 304
17.6.3 From Polymorphisms to Clinic ................ 306
A. Hirbod-Mobarakeh, MD 17.6.4 HLA Typing and HLA Association
Molecular Immunology Research Center, Studies: Lessons from the Past ................ 308
School of Medicine, Childrens Medical Center, 17.6.5 Typing Methods ....................................... 311
Tehran University of Medical Sciences, 17.6.6 Environmental Factors ............................. 311
Tehran 14194, Iran 17.6.7 Linkage Disequilibrium ........................... 311
e-mail: ahm.armin@yahoo.com
17.7 The Cytokine Network .......................... 312
A.A. Amirzargar, PhD (*) B. Nikbin, MD, PhD 17.7.1 Background .............................................. 312
M.H. Nicknam, MD, PhD 17.7.2 Interleukin-1 Superfamily ........................ 313
Molecular Immunology Research Center, and 17.7.3 Interleukin-4............................................. 316
Department of Immunology, School of Medicine, 17.7.4 Interleukin-6 (IL-6) .................................. 317
Tehran University of Medical Sciences, 17.7.5 Interleukin-8............................................. 318
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran 17.7.6 Interleukin-10........................................... 319
e-mail: amirzara@tums.ac.ir; 17.7.7 Interleukin-12........................................... 323
dnik@ams.ac.ir; nicknam_m@yahoo.com 17.7.8 Tumor Necrosis Factor-
A. Kutikhin, MD, PhD and Lymphotoxin- .................................. 324
Department of Epidemiology, Kemerovo State 17.7.9 Interferon Gamma (IFN-).................. 330
Medical Academy, Kemerovo, Russian Federation 17.7.10 Transforming Growth
e-mail: antonkutikhin@gmail.com Factor- (TGF-)...................................... 330
N. Rezaei, MD, MSc, PhD (*) 17.8 Concluding Remarks ............................. 333
Research Center for Immunodeciencies,
References ............................................................... 333
Childrens Medical Center, Pediatrics Center of
Excellence, Tehran University of Medical Sciences,
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran 17.1 Introduction
Department of Immunology, School of Medicine, and
Molecular Immunology Research Center, The inuence of genes in the development of
Tehran University of Medical Sciences,
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran cancers can be very high, very well depicted in
e-mail: rezaei_nima@tums.ac.ir numerous hereditary cancers or very low in some

296 A. Hirbod-Mobarakeh et al.
Fig. 17.1 Heritability 100 %

of cancers in different sites 90 %
based on the information 80 %
available from twin studies 70 %
60 %
50 %
40 %
30 %
20 % 42 36 35 31 28 27
10 % 26 22 21
0%
e s m h t ia
at ea ac as ng
ry
r
tu m
de
t r e Lu e
va
c
os nc re om Br uk
ad
O
Pr Pa ol
o St Le
Bl
C
Heritable factors Environmental factors
cancers. Although the roles played by genes in the development of cancers can be very high,
the pathophysiology and prognosis of the very well depicted in numerous hereditary can-
malignant transformation are highly variable in cers like familial adenomatous polyposis, or
different cancers, their role cannot be ignored. very low in some cancers like cancer of the cer-
For sure, polymorphisms in immune-related vix (Fig. 17.1) [5]. Although the roles played by
genes, known as immune polymorphisms, have genes in the pathophysiology and prognosis of
an undeniable role in shaping undeniable but the malignant transformation are highly variable
complex interactions between the immune sys- in different cancers, their role cannot be ignored
tem and malignancies which can signicantly [6, 7]. Malignant transformation is not just a
inuence the face of malignancy with respect to result of a cell-autonomous process and is
predisposition, nature, prognosis and response to shaped by intrinsic properties, but also its cross
treatment in each individual. talk with microenvironment governed by the
immune and endocrine systems, stroma, vascu-
lar system, and other systems [6]. Therefore,
17.2 Cancers: Why Are There this heritability results from additive effects of
Different Faces? low-penetrance genetic factors, each one con-
tributing a small amount of risk [6].
It has been long observed that individuals are
different with respect to predisposition nature,
prognosis, and response to treatment in cancer 17.3 Immune Polymorphism
[1, 2]. Since the rst observations, scientic
minds have been preoccupied with the question The role of immune system in defense against
that, what is the reason for this high inter-indi- malignancies was proposed in the early 1990s by
vidual variation. Nowadays, it is obvious that Paul Ehrlich [8]. So far this book, page by page,
behind the ugly scene of cancers, there is a has tried to show the undeniable but complex
complex interplay between genes and environ- interactions between the immune system and
ment and this question can be answered malignancies. This complex interaction mostly
straightforwardly by the high variability of results from the manipulation of the immune sys-
genetic and environmental factors for each indi- tem by cancer cells evoluting to prevent self-
vidual [1]. Although it is estimated that less destruction [8]. Four phenomena contribute to
than 0.1 % of the genome is different between the escape of malignant cells from the
any two individuals, this variability is equal to immunosurveillance:
at least several million nucleotide differences 1. Immunoedition: Natural selection of malignant
per individual [3, 4]. The inuence of genes in cells which are most successful in deceiving
17 Immunogenetics of Cancer 297
Microsatellite VNTR
SNP
A CA.CA ....CAGTAAGCGTTA...
2 repeats 15100 BASES
12 repeats
C CA.CA.CA.CA
....CAGTAAGCGTTA...
4 repeats
15100 BASES
16 repeats
Fig. 17.2 Different types of polymorphisms in the human genome
the immune system occurs by the pressure of Genetic polymorphisms are defined as
the immune system itself. This pressure leads variations in human genome present in at least
a somatic evolution toward variants procient 1 % of the population [16]. These polymor-
in immune escape in primary tumor lesions phisms were beneficiary either in their cross
[9, 10]. talk with certain environmental factors alone
Down regulation of the local immune system: or in combination with their associated poly-
Several tumors can manipulate the local defense morphisms, or they were at least neutral
by producing inhibitory molecules such as enough not to compromise the life of the indi-
indoleamine 2,3-dioxygenase (IDO) and differ- vidual bearing them; therefore they were not
ent cytokines or expression of apoptose- erased by the evolutionary pressure [14, 16,
inducing ligands such as Fas-ligand [9, 11] 17]. Immune response-associated genes are
2. Tolerance induction and losing immunogenic- not an exception, and they have an uncount-
ity: The absence of co-stimulatory molecules, able number of polymorphisms [14]. For
localization in natural environment of healthy example, HLA region includes the most poly-
cells and therefore absence of danger signals, morphic genes in the human genome [14]. This
losing human leukocyte antigen (HLA) class I high variety in immune-associated genes is a
molecules, and aberrant expression of immu- product of a long interaction with an environ-
nomodulatory non-classical HLA class I ment consisting of numerous ever-evolving
antigen (Ag) can all induce tolerance in the pathogens [14]. In this context, majority of
immune system [9, 11, 12]. polymorphisms had the chance to be benefi-
3. Host immunodeciency: Any deciency in the ciary in defense against some pathogens [15,
immune status of individuals can predispose 18, 19].
them to various malignancies. Single nucleotide polymorphisms (SNP),
In addition, once the immune escape occurred, variable number of tandem repeats (VNTRs) (a
the immune system can profoundly inuence the repeat unit includes 15100 nucleotides) and
prognosis, natural history, and response to differ- microsatellites are three important types of
ent therapies either by direct effects on malignant polymorphisms [20].
cells or indirect effects on angiogenesis and SNP is dened as a difference in a single
inammation [9, 1113]. nucleotide in the DNA sequence and is estimated
The immune system of each individual is to account for 90 % of the human genome varia-
subject to variability due to different environ- tions. Microsatellites, scattered through the
ments, different diets and nutritional status, and genome with an average density of one in every
different antigenic exposures and most impor- 2,000 pb, are variable tandem repeats of 28 bp,
tantly due to an uncountable number of polymor- most commonly CA dinucleotide, and their
phisms in genes governing the immune system alleles are differentiated by the number of repeats
elements and cells [14, 15]. (Fig. 17.2) [20, 21].
Polymorphisms are able to change the immune Although the term immunogenetics was
function at several levels from expression pat- used earlier [29], the rst milestone in the history
terns to posttranslation modications: of immunogenetics was coincident with the failed
1. Some polymorphisms might change DNA study of blood transfusion in 1952 [30]. This fail-
methylation and consequently chromatin ure resulted in the discovery of HLA system [14,
structure and expression patterns [22, 23]. 31], which attracted the attention of biomedical
2. Some polymorphisms may disrupt transcrip- researchers to interindividual differences in the
tion factor binding sites (TFBSs) and conse- immune system. From that point on, for decades,
quently inuence the expression [20, 24, 25]. investigators tried to associate different complex
3. mRNAs splicing patterns can be modied by diseases with various HLA types using serologi-
polymorphisms as a result of deletion of a cal methods [32, 33]. However, modern immuno-
splice site, creation of a new splice site, or genetics required more than one century of
modication of exon-splicing enhancers and biomedical advances remarked by Mendels laws
silencers [24]. of heredity in 1865 [16, 34], discovery of chro-
4. MicroRNAs (miRNAs) are important ele- mosomes as the cellular basis of heredity in 1902,
ments in gene regulation with various actions. discovery of DNA double helix as the molecular
Their binding sites might be disrupted as a basis of heredity in 1953 [35], decoding the
result of polymorphisms [24]. genetic codes, and last but not least the completion
5. Some polymorphisms can cause mRNA insta- of Human Genome Project in April 2003 [16, 36,
bility and its early destruction [20, 26]. 37]. Human Genome Project not only contributed
6. Polymorphisms may create premature termi- to the discovery of genetic polymorphisms but
nation codons [24]. also provided a infrastructure for other large-
7. Exonic polymorphisms can substitute an scale projects like International HapMap Project
amino acid in protein sequence, change pro- and 1,000 Genomes Project [38]. Discovery of
tein structure, and consequently alter protein approximately 2535 % of estimated nine to ten
function [20, 25, 26]. million SNPs is just one of the uncountable
8. Some polymorphisms may change post- achievements of such projects [14, 3739].
translational modication (PTM) site and Genetic polymorphisms in the immune system
consequently inuence posttranslational mod- contribute to a large part of the interindividual
ications [24]. variation in immune response and today, immu-
Therefore, it seems that this high genetic vari- nogenetic studies have provided a vast knowl-
ability in immune response associated genes edge of the effects of immune polymorphism on
known as immune polymorphism contributes to the host defense. However, just the estimation
the observed interindividual differences [14, 19]. that there is one SNP per every 290 bp shows that
there is much more to be brought to light [38, 39].
17.4 Immunogenetics
17.4.2 Immunogenetic Tools
17.4.1 Background
Along with the concert of conceptual advance-
Immunogenetics, as the meeting point of two ments, tools employed in this eld have changed
exciting elds of immunology and genetics, is a in order to gather immunogenetic information
new but rapidly expanding eld of science study- more accurately, in less time and less cost [14].
ing this immune polymorphism in order to under- Twin studies recruit twins in order to remark the
stand the governance of genetics on the immune importance of genetic component in susceptibil-
system [14, 27, 28]. ity to traits and diseases [16, 40]. The result of
such studies provides a rough estimation of block of neighboring SNPs; in 2002, the
genetic contribution to interindividual differ- International HapMap Project, as a global
ences in immune system by comparison of movement, began to identify these blocks
concordance rates of immune traits between (known as haplotypes) and pattern of LD in the
monozygotic and dizygotic twins [16, 33, 40]. human genome [38, 39]. LD results in organiza-
The higher the concordance difference is, the tion of genetic variation in haplotype blocks
greater the heritability [7, 16]. with strong LD separated by recombination
Upon introduction of immune polymorphism, hotspots [16, 39]. The information from this
several association studies tried to show the con- project provided the immunogenetic scientists
tribution of specic genes using the candidate with the most suitable SNPs for genotyping in
gene approach or hypothesis-driven approach order to indirectly gather as much as informa-
[16, 41]. This approach includes looking into the tion about the genome variation of an individual
differences between patients and controls in [16, 46]. These SNPs, which are representative
allele frequencies of SNPs in genes selected of a block of SNPs, are known as tagSNPs. The
based on the known pathophysiologic pathways extent of LD in a region determines the number
of the disease. These studies at rst employed of tagSNPs required to cover a region. The
restriction enzymes to identify specic SNPs lower the LD is in a region, the higher number
called restriction fragment length polymorphisms of tagSNPs are needed and therefore the higher
(RFLPs) in the restriction site of the enzyme [42]. the cost of genotyping the region is [47].
This approach is also known as a reductionist Nowadays, availability of high-throughput gene
approach, since studies employing this approach technologies such as gene chips or microarrays
investigate only a few genes and polymorphisms has enabled investigators to genotype cost-
at a time [16, 41, 43]. effectively, rapidly, and almost effortless hun-
In the early 1990s, discovery of hundreds of dreds of thousands to millions of SNPs at the
informative microsatellites provided the possibil- same time [4, 33, 41, 44]; therefore this approach
ity of a dramatic change in the approach of immu- is also known as nonreductionist approach
nogenetic studies from a hypothesis-driven [4]. These technological advancements were
approach to positional approach [4, 16, 44]. In employed in community-based and large-scale
this approach, studies known as genome-wide GWASs in order to identify trait-associated
association studies (GWAS) mainly aim to iden- regions with higher resolution. The results of
tify the genome regions bearing disease-associated such studies is a trait-associated SNP (TAS) as a
genes and to localize causal genetic variants of representative of the true casual variant which
disease as accurately as possible [44, 45]. might be each of the known and unknown vari-
Therefore, in this approach, new hypothesis are ants in whole TAS block. The TAS block is
generated after making thousands of unbiased dened as all known and unknown polymor-
observations [4, 33, 41, 44]. They are especially phisms in strong LD with the tagSNP [4, 16,
helpful in order to nd unexpected genes as repre- 48]. Therefore, LD along with technological
sentatives of unknown disease-related pathway advances turned SNPs, the most common and
[4, 16]. In mid-1990s, early GWASs employed more importantly the most stable genetic varia-
informative microsatellite markers distributed tions in human DNA, into application [49].
evenly in the 23 chromosomes and investigated However, there are major limitations in
their aggregation in multi-case families and large GWASs to be overcome.
pedigrees identied major susceptibility loci for 1. Generally, the genetic component of complex
complex diseases [4, 42, 44]. diseases originates from several major suscep-
By introduction of linkage disequilibrium tibility loci and a component of as many as a
(LD) dened as the coinheritance of alleles of a dozen minor susceptibility loci known as
Fig. 17.3 Immune

polymorphism component in ividual
inter-individual differences Inter-ind es
d if fe re nc
Genetie s
hism
polymorp
Immune s
hism
polymorp
es
Polygen
polygenes (Fig. 17.3). These polygenes and environmental factors. It is possible that
individually have small to medium impact on the effect of one variant depends on the pres-
the overall genetic component; therefore, ence of one or several specic alleles in
GWASs require a large study sample with another locus or even certain environmental
homogenous ethnicity and phenotype to have risk factors. Therefore, such susceptibility
enough high power to identify these poly- variants can be detected only in GWASs with
genes [4, 21, 48, 50]. This is a major problem samples of patients with particular genetic and
in immunogenetic studies of cancers as environmental background [6].
patients with cancers present with highly vari- 4. At least 10 % of SNPs within a range of
able phenotypes. As a result, the odds ratio for 1 kpb of hotspots are untaggable which means
each allele is typically below 1.5, and the P they dont have any LD with tagSNPs [47].
value should be less than 106 to show a The presence of these numerous untaggable
signicant association [6, 51]. SNPs always limits the power of GWASs in
2. The genetic component and therefore effect of nding all possible genetic associations [39].
any risk allele decreases by increased expo- Therefore, GWASs should employ additional
sure of populations to environmental risk fac- sequencing within known recombination
tors which is the reason why some results hotspots [39].
could not be replicated in different popula- 5. GWASs are less effective in some old popula-
tions [6]. For example, increased prevalence tion like African countries, since LD is gener-
of acquired immune deciency syndrome ally lower in these populations due to the
(AIDS) in some African populations predis- longer duration being affected by genetic
poses population to different cancers disre- recombination [4, 16, 48, 49].
garding their genetic background [52, 53]. 6. The different LD, hotspots, and haplotype
This is also the case in regard to some extreme patterns in different populations might com-
dietary patterns, smoking habits, and other plicate replication studies in different popula-
environmental factors [54, 55]. tions [49]. For example, in some population,
3. Some cancer susceptibility variants have the causal variant may be separated from the
nonadditive interactions with other genetic associated TAS block by a hotspot.
7. Sometimes the associated TAS block does not as a powerful statistical method is essential
include a causative allele but an allele [26, 63]. Meta-analysis by pooling the results of
beneciary for the affected individuals with old studies allows us to see the whole picture of
the disease, and therefore the natural selection the effect of a certain polymorphism [26].
has selected them instead of those affected Regardless of interspecies differences, there
individuals without the allele [16]. are similarities in cancer development between
8. Population stratication is another source of humans and rodents, and therefore mouse studies
bias in such studies as the association of the are a complementary tool for genetic association
trait and TAS block may be due to an ancient studies within human population [6, 64, 65].
branching of the population bearing both Numerous genetically engineered mouse (GEM)
causal trait alleles and the TAS block; models provide a simplied model of various
however, this bias can be minimized by the cancers with controllable genetic and environ-
careful selection of the control group or by mental background in which the effects of a
assessing population structure and correcting unique polymorphism on the malignancy can be
for it [16, 49, 56]. studied [6, 66].
9. If certain alleles are associated with a more Exact mechanism of action of polymor-
aggressive disease and lower survival, they phisms can be identied using different bioin-
are less presented in patients and may not be formatic tools and in vitro studies [24].
detected as a susceptibility allele [57]. Numerous bioinformatic online and ofine tools
After identication of associated TAS blocks are available which can predict the effect of
by GWASs, the actual functional variant in the polymorphisms by considering amino acid bio-
associated TAS block can be found by further physical properties, active site residues, metal
genetic association studies employing more and lipid binding sites of gene product, TFBSs,
accurate low-throughput technologies and other splice sites and its regulatory motifs, miRNA
SNP markers in order to nely map the associ- binding sites, and PTM sites (Table 17.1) [24].
ated genes and alleles in the associated TAS However, bioinformatics is limited by the extent
block [44]. In these studies, allele frequencies of of our knowledge [22, 24].
polymorphisms are compared in groups of cases Different in vitro methods are developed to
and controls. However, results of such associa- identify functional polymorphisms. The most
tion studies are often contradictory due to the het- important ones are reporter gene assay and elec-
erogeneous nature of the cancers, numerous trophoretic mobility shift assay (EMSA)
genegene and geneenvironment interactions (Figs. 17.4 and 17.5) [22]. The reporter gene
[58, 59]. In addition, another source of discrep- assay employs a reporter gene with a quantiable
ancy between these studies is the limitation in product and clones the promoter of interest in its
study design. For example, using hospital-based upstream [22, 67, 68]. Therefore, quantication
controls can result in a serious selection bias of reporter gene product can provide information
since polymorphisms under investigation might about the promoter strength [22, 67, 68]. On the
have association with the diseases that hospital- other hand, EMSA can measure the effect of dif-
based controls may have [60, 61]. Moreover, ferent polymorphisms on the afnity of TFBS
some association studies failed to consider other sequence for different transcription factors. In
genetic and environmental risk factors such as these studies, double-stranded oligonucleotide
socioeconomic status, nutritional statues, smok- containing the polymorphism of interest is mixed
ing patterns, etc. [60]. Lacking such information with nuclear extract with various transcription
may cause serious confounding bias [62]. factors [22, 69, 70]. Higher afnity for these fac-
Therefore, in order to get the most benet from tors results in the formation of more protein
results of genetic association studies and to sys- DNA complex resulting in retardation of mobility
tematize their ndings, employing meta-analyses in electrophoresis [22, 69, 70].
Table 17.1 A small example of different bioinformatics tool

Title Address Description
dbSNP http://www.ncbi.nlm.nih.gov/SNP/ A database for SNP information
Ensembl http://www.ensembl.org/ A database for genome information, comparative
genomics, variation, and regulatory data
HapMap http://www.hapmap.org/ A database for haplotype blocks
consortium
SNPper http://snpper.chip.org/ Online tool available for SNP analysis
SNP3D http://www.snps3d.org/ Online tool available for functional analysis of SNPs
based on structure and sequence analysis
SNPeffect http://snpeffect.vib.be/index.php A database for phenotyping human SNPs and for
nding information regarding SNPs effect on structure
stability functional sites, structural features, and
PTM sites
MutDB http://www.mutdb.org/ Online database for human variation data with protein
structural information and other functionally relevant
information
dbSNP http://www.ncbi.nlm.nih.gov/SNP/ A database for SNP information
Ensembl http://www.ensembl.org/ A database for genome information, comparative
genomics, variation, and regulatory data
HapMap http://www.hapmap.org/ A database for haplotype blocks
consortium
SNPper http://snpper.chip.org/ Online tool available for SNP analysis
SNP3D http://www.snps3d.org/ Online tool available for functional analysis of SNPs
based on structure and sequence analysis
SNPeffect http://snpeffect.vib.be/index.php A database for phenotyping human SNPs and for
nding information regarding SNPs effect on structure
stability functional sites, structural features, and
PTM sites
MutDB http://www.mutdb.org/ Online database for human variation data with protein
structural information and other functionally relevant
information
Construct of oligonucleotides with Mixture with

different alleles of the polymorphism transcription factors Electrophoresis
under study
Allele with higher affinity
Allele with lower affinity
polymorphism Transcription factor
Fig. 17.4 EMSA, an in vitro experiment to measure binding afnities of different TFBS for transcription factors
Promoter under study Reporter gene
Promoter with higher activity
Promoter with lower activity
Construct of reporter vector transcription and Quantification of

with the promoter under study translation reporter product
(measured by
light or enzyme
activity)
Fig. 17.5 Reporter gene assay, an in vitro tool to measure strength of different promoters
Fig. 17.6 Different methods

in immunogenetic studies are
pieces of a complex puzzle Meta analysis
Genetic
association
studies Mouse studies
GWAS
Bioinformatic
tools
Family studies
In vitro studies
Twin studies
The results from immunogenetic studies should 17.5 Immunogenetics:

always be interpreted with consideration of infor- A Champion in Fighting
mation from immunogenomics and immunopro- the Losing Battle Against
teomics [33]. It should be noted that information Cancer
from each type of study i.e., GWASs, genetic asso-
ciation studies, in vitro and mouse studies and bio- The application of immunogenetics in cancer is more
informatics, are just pieces of the complex puzzle of than promising. Some variations in immune poly-
immunogenetics and cancer. No individual method morphism reduce the immune capacity in clearing
is precise enough to see the nal picture (Fig. 17.6). either malignant transformations or cancer-inducing
infectious agents and predispose bearing individuals the surface of almost all nucleated cells and gen-
to various cancers as exaggerated in case of most erally present processed endogenous antigens to
primary immunodeciency diseases [4, 16, 19, 33]. CD8+ cells [13, 76]. Presentation of abnormal Ags
Although each individual variant has a little infor- derived from intracellular pathogens or malignant
mative potential for clinical application, understand- transformations potentially initiate a cytotoxic T
ing their interactions and therefore their cumulative lymphocyte (CTL) response and consequently
effect is of high clinical importance [6]. target cell lysis [77]. By their interaction with
Immunogenetic studies not only can help killer cell immunoglobulin-like receptors (KIRs)
clinicians in risk assessment of individuals for sus- on the surface of natural killer (NK) cells, HLA
ceptibility to certain cancers in order to employ class 1 antigens regulate lytic activity of NK cells.
preventive strategies but also may open new win- Therefore, any change in either in expression or
dows for treatment [4, 16, 19, 33, 48, 7173]. structure of HLA class 1 profoundly inuence T
GWASs might result in the identication of unex- and NK cell-mediated immunity [10].
pected genes which in turn result in identication On the other hand, HLA class 2 Ags are
of new pathways in pathophysiology of cancers exclusively expressed on the surface of profes-
[48]. These new pathways not only provide a sional antigen-presenting cells (APC) and pres-
broader insight into how and why of the cancers ent processed exogenous Ags to T helper (Th)
but also may suggest new molecular targets for cells. Following presentation of unfamiliar Ags
prevention and immunopharmacology and immu- and in the presence of appropriate costimulatory
notherapy [4, 16, 33, 42, 48]. Keeping in mind that molecules, Th cells activate effector elements of
immune system provides the only antineoplastic the immune system [13, 77].
reaction completely specic to cancer cells, it is Both classes of Ags comprise an intracellular,
vital to completely understand the genetic factors transmembrane, and an extracellular part which
governing the immune systemcancer interactions includes highly polymorphic antigen binding
and employ this knowledge in eliminating the can- groove. From the evolutionary view, this high
cers [4, 74]. In addition, this knowledge might variety favors the chance of heterozygosity and
begin a post-genomic era in individualized medi- consequently Ag presenting potential for each
cine [4, 33]. The presence of some variants in individual along with a signicant increase in the
immune associated genes might affect the success general repertoire of the whole specie for Ag
or failure in applying a particular therapy and presentation [14, 77].
immunogenetic information provides a way to pre-
dict toxicity and clinical effectiveness of different
immune-based therapies [4, 14, 20, 33]. Therefore, 17.6.2 Genes Behind HLA
employing the knowledge from immune polymor-
phism in prediction of treatment outcome may jus- HLA loci, located in 6p21.3 region, occupy only a
tify the application of an expensive partly effective small part of major histocompatibility complex
treatment option [4, 14, 33, 75]. (MHC) genetic system which is home to at least
220 genes [78, 79] (Fig. 17.7). MHC is divided into
three classes of genes distributed from centromere
17.6 Human Leukocyte Antigen to telomere. Class 2 with 0.9 mb is the nearest one
to the centromere; class I with 19 Mb is near telo-
17.6.1 Background mere, and class 3 with 07 Mb lies in between [80].
The rst two classes encode for HLA class 1 and 2
Human leukocyte antigens are specialized ele- and the third class consists of a group of genes
ments of the immune system in recognition of self encoding some members of the complement
from non-self. HLA is responsible for presenting system, some cytokines like tumor necrosis factor
Ags to T cells and therefore serves as a door to the alpha (TNF-), heat shock proteins (HSP) and an
specic immune system. HLA class 1 Ags are on enzyme called 21-OH hydroxylase [31, 80].
6p21. 1-21.3
DP DQ DR
B A B A B A MICA MICB B C E A G F
21-OH C4 BF C2 HSP70 LTB TNF LTA

HLA2 HLA3 HLA1
0.9 106 bp 0.7 106 bp 1.9 106 bp
B2
Exogenous HLA 1 microglobulin
antigen HLA2
chain
TCR
chain Endogenous
antigen
APC
CD 8+ cells Malignant NK cells
Th cells cell
Antigen presentation Absence of HLA 1 on

by HLA1 to CD8+ the surface in
cells interaction with NK cells
polarization and amplification of

the adaptive immune response
Fig. 17.7 HLA as the gate of adaptive immunity from genes to function
In class 1, there are three highly polymorphic tion within this gene guarantees that there is a
classic genes known as HLA-A, HLA-B and HLA- constant protection for healthy cells in most peo-
C, while there are numbers of nonclassical genes ple and provides a minimum safeguard for auto-
known as HLA-E, HLA-F and HLA-G [81, 82]. immunity [32, 84, 85]. Some of them like HLA-G
Class 1 genes encode the highly polymorphic are expressed on trophoblastic cells and placental
heavy chain of HLA class 1 (45 kDa) which later chorionic endothelium and induce immune
joins the non-polymorphic B2 microglobulin tolerance during pregnancy [81, 8690].
encoded by chromosome 15 [81, 82]. Classic Class 2 consists of classic genes called DP, DQ
genes consist of eight exons, but the most and DR and nonclassic genes known as DM and
important exons are exons 2 and 3 encoding for DO. Classic genes encode for one highly poly-
peptide binding groove. Other exons encode for morphic beta chain (2628 kDa) and a less poly-
transmembrane region and cytoplasmic tail [31, morphic alpha chain (3335 kDa) [80]. Therefore,
83]. Beside these highly polymorphic classic there are six classic D genes in this region. Genes
HLA class 1 genes, there are three other HLA for alpha chain consist of ve exons, while beta
genes in class 1 known as HLA-E, HLA-F and chains are encoded by six exons. The exons 2 and
HLA-G which are more conserved. Most 3 in both set of genes are responsible for encoding
probably, they are not involved in Ag presenta- peptide binding domains [31].
tion but in interaction with more conserved parts HLA class 1 and 2 genes are the most poly-
of the immune system. For example, HLA-E, morphic genes in the human genome with 2,365,
which is minimally polymorphic, regulates cyto- 3,005 and 1,848 alleles for HLA-A, HLA-B and
toxic activity of NK cells by interacting with HLA-C, respectively, and 2,156 alleles for class 2
CD94/NKG2 lectin-like receptors. The conserva- genes (based on IMGT/HLA database, release
3.13 on July 2013) [91]. This high polymorphism 2. Differentiation Ags are normally expressed in
is mostly clustered in several hypervariable the tissue of origin of the tumor, like Melan-A,
blocks in exons 2 and 3 which are responsible for and tyrosinase in melanomas
encoding antigen binding groove. Therefore, a 3. Unique tumor Ags are products of mutated
unique combination of sequence motifs in these tumor suppressor genes and oncogenes like
hypervariable regions determine each allele [13]. abnormal product of RAS or p53. Fusion
This genetic structure is accompanied by high proteins as a result of chromosomal aberra-
LD not only between HLA genes but also non- tions are also included in this group.
HLA genes constituting extended haplotypes 4. Infectious tumor Ags are expressed by onco-
[92]. The majority of polymorphisms in hyper- genic viruses associated with some malig-
variable regions result in amino acid substitu- nancies. The examples are latent membrane
tions in peptidebinding grooves, which in turn proteins 1 and 2 (LMP-1 and LMP-2) in
dramatically changes Ag binding afnity of the Epstein-Barr Virus (EBV)-associated Hodgkin
nal product [13]; on the other hand, variants in lymphoma (HL) and E6 and E7 associated
noncoding regions, inuence transcription, with human papillomavirus (HPV)-associated
translation, and splicing and thereby expression cervical cancer.
levels [77]. Nowadays, hundreds of HLA association
Nowadays, with a few exceptions, HLA alleles studies prove that HLA alleles are important ele-
are named by six or even eight digits. The rst ments in predisposition to cancer. Seven mecha-
two digits are representative of the serological nisms are suggested for complex relationship of
family the allele belongs to, while the third and HLA genotypes and susceptibility, prognosis,
fourth digits distinguish between different recurrence, and clinical response to immunother-
sequences affecting amino acid sequences. The apy and tumor vaccines:
next two digits are identiers of synonymous 1. Efciency in TAA presentation: One of the
polymorphisms, and seventh and eighth digits are major factors in Ag presenting ability of dif-
used to distinguish intronic polymorphisms or ferent HLA is the afnity of their Ag binding
ones located into untranslated regions [93]. grooves to different epitopes. This afnity is
highly dependent on the amino acid sequence
in the hypervariable regions. Even one change
17.6.3 From Polymorphisms to Clinic in this sequence due to polymorphisms pro-
foundly inuences binding afnities to TAAs
HLAs are involved in cancer immunity and and Ags used in tumor vaccines and therefore
therefore in susceptibility and prognosis mainly susceptibility prognosis and response to tumor
by presenting certain Ags known as tumor- vaccines [32, 82, 9597]. For instance, HLA-
associated antigens (TAA). TAA are the rst con- A*0207 is associated with susceptibility to
tact of malignant cells with adaptive immunity. EBV-associated lymphoma in East Asian pop-
Since introduction of the rst TAA in melanoma ulation, while HLA-A*0201 is a protective
patients in 1991, a broad heterogeneous group of factor; however, this huge difference at the
Ags were discovered and associated with differ- clinical level is a result of a single amino acid
ent malignancies. This heterogeneous group can change (Y99 to C) at the protein level [98, 99].
be divided in to four classes of Ags [8, 94]: 2. Interaction with T cells and NK cells: Change
1. Cancertestis Ags are a result of epigenetic in variable regions and constant regions
alterations leading to reactivation of silence involved in interaction with T cells and NK
genes. One of the famous examples is Ags cells can change HLA potential for inducing
from MAGE family. These Ags are not exclu- an effective immune response [96, 100].
sive to just one type of cancer. The reason for 3. Efciency in inducing immune response to
this naming is that they are normally expressed infectious agents: Antigen binding abilities of
in MHC-negative testicular germ cells and different HLA alleles inuence immune reaction
placental trophoblasts. to infectious agents associated with malignant
transformation. For example, EBV is frequently In addition, chronic immune stimulation of

emphasized as an important environmental fac- B cells and prolonged and repeated DNA
tor in the pathogenesis of HL and nasopharyn- double-strand breaks associated with somatic
geal carcinoma (NPC) [101]. Latent membrane hypermutation (SHM) and class switch recom-
protein-1 (LMP-1) and Epstein-Barr virus bination (CSR) signicantly increase the chance
nuclear antigen (EBNA-4 and EBNA-6) proof malignant transformation, and therefore,
teins produced during latent infection by EBV, autoimmunity and chronic infection are impor-
are efciently presented by A*0201 and A*1101 tant risk factors for some hematological malig-
respectively [83]. Therefore, these alleles can nancies like non-Hodgkin lymphoma (NHL)
induce a strong immune response which conse- [107]. In these cases, HLA alleles can affect the
quently results in resolving the infection and extent of immune reaction and stimulation of B
lower chance of malignant transformation. cells [107]. For instance, HLA-DRB1*0301,
Another example is the protective effect of HLA-B*0801 HLA-DRB1*0101, and HLA-
DQB1*0301 allele on hepatitis C virus (HCV) DRB1*0401, the susceptibility alleles of NHL
infection, HCV-associated liver cirrhosis, and is associated with autoimmune diseases such as
HCV-associated Hepatocellular carcinoma systemic lupus erythematosus (SLE), rheuma-
(HCC). This allele can efciently present major- toid arthritis (RA), Sjgrens syndrome, and
ity of immunodominant epitopes of HCV [102]. celiac disease [97, 102, 108]. The more promi-
4. Change in HLA expression patterns: In some nent example is the paradoxical relationship of
malignancies like melanoma, Burkitts DQB1*0301 with HCV infection and HCV-
lymphoma, and carcinoma of the cervix and related B-cell lymphoma. While DQB1*0301 is
lung, HLA expression and Ag processing associated with a better immunologic control of
machinery are disturbed in order to prevent HCV and a self-limiting infection, it is a suscep-
TAA presentation and consequently immune tibility factor for HCV-related NHL. In this
recognition of malignant cells. This mechanism case, efcient presentation of viral antigens by
is one of the major pathways for the immune DQB1*0301 in the context of persistent HCV
escape of tumoral cells [10]. Some polymor- infection results in CD4+-dependent chronic
phisms within the noncoding regions can inu- stimulation of B cells [102].
ence expression levels [32]. In addition, some 6. Sensitivity to mutation: It is suggested that
HLA alleles are specically lost during malig- some HLA alleles are more susceptible to
nant transformation [103]. Loss of HLA-A2 in mutations like rearrangements of the DNA
colorectal cancers, breast cancer, and cervical material and crossover. Such dramatic altera-
cancer or lower expression levels of HLA-DR4 tions might inuence the function of onco-
and HLA-DR6 in melanoma is a good example genes or tumor suppressors in the proximity of
for these phenomena [104, 105]. On the con- HLA genes. An example of such an oncogene
trary, some alleles like HLA-B*4405 are not is Waf1/p21 gene, located in 6p21.1 [100].
dependent on some elements of the regular Ag 7. Linkage disequilibrium: LD with non-HLA
processing machinery like transporter associ- genes of class 3 or even nonclassical HLA in
ated with Ag presentation (TAP) and therefore, the form of extended haplotypes can justify
can present antigens without susceptibility to some of the founded associations. Some clas-
viral-induced diminished TAP function [106]. sical genes are in LD with certain HLA-G and
5. Increased susceptibility to chronic infections HLA-E alleles which are both involved in
or autoimmunity: Some HLA haplotypes and suppression of NK cell-mediated immunity
alleles are associated with various chronic against tumors [73]. LD with non-HLA genes
inammatory diseases which in turn predispose like TNF-, in context with extended haplo-
individuals to various cancers [75, 107]. Excess type, can inuence the relationship between
growth factors and prolonged proliferation in toxicity of immunotherapy and HLA alleles.
the background of chronic destruction increase For example, high TNF- increases the IL-2
the risk of malignant transformation [107]. toxicity in patients with melanoma [109, 110].
17.6.4 HLA Typing and HLA alleles. In this method which is based on dye termi-
Association Studies: nator chemistry, dye bounded 2,3 dideoxynucleo-
Lessons from the Past tides are used as substrates for PCR process.
Randomly addition of labeled dideoxynucleotides,
HLA has a history as long as immmunogenetics and consequently, a stop in elongation of DNA
itself. An observation of transfusion failures in chain result in the development of numerous DNA
1952 paved the road to the discovery of the rst fragments with different sizes. These DNA frag-
HLA allele by Jean Dausset in 1958 [111]. Since ments can easily be separated by capillary electro-
1958, there was a continuous international effort phoresis, and the ending dideoxynucleotides can
in order to share experimental data and HLA typ- be identied by specic uorescence emitted from
ing technologies, identify new HLA alleles and the related dye.
serotypes, and uncover the role of HLA system in In parallel, huge efforts were made to under-
pathogenesis of numerous diseases [31]. The stand the role of these alleles in etiology and nat-
result of such effort was the identication of over ural history of several diseases. In oncology, the
9,500 alleles for HLA class 1 and 2 over a short rst association was found in HL in 1967 [32].
period of four decades [31]. Along with the dis- This nding triggered a series of HLA associa-
covery of new alleles, the rst nomenclature tion studies on different cancers worldwide. The
committee was held in 1987 followed by several fruit of this global movement was nding associ-
nomenclature committees to unify the nomencla- ation between HLA alleles and susceptibility to
ture and classication [31]. several hematological malignancy including HL,
Early studies employed low-resolution serolog- NHL, childhood acute lymphoblastic leukemia,
ical methods which detected HLA on T cells or B Kaposis sarcoma, chronic myeloid leukemia
cells [112]. Although these serological methods (CML), and also non-hematological malignan-
were subject to huge development in detection cies including nasopharyngeal carcinoma, thy-
methods from complement-dependent cytotoxicity roid cancer, renal cell carcinoma (RCC), cervical
test to ELISA method, ow cytometry, and cancer, and both melanoma and non-melanoma
Luminex technique, the real breakthrough in HLA skin cancers [13, 115]. Moreover, investigations
association studies was the introduction of PCR on natural history of cancers showed relationship
and high resolution DNA-based typing methods of several alleles from both classes with mortality
[31]. This technology allowed not only detection in ovarian cancer, non-small cell lung carcinoma,
of high HLA polymorphisms with higher sensitiv- head and neck squamous carcinoma, and local
ity and specicity but also the detection of new recurrence in melanoma [73, 96, 100]. Several
alleles with more exibility by simply adding new studies showed importance of HLA context in the
probes to the old panels [113]. Nowadays, the old outcome of immunotherapy and tumor vaccines
DNA-based method employing PCR-RFLP has in melanoma, RCC, cervical carcinoma and CML
been replaced by more rapid tests [113]. Generally, [73, 95, 110, 116].
they either identify PCR products containing Although the result of such studies was
hypervariable regions by hybridization with inconsistent in some cases, most studies pointed
sequence-specic probes (SSO) or employ to the undeniable role of HLA polymorphism
sequence-specic primers (SSP) to identify vari- in susceptibility, prognosis, natural history, and
ants as part of PCR process itself [13, 31, 114]. The response to immunotherapy in different cancers
latter was extensively used back in mid-1990 [13, [32]. These past experiences emphasize that a
31, 114]. Even though aberrant typing as a sign of prestigious HLA association study is a complex
new allele can be followed by direct DNA sequenc- art rather than a simple case-control study and
ing, both methods are ineffective in case there is a several factors should be considered in interpret-
new allele [13]. Later this limitation was overcome ing their results. In this regard, results of meta-
by polymerase chain reaction-sequence-based typ- analysis of these association studies are more
ing which can directly detect the sequence of reliable (Table 17.2).
Table 17.2 Signicant results from published meta-analysis of HLA associations with cancers
Total number Total number
Alleles Cancer site of cases of controls OR 95 % CI Population included Reference
DQB1*03 Hepatocellular carcinoma 398 593 0.65 (0.480.89) China, Italy, Spain, Egypt Xin et al. [117]
DQB 1*02 Hepatocellular carcinoma 398 593 1.78 (1.053.03) China, Italy, Spain, Egypt Xin et al. [117]
DQB1*0502 Hepatocellular carcinoma 257 349 1.82 (1.142.92) China, Spain Xin et al. [117]
DQB1*0602 Hepatocellular carcinoma 173 226 0.58 (0.360.95) China, Spain Xin et al. [117]
HLA-DRB1*07 Hepatocellular carcinoma 281 466 1.65 (1.082.51) China, Italy, Spain, Egypt Lin et al. [118]
156 224 2.1 (1.064.14) China Lin et al. [118]
17 Immunogenetics of Cancer
125 242 1.41 (0.832.42) Italy, Spain, Egypt Lin et al. [118]
HLA-DRB1*12 Hepatocellular carcinoma 281 516 1.59 (1.092.32) China, Italy, Spain, Thailand Lin et al. [118]
206 324 1.73 (1.172.57) China, Taiwan Lin et al. [118]
75 192 0.3 (0.042.47) Spain, Italy Lin et al. [118]
HLA-DRB1*15 Hepatocellular carcinoma 281 466 1.7 (0.83.59) China, Italy, Spain, Egypt Lin et al. [118]
156 224 3.22 (1.636.37) China Lin et al. [118]
125 242 0.8 (0.341.89) Spain, Egypt, Italy Lin et al. [118]
HLA-DRB1* 0701 Cervical squamous cell 1,445 2,206 1.59 (1.092.35) Iran, USA, England, Sweden, France, Brazil Yang et al. [119]
carcinoma 1,083 1,248 1.29 (1.021.63) Caucasians Yang et al. [119]
carcinoma 2,191 2,628 1.22 (1.011.47) Yang et al. [119]
carcinoma
(continued)
309
310

Alleles Cancer site of cases of controls OR 95 % CI Population included Reference
HLA-DRB1* 1503 Cervical squamous cell 432 894 3.4 (1.696.87) Iran, USA, England, Sweden, France, Brazil Yang et al. [119]
carcinoma
carcinoma
HLA-DRB1* 0403 Cervical squamous cell 1,796 2,050 2.05 (1.024.12) Caucasians Yang et al. [119]
carcinoma
carcinoma
carcinoma
carcinoma
A. Hirbod-Mobarakeh et al.
17.6.5 Typing Methods is not the same in different populations. Each

strain is best presented by certain HLA alleles.
Indeed, immunogenetic studies are deeply Therefore, one HLA allele efcient for present-
inuenced by technological advances. Low- ing Ags of one populations prevalent strain may
resolution serologic HLA typing was one of the not present Ags of another populations prevalent
major limitations in early studies [83]. Serologic strain efciently [83]. Such a phenomenon might
typing is only enabled to identify the family of be extended to other environmental factors like
alleles. This family often comprises a heteroge- virus prevalence, viral load, diet, cigarette smok-
neous group of alleles with different afnities ing, and socioeconomic status, all of which are
and different potential for Ag presentation. Since highly dependent on the population under study
distribution of alleles belonging to the same sero- [74, 127]. For instance, pathogenesis of cervical
type is different in various populations, such cancer is dependent on persistent infection with
studies often obtained conicting results in dif- high-risk human papillomavirus (HPV) and this
ferent populations. One of the best historical risk factor itself is highly related to socioeco-
examples is HLA association studies in nasopha- nomic status, sexual relationship, and prevalence
ryngeal carcinoma (NPC). of high risk variants in the region [127, 128].
NPC, as an epithelial carcinoma of the head
and neck origin, was one of the main focuses of
early HLA association studies. Early serological 17.6.7 Linkage Disequilibrium
studies showed an association between HLA-A2
and NPC-in Chinese population, while studies in MHC region is home to more than 200 genes
Caucasians found HLA-A2 as a protective allele beside classic HLA genes. Due to the low recom-
for both NPC and EBV-associated HLA [106, bination rates, these genes are often in strong
120124]. Later, higher-resolution studies linkage disequilibrium together [78]. This strong
showed HLA-A*02:07, a common allele in LD can complicate nding the actual causal
Chinese population but rare among Caucasians, allele. The problem gets worse when the causal
as the main risk factor, while HLA-A*02:01, a allele is an unknown allele in strong LD with the
common allele in Caucasians, was shown to be associated allele. This limitation can be over-
the actual protective factor in this population come by whole genome sequencing (WGS) of
[125, 126]. It is possible that future studies the region in close proximity of the associated
employing higher-resolution methods reveal allele [101]. One example is the association of
even new causal variants within the current NPC with HLA-A*0207 and HLA-B*4601
associations. which are in strong LD. In this case, either allele,
both of them, or even a third allele in LD with
both of them might inuence the pathogenesis of
17.6.6 Environmental Factors NPC [126].
Some studies reported extraordinary LD in
Various environmental and genetic factors play MHC region between alleles from one class and
roles behind scenario of cancer, and malignant alleles of other classes and even non-HLA genes.
transformation is the result of a complex interac- This extraordinary haplotypes are known as
tion between these factors. It is often the case that extended haplotypes [83]. Thus, in interpreting
certain genetic factors need certain environmen- results of HLA association studies or design of
tal factors to play their role in pathogenesis of one, non-HLA genes such as the transporter
cancer. The role of environmental factors in HLA associated with Ag processing (TAP) MHC class
association studies is more prominent in virus- I chain-related A (MIC-A), heat shock proteins
associated malignancies like HL, NPC and cervi- (HSP), and TNF- which are located nearby or
cal cancer. Each virus has different strains with within the classic HLA genes should be consid-
different Ags and the prevalence of these strains ered [78, 83]. These extended haplotypes are
especially of importance in immunogenetic this haplotype might be absent [57]. One of

studies of cancers, since numerous elements of such associations has been reported between
the immune system are in the front line of defense cervical squamous cell carcinoma and
against cancer. multi-locus haplotype of B*4402-Cw*0501-
For instance, the ancestral haplotype 8.1 (AH DRB1*0401-DQB1*0301 [57].
8.1: HLA-A*01-B*08-Cw*07-DRB1*03-TNF-
G308A), in which HLA alleles are in LD with
TNF-, is the most frequent extended MHC hap- 17.7 The Cytokine Network
lotype in Caucasian populations [109]. Primarily,
this extended haplotype was associated with 17.7.1 Background
clinical course of NHL [75, 109]; however, later
studies showed that polymorphism in TNF- Cytokines are a group of soluble regulatory fac-
gene has a more prominent effect in this associa- tors by which the immune system controls and
tion compared to Cw*07 and DRB1*03 alleles modulates different activities of its cells. Each
[8, 75]. In this case, polymorphisms in TNF- cytokine triggers certain cascade of events in
promoter inuence TNF- expression levels. their target cells by binding to their receptors and
TNF- level consequently affects the extent of activating intracellular signal transduction path-
immune activation upon tumor challenge. In way [14, 20]. Cytokine network is responsible for
addition, increased TNF- impairs Ag presenta- coordination of effector actions of different ele-
tion potential of APCs and by its effect on cyto- ments of the immune system, as well as the dif-
kine prole results in a bias toward Th2 immune ferentiation and proliferation of different immune
responses [75]. All these factors can contribute to cells. In addition, secretion of antibodies and
the exacerbation of systemic symptoms, anemia, inammation is tightly regulated by complex
hypoalbuminemia, and poor outcome [8]. interaction between these cytokines [13, 23, 26].
Another example is the association of HLA- Chronic inammation, by inducing chronic
A*03 and chronic myeloid leukemia (CML) tissue damage and compensatory cell prolifera-
[78]. A translocation between t(9;22)(q34;q11) tion, is a considered a major promoter of malig-
creating a truncated chromosome 22 known as nant transformations. As an example, nitric
Philadelphia chromosome is present in majority oxide, produced during inammation, might
of patients with CML [129]. Depending on the damage DNA structure in different tumor sup-
precise location of the fusion, different fusion pressor genes and oncogenes [130]. Therefore,
proteins are encoded. Keeping this in mind and any dysregulation in cytokine network can result
the absence of costimulatory molecules on in excessive production of tumor-inducing fac-
CML cells, it is improbable that the association tors, DNA damage, angiogenesis, and dysplasia
of HLA-A*03 is due to its efciency in present- and consequent development of various inam-
ing fusion proteins and its ability to induce an matory diseases including different cancers [26,
effective immune response [78]. However, this 131]. Cytokine network is a determinant factor
allele is in with the C282Y mutation of the in the development of metastasis and natural
hemochromatosis gene, a susceptibility marker history of cancers [26]. In some cancers, malig-
for CML [78]. nant cells can manipulate cytokine network in
In some cases, an optimal immune response order to escape immunosurveillance or promote
is dependent on optimal Ag presentation by their own proliferation [130, 132]. In addition,
both HLA classes and the presence of certain cytokine network can inuence the outcome and
alleles in non-HLA genes. An absence of one of toxicity of different immunotherapy methods
these optimal alleles may result in anergy and [13, 20, 133]. Several cancers including hepato-
immune escape. In some populations, these cellular carcinoma (HCC), oral squamous cell
alleles might be in LD in form of an unknown carcinoma, melanoma, the gastric, pancreatic,
extended haplotype, while in other populations and prostate cancer were associated with high
Table 17.3 Genotype details for SNPS of IL-1

Change at Change at Effect on
SNP GMAFa [137] Population diversityb [138] DNA level protein level cytokine level
rs1800587 T = 0.253 CEU
HCB 889 C>T NAc T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs17561 T = 0.203 CEU
HCB +4845 G>T Ala114Ser T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(G;G) (G;T) (T;T)

a
GMAF: the minor allele frequency in 1,094 worldwide individuals provided from 1,000 genome phase 1 genotype data
b
CEU European, CHB Han Chinese, JPT Japanese Tokyo, YRI Yoruba African, AVG Mathematical average of all
samples
c
NA not applicable
levels of certain proinammatory or antiinam- B gene, one of the most important transcription
matory cytokines [26]. factors, can result in extensive changes in the
Cytokine levels are not the same in all cytokine network by altering transcription of
individuals. Interindividual differences in cyto- TNF-a, IL-1, IL-6, and IL-8 [20]. Although the
kine levels in both baseline and stimulated phases exact roles of these polymorphisms in tumor
are a result of both genetic and environmental immunology are less clear, the relevance of this
factors [133]. Since there is no an intracellular role is becoming more and more apparent in
storage for cytokines, their secretion is dependent recent years [20].
on the transcriptional and translational rates of
their genes [14, 26]. Not surprisingly, genes
responsible for encoding cytokines and their 17.7.2 Interleukin-1 Superfamily
receptors are relatively polymorphic [13, 20, 23].
Several polymorphisms in their gene can affect IL-1 and IL-1 and their antagonist IL-1Ra
their expression, structure, and activity [20, 23, are members of this superfamily with pleiotro-
26, 130, 134]. Most of these polymorphisms are pic effects on inammation, immunity, and
in non-coding regions including promoter or hemopoietic system. High levels of IL-1 are
intronic sequences and exonic regions are usually found in tumor sites, however IL-1 family plays
highly conserved [13, 14]. So far, numerous an ambivalent role in tumor immunity. IL-1
genetic association studies have been suggested induces cytokine secretion from T cells to
as associations of these SNPs with various can- potentiate the differentiation and function of
cers in different populations. However, results of immunosurveillance cells. On the other hand,
such studies were often inconsistent, and the IL-1 induces the expression of adhesion mole-
reported associations varied not only in different cules, matrix metalloproteinases, growth fac-
populations but also in different cancers and even tors, and angiogenic factors and promotes
in their different subtypes [131]. Therefore, a invasiveness and metastasis of malignant cells
meta-analysis of these studies can show some [135, 136].
more conclusive evidence of these associations.
In addition to polymorphisms of cytokine 17.7.2.1 Interleukin-1
genes, there are other polymorphic elements such IL-1 is encoded by seven exons of a gene located
as various transcription factors and cytokine- in 2q14. Variant889 C>T (rs1800587) is one of
specic receptors which are involved in actions the common promoter variants of IL-1 gene
of cytokine network [20, 26]. For instance, (Table 17.3). Although, the promoter containing
polymorphisms in the NF-B nuclear factor-kappa T allele has been shown to result in a marginally

SNP GMAF [137] Population diversity [138] DNA level protein level cytokine level
rs16944 C = 0.465 CEU
HCB 598 T>C NA C allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs1143627 C = 0.4808 CEU
HCB 31 C>T NA T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs1143634 T = 0.146 CEU
HCB +3954 C>T NA UAa
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

a
UA unavailable
higher level of expression, at the protein level, T haplotype [23, 141]. In the same line, in vitro
allele was associated with signicantly increased studies like luciferase reporter assay showed
IL-1 levels which could not be justied by only higher expression of luciferase gene with pro-
different expression patterns. Further studies moter containing T allele in 31 C>T (rs1143627)
showed that this SNP has high LD with an exonic [23]. Results of EMSA studies suggested that this
SNP in +4845 G>T (rs17561) resulting in substi- higher expression is a result of higher afnity for
tution of alanine with serine at the position of 114 several transcription factors as a result of a
which results in more efcient process of pre- change in a TATA-box motif [23].
IL-1 comparing to Ala114 and consequently T allele in rs1143634 was associated with
higher release of IL-1 [23]. increased IL-1 secretion and several inamma-
tory diseases [132]. However, no evidence on the
17.7.2.2 Interleukin-1 functionality of +3954 C>T (rs1143634) is avail-
High levels of IL-1 have been shown to be asso- able, and it seems that +3954 C>T (rs1143634) is
ciated with increased risk of most human cancers just a marker for a functional polymorphism such
and also poor prognosis in cancer patients [130, as 31 T>C (rs1143627) [23, 26].
132, 139]. IL-1 is encoded by a 7.5 kb gene with One recent meta-analysis of 81 case-control
seven exons located on 2q14. Its expression is studies with 19,547 patients with HCC, gastric,
regulated by two distal and proximal promoter lung, blood, cervical, esophageal, prostate,
elements [140, 141]. So far, several polymor- breast, and skin cancers and 23,935 controls
phisms have been identied in this gene. showed that, overall, 598T>C (rs16944) has no
598T>C (rs16944) and 31 C>T (rs1143627) signicant association with cancers [132], while
are two common variants in the promoter region, another meta-analysis of 26 studies with 8,083
and +3954 C>T (rs1143634) is a common syn- patients with cancer and 9,183 controls showed a
onymous polymorphism in coding region of signicant association of +3954 C>T (rs1143634)
IL-1 gene (Table 17.4) [26]. with increased risk of cancers in a dominant
In northern and western European ancestry model which is in accordance with the results of
(CEU), 598T>C (rs16944) and 31 C>T another metaanalysis of 33 studies (Table 17.5)
(rs1143627) had strong LD (r2 = 0.94) [26, 132]. [132, 145].
In vivo, 598C/31T haplotype has been associ- A meta-analysis of studies on associations
ated with higher IL-1 levels in the lungs and between IL-1 gene polymorphisms and gas-
gastric mucosa. It is suggested that 31 C>T tric cancer published from January 2000 to
(rs1143627) is the causal variant of this December 2009 (including 18 studies with 4,111
Table 17.5 Signicant results from published meta-analysis of associations of IL-1 polymorphisms with cancers
Alleles Cancer site of cases of controls Analysis type OR 95 % CI Population included Reference
rs16944 Gastric cancer 2,041 2,441 TT + CT vs. CC 1.23 (1.091.37) Italy, Japan, China, Korea, Vincenzi et al. [142]
Portugal, UK, mixed Asian
Cervical cancer 836 980 TT vs. CC 1.74 (1.282.36) Egypt, Korea, India, China Xu et al. [132]
CT vs. CC 1.71 (1.322.23)
TT + CT vs. CC 1.74 (1.352.23)
Hepatocellular 890 821 CT vs. CC 0.75 (0.600.94) Japan, Taiwan, Thailand Xu et al. [132]
carcinoma TT + CT vs. CC 0.68 (0.470.99)
Blood cancers 3,839 3,762 CC + CT vs. TT 1.19 (1.041.37) Italy, Spain, Germany, USA, Xu et al. [132]
Canada, Greece
rs1143627 Lung cancer 3,435 4,719 TT + TC vs. CC 1.23 (1.061.43) China, Italy, mixed European, Peng et al. [143]
Denmark
Gastric cancer 1,535 2,585 TT + TC vs. CC 1.16 (1.011.33) Korea, Mexico, China, Brazil, Vincenzi et al. [142]
Italy, USA
Hepatocellular 1,039 1,588 CC + CT vs. TT 1.31 (1.091.57) Japan, Taiwan, Morocco Jin et al. [144]
carcinoma
rs1143634 Malignancy 8,083 9,183 TT + CT vs. CC 1.15 (1.011.30) Sweden, Poland, China, UK, Xu et al. [132]
Germany, Tunisia, Costa Rica,
Oman, USA, Greece, Netherlands,
Norway, Japan
Gastric cancer 2,359 3,613 CT vs. CC 1.16 (1.031.32) USA, China, UK, Germany, Italy, Zhang et al. [145]
Japan, India, Sweden, Oman
Oral cancer 346 417 CT vs. CC 0.65 (0.450.94) Greece, China Zhang et al. [145]
TT + CT vs. CC 0.69 (0.490.98)
315
controls and 3,295 cases for 598T>C (rs16944), prolonged proinammatory immune response
21 studies with 5,883 controls and 3,786 cases [23]. Although, intronic VNTR contains poten-
for 31 T>C (rs1143627) polymorphism, 10 tial binding sites for an interferon- silencer, an
studies with 3,610 controls and 1,559 cases for interferon- silencer, and an acute-phase
+3954 C>T (rs1143634)) showed signicantly response element, all leading to its functional
increased risk of cancer in individuals with IL-1 importance, these associations are suggested to
598T allele. In stratied analysis for different be a result of LD with other variants [140, 152].
ethnicities, such an association was present in Some authors suggested that the enhancing effect
Caucasians but not in Asians or in Hispanics. of IL-1RN*2 on IL-1RA levels is dependent on
This study also showed such an association for the presence of 511T allele or the absence of
intestinal-subtype and noncardia gastric cancer +3954T in IL-1 [23].
[146, 147]. However, this study didnt show any A meta-analysis of 71 case-control studies
signicant associations between gastric cancer (including 37 studies on gastric cancer, six stud-
risk and 31 T>C (rs1143627) and +3954 C>T ies on HCC, four on cervical cancer, four on
(rs1143634) [146]. Older studies conducted on breast cancer, four on lung cancer, and 16 studies
2005 and 2007 more or less showed such pattern on other cancers) with 14,854 cases and 19,337
for this SNP [142, 147]. However, a meta-analy- controls showed that overall carriers of IL-1RN*2
sis of ve studies published up to September 2008 are signicantly more susceptible to cancer
showed association of +3954 C>T (rs1143634) (Table 17.6) [145].
and gastric cancer risk in Chinese and Japanese
population [148].
Another systematic review evaluating associa- 17.7.3 Interleukin-4
tions of HCC with polymorphisms of IL-1 gene
(reported up to September 2010) and a meta- Interleukin-4 (IL-4) is a pleiotropic cytokine with
analysis of 1,279 patients with lung cancer and major roles in regulation of humoral immunity by
2,248 controls failed to support any signicant its various effects on production of several other
increased risk for 598T>C (rs16944) and 31 cytokines and dedifferentiation of B cells and
C>T (rs1143627) [143, 149]. promoting expression of class II MHC Ags [26,
130]. It also has potent antitumor activity against
17.7.2.3 Interleukin-1Ra (IL-1Ra) various tumors by its inhibitory effect on the
IL-1RA has antiinammatory properties by com- growth of tumor cells and its growth stimulatory
peting with IL-1 cytokines in binding to their effect on lymphocytes [153, 154].
receptors. This cytokine is encoded by IL-1RN IL-4 gene is located on the long arm of chro-
gene located on 2q14.2. Its transcript may con- mosome 5 (5q31.1), and through recent years,
tain six, ve, or four exons [23, 130]. There is an many variants identied on this gene. Among
86-bp variable number tandem repeat (VNTR) in these variants, 589 C>T (rs2243250) is a pro-
intron 2 of this gene [23]. The short allele of this moter SNP of which T allele is associated with
VNTR contain only two repeats (IL-1RN*2), increased production of IL-4 in in-vivo studies
while long alleles may have three to six repeats [26, 155]. The other variant of this gene is a
(IL-1RN L) [58, 146]. The more prevalent allele 70-bp VNTR at intron 3 (Table 17.7) [155].
containing four repeats is named IL1RN*1 [150]. A meta-analysis of 8,715 patients with various
In vitro and in vivo studies have shown extensive cancers and 9,532 controls presented in 23 case-
associations of this variant with the members of control studies found no signicant association
IL-1 superfamily. IL-1RN*2 was associated with between this SNP and overall cancer susceptibil-
not only higher IL-1RA levels but also enhanced ity. This study also didnt nd any signicant
IL-1 production and decreased IL-1 produc- relationship in stratied analysis for ethnicity or
tion [151]. However, the nal result of IL-1RN*2 different cancer types [156]. However, another
was decreased IL-1RA/IL-1 ratio, followed by meta-analysis of 14 studies involving 3,562
Table 17.6 Signicant results from published meta-analysis of associations of IL-1RN VNTR with cancers
Total Total
number number of
Cancer site of cases controls Analysis type OR 95 % CI Population included Reference
Malignancy 14,854 19,337 22 vs. LL 1.37 (1.071.75) 40 studies of Asian Zhang
2 L vs. LL 1.19 (1.071.32) descendents, 29 of et al.
22 + 2 L vs. LL 1.25 (1.121.41) Caucasian descendents, [145]
and two with mixed
2 vs. L 1.23 (1.101.38)
ethnicity
Breast 1,145 1,102 2 L vs. LL 0.74 (0.580.93) Japan, Germany, Zhang
cancer 22 + 2 L vs. LL 0.78 (0.620.97) Korea, India et al.
[145]
Gastric 3,209 4,856 2 L vs. LL 1.22 (1.051.41) Portugal, China, Zhang
cancer 22 + 2 L vs. LL 1.25 (1.091.43) Germany, Brazil, et al.
2 vs. L 1.20 (1.051.38) Taiwan, Thailand, [145]
UK, Italy
3,418 5,789 22 + 2 L vs. LL 1.26 (1.061.51) Arab, Brazil, Xue et al.
Netherland, Korea, [146]
USA, China, Italy,
Mexico, South Korea,
Germany, Taiwan,
Portugal, Poland

Change Change at Effect on
SNP GMAF [137] Population diversity [138] at DNA level protein level cytokine level
rs2243250 T = 0.484 CEU
HCB 589C>T NA T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)
cancer cases found that T allele was signicantly and breast cancer [23, 26]. This cytokine is
associated with decreased oral cancer risk and encoded by a gene on chromosome 7p21 with
increased risk of RCC (for oral cancer, TT vs. ve exons [159]. Two common promoter variants
CC: OR = 0.40, 95 % CI = 0.190.84; TT + CT vs. of IL-6, 174G>C (rs1800795) and 572G>C
CC: OR = 0.45, 95 % CI 0.220.94; and for renal (rs1800796), were extensively studied in different
cell carcinoma, TT vs. CC: OR = 1.98, 95 % inammatory diseases (Table 17.8). 174G>C
CI = 1.063.69; TT vs. CC + CT OR = 1.43, 95 % (rs1800795) is the rst identied common pro-
CI = 1.051.95) [157]. moter variant of IL-6 [23]. C allele in both of these
variants was associated with lower IL-6 levels in
several studies [134, 138, 160165]. However,
17.7.4 Interleukin-6 (IL-6) such an effect on IL-6 levels was not conrmed
by some studies on 174G>C (rs1800795) [134,
IL-6, a 23.7 kD proinammatory cytokine, is 160164]; therefore, this inconsistency might be
involved in inducing acute-phase response, the result of partial LD between this SNP and
differentiation of monocytes to macrophages, an actual functional SNP [23]. EMSA studies
proliferation of T cells and Th2 cytokine pro- showed that 572G>C (rs1800796) is not in a
duction [158]. It has been previously shown to TFBS; therefore, its inuence on IL-6 serum lev-
be of importance in susceptibility, natural his- els probably results from strong LD with a func-
tory, and prognosis of several malignancies tional variant such as 6331 T>C (rs10499563)
including prostate cancer, colorectal carcinoma, [160]. C allele in 572G>C (rs1800796) is

rs1800795 C = 0.185 CEU
HCB 174G>C NA C allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;G) (G;G)

rs1800796 C = 0.290 CEU
HCB
572G>C NA C allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;G) (G;G)
Table 17.9 Signicant results from published meta-analysis of associations of 174G>C (rs1800795) in IL-6 gene
with cancers
Total
number of Total number OR 95 % Population
Cancer site cases of controls Analysis type CI included Reference
Colorectal cancer 3,061 4,024 GC/CC vs. 0.75 Individuals from Yu et al. [166]
GG (0.640.88) Denmark, USA,
and Spain who
regularly or
currently took
NSAIDs
highly associated with T allele in 6331 T>C [166]. This study didnt show any signicant
(rs10499563) [160]. Interestingly, T allele in this association between colorectal cancer and
SNP is associated with higher expression of IL-6 572G>C (rs1800796) in 2,574 cases and 3,344
gene [160]. 6331 T>C (rs10499563) is near the controls [166]. In line with this, two recent
distal promoter of IL-6 located between 5202 meta-analyses on gastric cancer patients did not
and 5307. EMSA studies showed that T allele in conrm any effect of these two SNPs on suscep-
6331 T>C (rs10499563) resulted in more afn- tibility to cancer [167, 168]. The most recent one
ity for Oct-1 of which binding changes the chro- evaluated 13 studies reporting associations of
matin structure and locates the distal promoter to 174G>C (rs1800795) (1,581 gastric cancer
the transcription start site [23]. patients and 2,563 controls) and four studies on
A systematic review of 12 case-control studies 572G>C (rs1800796) [167]. In addition, a sys-
on breast cancer (published till December 2009) tematic review of 2,949 patients with lung cancer
with 10,137 cases and 15,566 controls found no and 3,375 controls did not show any signicant
signicant association between 174G>C association between 174G>C (rs1800795) and
(rs1800795) and susceptibility to breast cancer lung cancer [143].
[134]. Similarly, another meta-analysis of 7,210
patients with colorectal cancer and 9,467 controls
did not show any signicant association in any 17.7.5 Interleukin-8
genetic model between 174G>C (rs1800795)
and colorectal cancer [166]. However, in strati- IL-8, a member of human -chemokine subfam-
ed analysis in a subgroup of patients with the ily, has a major inuence on tumor invasion and
history of current or habitual use of NSAIDs metastasis by its stimulatory properties on angio-
(3,061 cases and 4,024 controls), carriers of C genesis and inammation [23, 26, 59, 169, 170].
allele in 174G>C (rs1800795) had signicantly A gene located on chromosome 4q13q21 with
lower risk for colorectal cancer (Table 17.9) four exons is responsible for encoding this

rs4073 T = 0.497 UA 251A/T NA A allele:
rs2227306 T = 0.294 CEU
HCB +781C/T NA T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs2227307 G = 0.422 UA +396T/G NA UA
cytokine [169]. Fifteen functional SNPs have in Asian population. In stratied analysis for
been identied within this gene including tumor location and histology, this association
251A>T (rs4073), +396T>G (rs2227307), and remained signicant only in the cardia gastric
+781C>T (rs2227306) (Table 17.10) [26]. cancer and diffused type [59]. A more recent
251A>T (rs4073), located in the promoter meta-analysis evaluating papers on gastric can-
region, was identied in 2000. Although there cer published from January 2000 to January
was little evidence on the functionality of this 2011 (18 papers including 6,554 controls and
SNP in vitro, several in vivo studies showed 4,163 cases) also found such an association in
higher levels of IL-8 in carriers of A allele [23]. Asians but not in Caucasians. However, unlike
On the contrary, one study showed higher tran- the previous study, when stratifying for pathol-
scription for T allele in gastric carcinoma cell ogy types, the association remained signicant
line [131, 171]. EMSA studies showed that T only in intestinal-type cancer but not in the dif-
allele in +781 C>T allele (rs2227306) is associ- fused type [174].
ated with higher binding ability for a transcrip- A systematic review of 1,324 patients with
tion factor (C/EBPb) [23]. Several studies oral cancer and 1,879 controls reported in six
showed associations of 251 A>T (rs4073) with studies (published till October 2012) also showed
lung, gastric, colorectal, bladder, and prostate higher risks of oral cancer in carriers of A allele
cancer in different populations (Table 17.11) in 251A>T (rs4073). In subgroup analysis for
[155]. A meta-analysis of 13,189 patients with ethnicity, there were only signicant associations
lung, prostate, breast, colorectal, and nasopha- among Caucasians but not in Asians [172].
ryngeal cancers and 16,828 controls showed that On the contrary, T allele in this SNP was asso-
carriers of A allele in 251 A>T (rs4073) which ciated with an increased risk of breast cancer in
were more susceptible to different cancers [131]. Asian and African populations. However, this
Another study reviewed results of 45 studies study showed no signicant associations between
including 14,876 cases and 18,465 controls this SNP and breast cancer in 1,880 breast cancer
and showed such an association only among patients and 2,013 controls [173]. There were not
hospital-based studies and surprisingly showed any signicant associations between this SNP
signicantly decreased risk of cancers for AA and colorectal cancer in a meta-analysis of nine
genotype among population-based studies [171]. case-control studies with 3,019 cases and 3,984
It should be noted that hospital-based studies controls [175].
have an increased chance of a selection bias
since hospital-based controls might have disease
conditions under the inuence of the studied 17.7.6 Interleukin-10
polymorphism [169].
Another systematic review of ten papers IL-10 is a pleiotropic, immunoregulatory
including 2,195 gastric cancer patients and 3,505 cytokine which can affect both the innate and
controls conrmed that AA genotype was a risk adaptive immune systems [176]. IL-10 has
factor for gastric cancer in whole population and pleiotropic effects on tumor immunology. It
320
Table 17.11 Signicant results from published meta-analysis of associations of 251T/A (rs4073) in IL-8 gene with cancers
Total Total
number number
Cancer site of cases of controls Analysis type OR 95 % CI Population included Reference
Malignancy 13,189 16,828 AA vs. TT 1.21 (1.081.36) Tunisia, Iran, Denmark, UK, Croatia, Germany, Wang et al. [131]
(AA + TA) vs. TT 1.12 (1.031.23) USA, Greece, China, Japan, Portugal, Spain,
Mexico, Finland, France, Norway, Poland, Korea,
India, Netherlands
5,633 8,240 (AA + TA) vs. TT 0.90 (0.830.97) (Population-based studies) Gao et al. [171]
Gastric cancer 3,036 3,082 AA vs. TT 1.48 (1.131.95 (Asian population) Wang et al. [131]
TA vs. TT 1.20 (1.041.40)
(AA + TA) vs. TT 1.27 (1.081.48)
4,274 6,498 AA vs TT 1.28 (1.021.62) Japan, Iran, China, Korea, Finland, Spain, Mexico, Wang et al. [131]
(AA + TA) vs. TT 1.17 (1.011.36) Poland
Nasopharyngeal 440 459 AA vs. TT 2.04 (1.382.99) Tunisia, China Wang et al. [131]
cancer TA vs. TT 1.59 (1.192.13)
(AA + TA) vs. TT 1.70 (1.302.24)
545 568 (AA + TA) vs. TT 1.48 (1.161.89) Tunisia, China Gao et al. [171]
Oral cancer 1,324 1,879 AA vs. (AA + TA) 1.23 (1.031.46) China, Taiwan, Thailand, Greece, Japan, France Wang et al. [172]
AT vs. TT 1.25 (1.071.47)
Breast cancer 683 880 TA vs. AA 1.444 (1.0921.908) Iran, China Xue et al. [173]
AA vs. (AA + TA) 1.435 (1.1071.861)
717 537 TA vs. AA 0.541 (0.3960.741) Tunisia Xue et al. [173]
AA vs. (AA + TA) 0.737 (0.5700.953)
1,262 1,419 TT vs. (AA + TA) 0.692 (0.5660.861) (Population-based) Tunisia, China, UK Xue et al. [173]

rs1800896 G = 0.303 CEU
HCB 1082 A>G NA G allele:
JPT
YRI
AVG
0 20 40 60 80 100
(A;A) (A;G) (G;G)

rs1800871 T = 0.409 CEU
HCB 819 C>T NA UA
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

CEU
rs1800872 C = 0.409 HCB 592 A>C NA UA
JPT
YRI
AVG
0 20 40 60 80 100
(A;A) (A;C) (C;C)
plays an antiinammatory role by inhibiting with various cancers including lung cancer, breast
production of proinammatory mediators such cancer, cervical cancer, gastric cancer, melanoma
as IL-1, IL-1, IL-6, IL-8, IL-12, TNF- and and nasopharyngeal cancer, and prostate cancer
IFN- [23, 63]; in addition, IL-10 inhibits [62, 63]. A systematic review evaluated associa-
presentation of tumor Ags by suppressing the tions of 1082 A>G (rs1800896) with risk of
expression of HLA molecules [130, 133]. On the malignancy by reviewing results of 61 articles
other hand, IL-10 induces proliferation in B cells (published up to September 2010) with a total of
and T cells and regulates angiogenesis in various 14,499 cancer patients and 16,967 controls. This
cancers [26, 177]. study found no signicant association between
Twin studies demonstrated that IL-10 levels alleles of this SNP and overall susceptibility to
are signicantly inuenced by genetic factors cancers. However, carriers of G allele in Asian
with a heritability of 74 % [23, 178]. IL-10 is population had signicantly more susceptibility
encoded by ve exons of a gene located on to various cancers. In stratied analysis for can-
1q31-1q32. At least 40 SNPs have been identied cer types, there was increased risk of lung can-
in this gene [62, 63, 179]. Several common vari- cer and NHL in carriers of G allele (Table 17.13)
ants including 1082 A>G (rs1800896), 819 [62]. The rst systematic reviews of gastric
C>T (rs1800871), and 592 A>C (also called cancer studies showed signicant associations
571 rs1800872) have been identied within the between 1082 A>G (rs1800896) and gastric
promoter region of this gene (Table 17.12) [177]. cancer not in overall population but only when
In vivo studies showed higher levels of the analysis was limited to the Asian populations
IL-10 in individuals with GCC haplotype of these [184]. However, a more recent systematic review
three SNPs, while ATA haplotype was associ- of 22 studies with 4,289 patients and 5,965 con-
ated with the lowest levels of IL-10 [23, 133]. It trols evaluated the association of 1082 A>G
is suggested that 1082 A>G (rs1800896) is the (rs1800896) with susceptibility to gastric can-
most functional SNP of these three variants and cer. This meta-analysis showed that carriers of G
G allele in this SNP results in higher IL-10 levels allele has signicantly increased the risk for gas-
[23]. EMSA studies showed different afnities of tric cancer especially in Caucasian populations
alleles of this SNP for a nuclear protein identi- [177]. Another meta-analysis with 3,631 patients
ed as poly ADP-ribose polymerase1 (PARP- and 6,431 controls showed similar results; none-
1) which acts as a transcription repressor [23, theless, results remained signicant in Asian
62]. So far, several studies have evaluated the population but not in Caucasians. This study, in
associations of different IL-10 polymorphisms stratied analysis, showed that this association is
322
Table 17.13 Signicant results from published meta-analysis of associations of polymorphisms of IL-10 gene with cancers
rs1800872 HCC 354 1,683 CC vs. (AA + AC) 1.68 (1.252.26) Taiwan, Korea, China, Japan Wei et al. [63]
C vs. A 1.29 (1.121.49)
Cervical cancer 2,396 1,388 A vs. C 1.16 (1.041.31) Korea, Netherlands, Sweden, India, Ni et al. [180]
(AA + AC) vs. CC 1.18 (1.011.39) China
Gastric cancer 1,526 2,538 AA vs. (CC + AC) 0.81 (0.680.97) (Asians) China, Korea, Japan Xue et al. [181]
Lung cancer 601 1,008 (CC + AC) vs. AA 1.8 (1.282.54) Chinese, Denmark, Germany Xue et al. [143]
CC vs. AA 2 (1.243.23)
rs1800871 Gastric cancer 989 2,350 TT vs. (CT + CC) 0.82 (0.70.96) (Asians) China, Korea, Japan Xue et al. [182]
Lung cancer 311 507 C vs. T 1.27 (1.011.58) China, Denmark, Germany Peng et al. [143]
CC vs. TT 2.27 (1.323.89)
rs1800896 Gastric cancer 4,289 5,965 A vs. G 0.489 (0.3350.713) China, USA, Italy, Korea, Costa Rica, Pan et al. [177]
Honduras, Finland, Japan, Spain
3,631 6,431 (GG + GA) vs. AA 1.41 (1.131.76) China, USA, Italy, Korea, Costa Rica, Ni et al. [183]
Honduras, Finland, Japan, Spain,
mixed European
Lung cancer 315 507 GA vs. AA 3.16 (1.168.63) Taiwan, Germany, Turkey Peng et al. [143]
GG vs. AA 2.07 (1.163.70)
(GG + GA) vs. AA 3.17 (1.317.68)
Non-Hodgkin 2,338 1,999 GA vs. AA 1.18 (1.021.36) Australia, Maryland, Sweden, France, Wang et al. [62]
lymphoma (GG + GA) vs. AA 1.17 (1.021.35) Athens, Germany
Malignancy 1,733 2,003 GA vs. AA 1.80 (1.172.76) (Asian) China, Taiwan, Kentucky, Wang et al. [62]
GG vs. AA 3.32 (1.626.82) Korea, Japan
(GG + GA) vs. AA 1.67 (1.072.60)
GG vs. (AA + GA) 2.93 (1.436.03)
signicant in cardiac subtype and intestinal-type studies published up to June 2012. The same
but not in noncardia subtype or diffuse-type can- review indicated signicant increased suscepti-
cer [183]. Regarding 819 C>T (rs1800871), a bility to cervical cancer in carriers of A allele in
systematic review based on 11 studies and 4,008 592 A>C (rs1800872) [180].
controls and 1,490 cases showed signicantly
increased risk for carriers of C allele among
Asians but not Caucasians. Such increased risk 17.7.7 Interleukin-12
was also noted for diffuse-subtype cancer but not
for intestinal-subtype [182]. Interleukin-12 (IL-12) is a proinammatory cyto-
A systematic review of studies on 592 A>C kine with several functions including differentia-
(rs1800872) found signicantly increased risk of tion of Th1 pathway, the critical pathway involved
gastric cancer in carriers of C allele only in Asian in protection against malignancy [23]. It can also
populations but not in Caucasians and Latinos. induce IFN- production by T and NK cells and
In stratied analysis for non-cardia and cardia therefore suppress angiogenesis. In addition,
subtypes or intestinal, diffuse, or mixed subtypes, IL-12 has a major role in the reactivation and sur-
no signicant association was found [181]. vival of memory CD4+ T cells which results in
A meta-analysis of seven articles published on repolarization of CD4+ T cells from dysfunc-
association of 1082 A>G (rs1800896) and HCC tional antitumor Th2 into Th1 cells [187, 188].
with 1,012 HCC cases and 2,308 controls showed IL-12 is composed of two parts, a p35 unit
no association between this SNP and susceptibil- which is encoded by IL-12a on 3q25.33 and a
ity to HCC. The same systematic review based on p40 unit encoded by IL12b on 5q33.3 [23]. One
the results of four studies showed carriers of C common variant in IL-12b gene, including
allele in 592 A>C (rs1800872) had an increased +1188A>C (rs3212227) in 3 UTR, and three
risk of HCC. This study also showed no signi- common variants of IL-12a including +277 G>A
cant association between 819 C>T (rs1800871) (rs568408) in 3 UTR, IVS2 T>A (798 T>A;
and HCC based on results of three studies [63]. rs582054), and 564 T>G (rs2243115) in 5UTR
A meta-analysis reviewed the results of 13 have been extensively studied previously
studies with 9,692 patients with prostate can- (Table 17.14) [189]. In vitro and in vivo studies
cer and 10,488 healthy individuals as controls. showed that A allele in +1188A>C (rs3212227)
However, this review did not show any signi- was associated with higher expression and greater
cant association for the three SNPs which was mRNA stability [23, 190]. It is suggested that
in accordance with the results of an older review +277 G>A (rs568408) may disrupt exon-splicing
on the basis of ten studies [179, 185]. Another enhancers and miRNAs binding and therefore
review, which analyzed results of eight studies results in an unstable IL-12 mRNA and lower
with 1,636 breast cancer patients and 1,670 con- IL-12 secretion [191].
trols did not show any altered risk of breast cancer One meta-analysis of ten studies involving
for different alleles of 1082 A>G (rs1800896). 2,954 cancer patients and 3,276 controls showed
This review also showed no signicant associa- signicant associations between +1188A>C
tions between 592 A>C (rs1800872) and breast (rs3212227) and susceptibility to cancer
cancer in any genetic model [186]. (Table 17.15). In addition, by stratied analysis
In addition to its regulating effects on the for cancer type, this study showed signicant
immune system, IL-10 can induce transcription increased susceptibility to cervical cancer and
of one of the promoters of HPV [180]. Therefore, nasopharyngeal cancer in C allele carriers [190].
polymorphisms of this cytokine were under focus A recent meta-analysis of 18 studies evaluated
of researchers in the eld of cervical cancer. the associations of polymorphisms of both IL-12
However, no signicant association was found genes and cancer susceptibility. This study
between 1082 G>A (rs1800896) and suscepti- reviewed results of 13 studies on +1188A>C
bility to cervical cancer in a meta-analysis of (rs3212227), including nine studies in Asians,

rs3212227 C = 0.338 CEU
HCB
+1188A>C NA A allele:
JPT
YRI
AVG
0 20 40 60 80 100
(A;A) (A;C) (C;C)

rs568408 A = 0.128 CEU
HCB
+277 G>A NA G allele:
JPT
YRI
AVG
0 20 40 60 80 100
(A;A) (A;G) (G;G)

rs582054 A = 0.489 UA +798 T>A NA UA
CEU
rs2243115 G = 0.107 HCB 564 T>G NA UA
JPT
YRI
AVG
0 20 40 60 80 100
(G;G) (G;T) (T;T)
Table 17.15 Signicant results from published meta-analysis of associations of 1188A>C (rs3212227) in IL-12b with
cancers
Total number Total number Population
Cancer site of cases of controls Analysis type OR 95 % CI included Reference
Malignancy 2,954 3,276 (CC + AC) vs. AA 1.32 (1.061.63) UK, Bulgaria, Chen et al.
AC vs. AA 1.30 (1.071.57) China, France [190]
CC vs. AA 1.39 (1.051.86)
CC vs. AC + AA 1.17 (1.021.33)
10,404 10,861 C vs. A 1.14 (1.021.27) UK, USA, Italy, Zhou et al.
(AC + CC) vs. AA 1.20 (1.011.15) China, Russia, [189]
Korea, Bulgaria,
Tunisia
three studies in Caucasians, and one in Africans, and other mediators, is one of the most important
and showed increased risk of all cancers in C pro-inammatory cytokines in the maintenance
allele carriers. This association remained signi- and homeostasis of the immune system, inam-
cant in Asian population but not in Caucasians mation, and host defense [192]. TNF- has both
[189]. This study like the previous one showed procarcinogenic and anticarcinogenic properties,
increased susceptibility to cervical and nasopha- and its importance in cancer is evidenced by
ryngeal cancer in carriers of C allele. However, previous studies which repeatedly reported high
no signicant association was found between levels of TNF- in cancer patients [193195].
cancer susceptibility and +277 G>A (rs568408). Some tumor cells can even produce TNF- in an
Also, there was no signicant association for autocrine manner [130]. Consistent with its
+564 T>G (rs2243115) and IVS2 T>A (rs582054) name, high levels of TNF- result in tumor
of IL-12a [189]. necrosis, but low levels of this cytokine impair
antitumor immune response and induce tumor
angiogenesis and therefore is associated with
17.7.8 Tumor Necrosis Factor- increased tumor growth, progression, invasion,
and Lymphotoxin- and metastasis of tumor cells [193196]. In addi-
tion, TNF- levels can inuence weight loss
Tumor necrosis factor- (TNF-), by its trigger- cachexia, and anemia in the host and also its
ing effect on the cytokine cascade of IL-1, IL-6 response to treatment [197].
Table 17.16 Genotype details for SNPS of TNF- and Lymphotoxin-

Change Change at Effect on
SNP GMAF [137] Population diversity [138] at DNA level protein level cytokine level
rs1800629 A = 0.096 CEU
HCB
308G>A NA A allele:
JPT
AVG
0 20 40 60 80 100
(A;A) (A;G) (G;G)

rs361525 A = 0.051 UA 238 G>A NA G allele:
rs1799964 C = 0.200 CEU
HCB
1031 C>T NA C allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs1800630 A = 0.145 CEU
HCB
863 C>A NA A allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs1799724 T = 0.097 CEU
CHB
857 C>T NA T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs1800610 A = 0.102 UA IVS1 + 123G>A NA UA
rs1800750 A = 0.013 CEU
HCB
376 G>A NA A allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)

rs909253 C = 0.398 CEU
HCB
+252 A>G NA G allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)
Lymphotoxin- (LTA), another cytokine of functionality for this SNP, some authors
the TNF family, is similar to TNF- with respect suggested that this allele had more afnity for a
to amino acid sequence, receptors, and biologic transcriptional activator and another study
activities [193196]. showed that A allele disrupts a 10-bp binding
TNF- is encoded by a gene located on region for activator protein-2 (AP-2) (a repressor
chromosome 6 (region p21.3) and is a member of protein) [23, 197]. Of interest, 308G>A
HLA class 3. 308G>A (rs1800629) and 238 (rs1800629) is in high LD with +252G>A, a
G>A (rs361525) are two common promoter vari- functional SNP in lymphotoxin alpha gene, and
ants of TNF- gene [23]. Other variants include other HLA genes within ancestral haplotype,
1031 C>T (rs1799964), 863 C>A (rs1800630) HLA A1-B8-DR3-DQ2-TNF_308A-LT_252A
and 857 C>T (rs1799724), 376 G>A [197, 199, 200].
(rs1800750), and IVS1 + 123G>A (rs1800610) An allele of 238 G>A (rs361525) was asso-
(Table 17.16) [23]. The LTA gene is located in the ciated with lower levels of TNF- in peripheral
same region and has an NcoI restriction fragment blood mononuclear cells carrying TNF--238A
length polymorphism (+252 A>G) in its rst allele [193]. However, several in vitro studies did
intron (rs909253). not provide any evidence on the functionality of
A allele of 308G>A (rs1800629) is associ- this SNP [23].
ated with higher levels of TNF- [198]. While A Japanese in vitro study showed that C
several in vitro studies did not show any allele in rs1799964 is associated with higher
production of TNF- by concanavalin A signicant association between breast cancer

(Con A)-activated peripheral blood mononuclear and 863 C>A (rs1800630) and 857 C>T
cells [201]. Reporter assays showed increased (rs1799724), 1,031 C>T (rs1799964) polymor-
promoter activity for A allele of 376 G>A phisms, which may be due to the fact that the
(rs1800750), and EMSA studies showed more overall sample analyzed for these polymorphisms
afnity of this allele for Oct-1 transcription factor was very small [192]. Consistent with the previ-
comparing to other allele [20, 23]. In vivo studies ous study, a meta-analysis of 11 studies on 10,184
showed that individuals carrying at least one patients with breast cancer and 12,911 controls
allele out of three (1031C, 863A, 857T) had found that G allele in 308G>A (rs1800629) is
higher TNF- production and higher transcrip- associated with signicantly increased risk of
tional activity [20, 23, 202]. In the same line, breast cancer [196]. Another meta-analysis eval-
minor alleles of 863 C>A (rs1800630) and 857 uated 10,236 breast cancer cases and 13,143 con-
C>T (rs1799724) were associated with higher trols presented in 13 studies [212]. This study
promoter activity and more afnity for oct-1 tran- could conrm such a decreased breast cancer risk
scription factor [20, 23, 202]. On the contrary, in carriers of 308A allele only in Caucasians
one study showed that 863A allele had less [212]. However, no signicant association
afnity for NF-B [20, 23, 203]. between breast cancer susceptibility and other
In vitro studies showed that phytohemagglu- polymorphisms of TNF- was found [212]. A
tinin-activated mononuclear cells having +252G meta-analysis of 4,625 breast cancer patients and
allele (rs909253) produce more LTA and interest- 4,373 controls for LTA-252 A>G (results from
ingly TNF- [204, 205]. seven studies published up to January 2012) did
Previously, several associations have been not nd any signicant association between gen-
reported between TNF- polymorphisms and otypes of this polymorphism and breast cancer.
susceptibility to NHL, gastric carcinoma, breast However, in stratied analysis for ethnicity, carri-
cancers, prostate, uterine endometrium, lung, ers of G allele had signicantly increased risk of
cervix, and nasopharynx. However, a meta- breast cancer in Asian population [213]. A sys-
analysis reviewed 34 studies (published up to tematic review of 11 studies with 3,094 cervical
March 2011) including 34,679 cancer patients cancer cases and 3,037 controls found that carri-
and 41,186 controls and found no signicant ers of AA genotype for 308G>A (rs1800629)
association between 238 G>A (rs361525) poly- had 39 % increased risk of cervical cancer com-
morphism and susceptibility to cancer [206]. In pared with 308GA/GG genotypes [195]. In
line with this, a meta-analysis of 30,000 breast addition, in stratied analysis, such an associa-
cancer cases and 30,000 controls from 30 studies tion remained signicant in Asian population
of the breast cancer association consortium could [195]. This meta-analysis by its review on 1,190
not nd any signicant association between 238 cases and 1,784 controls showed decreased risk
G>A (rs361525) and susceptibility to breast of cervical cancer in carriers of A allele in 238
cancer [207]. G>A (rs361525) [195]. In a meta-analysis of 13
A review of 18 studies with 11,320 breast studies reported up to October 2011 which
cancer patients and 14,112 controls found a involved 3,294 cervical cancer patients and 3,468
signicant relationship between 308G>A controls, no association was found between
(rs1800629) polymorphism and breast cancer 308G>A (rs1800629) and cervical cancer [189].
only in Caucasian population (Table 17.17) However, in Caucasian and African population,
[192]. In addition, after excluding hospital-based signicantly increased risk of cervical cancer
studies a signicant decreased risk in carriers of was observed in carriers of A allele in this
A allele was found. This study also reviewed SNP. This study also meta-analyzed results of six
33,112 patients and 35,814 (reported in 35 stud- studies on 238 G>A (rs361525) (2,416 cases
ies) and found no signicant association for 238 and 2,010 controls) and found that carriers of
G>A (rs361525). This study also did not nd any 238A allele had lower risk of cervical cancer
Table 17.17 Signicant results from published meta-analysis of associations of polymorphisms of TNF- with cancers
rs1800629 Cervical cancer 2,710 2,877 AA vs. GG 2.09 (1.343.25) (Caucasian) India, USA, Portugal, Zhou et al. [189]
AA vs. GA + GG 2.09 (1.353.25) Costa Rica, Sweden
3,094 3,037 AA vs. GG 1.41 (1.031.92) Sweden, India, Costa Rica, South Liu et al. [195]
AA vs. GA + GG 1.39 (1.021.90) Africa, Mexico, Portugal,
Zimbabwe, USA, South Korea
Breast cancer 10,184 12,911 G vs. A 1.08 (1.021.14) Italy, Tunisia, UK, Iran, USA, Fang et al. [196]
AA vs. GA + GG 1.10 (1.041.17) Poland, Croatia, Russia, Germany
10,254 12,926 AA + AG vs. GG 0.91 (0.850.97) (Caucasians) Italy, USA, Poland, UK, Yang et al. [192]
Russian, Croatia, Germany
HCC 2,357 3,161 AA vs. GG 1.97 (1.013.83) USA, Turkey, China, Japan, Thailand, Wei et al. [203]
AG vs. GG 1.88 (1.232.88) Italy
AA + AG vs. GG 1.80 (1.192.72)
1,665 2,177 AA + AG vs. GG 1.74 (1.122.72) Israel, Turkey, China, Italy, Thailand, Yang et al. [149]
USA, Japan
Gastric cancer 4,399 6,855 AA vs. GG 1.49 (1.111.99) South Korea, Taiwan, USA, Gorouhi et al. [208]
GA vs. GG 1.14 (1.021.27) Portugal, Colombia, China,
Germany, Japan, Mexico, Brazil,
Italy, Honduras, Poland, Finland,
Spain
3,335 5,286 A vs. G 1.23 (1.11, 1.36) USA, Spain, Korea, China, Finland,
AA vs. GG 1.78 (1.28, 2.48) Germany, Mexico, Portugal,
AA vs. GG + GA 1.65 (1.21, 2.25) Honduras, Italy, Brazil, Japan
AA + GA vs. GG 1.21 (1.08, 1.36)
UADT cancer 1,751 3,345 AA vs. GA + GG 1.54 (1.072.21) China, India, USA, Australia Wang et al. [209]
Oropharynx 944 1,712 AA vs. GA + GG 2.68 (1.345.35) China, India, USA Wang et al. [209]
AA vs. GG 2.70 (1.355.36)
AA vs. GA 2.59 (1.235.46)
(continued)
327
328

rs361525 Cervical cancer 2,416 2,010 A vs. G 0.61 (0.470.78) South Korea, USA, India, Sweden, Zhou et al. [189]
GA vs. GG 0.59 (0.450.77) Costa Rica
GA + AA vs. GG 0.59 (0.460.77)
1,190 1,784 GA vs. GG 0.54 (0.400.73) Costa Rica, Mexico, USA, India, Liu et al. [195]
GA + AA vs. GG 0.55 (0.410.74) Korea
HCC 1,572 1,875 A vs. G 1.32 (1.041.69) China, Thailand, Italy, Taiwan, South Cheng et al. [210]
AG vs. GG 1.32 (1.021.71) Korea, China
AA + AG vs. GG 1.33 (1.031.72)
938 1,370 AG vs. GG 1.63 (1.172.26) China, Korea, Thailand, Italy, Japan Wei et al. [203]
AA + AG vs. GG 1.62 (1.182.22)
rs1800630 HCC 627 1,004 AC vs. CC 1.72 (1.032.88) China, Korea, Thailand, Italy, Japan Wei et al. [203]
AA + AC vs. CC 1.65 (1.062.57)
rs1799724 Gastric cancer 1,118 1,591 T vs. C 1.17 (1.011.35) China, Japan Zhang et al. [211]
which remained signicant in Caucasian 857 C>T (rs1799724) and GC risk compared
populations [189]. A recent meta-analysis with the C allele [211].
reviewed results of 12 case-control studies Several systematic reviews have been pub-
including 1,751 cases with upper aerodigestive lished on the associations of TNF- polymor-
tract (UADT) cancer and 3,345 controls [209]. phisms and susceptibility to HCC. The most
Oropharynx cancer was investigated in six of recent one evaluated results of 11 case-control
these studies, while ve studies investigated studies (reported up to July, 2012) with a total of
esophagus cancer and one investigated larynx 1,572 HCC cases and 1,875 controls revealed an
cancer. Squamous cell carcinoma and adenocar- increased risk of HCC in carriers of A allele in
cinoma were investigated in nine and two studies, 238 G>A (rs361525) [210]. In stratied analy-
respectively, and one study investigated both can- sis, this association remained signicant only in
cer types. This study overall found a signicant Asian populations [210]. Another meta-analysis
increased risk of UADT cancer in carriers of AA included 2,357 cases and 3,161 controls pre-
genotype in 308G>A (rs1800629) compared to sented in 17 studies published till November
individuals who had GA or GG genotypes [209]. 2010 [203]. This study showed that A allele in
In addition, signicantly increased risks were both 238 G>A (rs361525) and 308G>A
found in oropharynx cancer but not in esophagus (rs1800629) was associated with an increased
cancer or larynx cancer. In the subgroup analysis risk of HCC. In stratied analysis for ethnicity,
for histologic type, this association remained sig- these associations remained signicant in Asians
nicant only for squamous cell carcinoma, but but not in Caucasians [203]. AA and AC geno-
not for adenocarcinoma [209]. types in 863 C>A (rs1800630) were also associ-
The most recent meta-analysis on gastric can- ated with increased HCC risk compared to CC
cer and 308G>A (rs1800629) reviewed 5,225 genotype. However, this study did not nd any
patients and 8,473 controls in 26 papers. This signicant association for 857 C>T (rs1799724)
study found a signicant increased risk of gastric and 1031 C>T (rs1799964) polymorphisms
cancer in carriers of A allele in comparison with [203]. The pattern for 238 G>A (rs361525) and
G allele [214]. Another meta-analysis on gastric 308G>A (rs1800629) was also repeated in other
cancer evaluated 4,399 cases and 6,855 controls systematic reviews [149, 215, 216].
presented in 24 studies published up to October A meta-analysis of seven case-control studies
2007 [208]. This study found a signicant with 1,311 bladder cancer cases and 1,436 con-
increased risk of gastric cancer in carriers of AA trols found that carriers of A allele in 308G>A
genotype in 308G>A (rs1800629) polymor- (rs1800629) had an increased risk of bladder can-
phism. In stratied analysis, AA genotype was cer [217]. A multicenter study investigated asso-
signicantly associated with an increased risk of ciations between six polymorphisms of TNF-
noncardia cancers and intestinal type of gastric (rs1799964, rs1800630, rs1799724, rs1800629,
cancer compared to the GG genotype [208]. rs361525, rs1800610) and prostate cancer risk in
Another meta-analysis on gastric cancer and 2,321 cases and 2,560 controls from two nested
308G>A (rs1800629) polymorphism included case-control studies within the Prostate, Lung,
19 studies with 3,335 GC patients and 5,286 con- Colorectal, and Ovarian Cancer Screening Trials
trols [211]. In addition, this study included ve and the Cancer Prevention Study II Nutrition
studies with 1,118 GC patients and 1,591 con- Cohort for [218]. Overall, this study found no
trols for 857 C>T (rs1799724). This study also signicant association between these polymor-
found a signicant increased risk of gastric can- phisms and prostate cancer risk. But this study
cer in carriers of A allele and AA genotype in found a signicant decreased risk in carriers of
308G>A (rs1800629) compared with G allele in T-C-T-G-A haplotype in rs1799964, rs1800630,
the whole population and in Caucasians but not rs1799724, rs1800629, and rs1800610 compar-
in East Asian [211]. This study also found a weak ing to the most frequent haplotype (T-C-C-G-G)
but signicant association between T allele of [218]. In subgroup analysis, T allele in 1036
C>T (rs1799724) in individuals who did not of HLA molecules [130]. In addition, it inhibits
regularly use NSAID was associated with signi- angiogenesis in various tumors [61, 223].
cantly less susceptibility to prostate cancer IFN- gene with four exons and a length of
compared to the CC genotype. In addition, when 5.4 kb is located on chromosome 12q24 [223].
limiting analysis to non-advanced tumors, carri- Two common SNPs including an intronic SNP
ers of 1036T or A allele in IVS1 + 123G>A (+874 T>A (rs2430561)) and a promoter variant
(rs1800610) had a signicantly decreased chance in (179 T>G (rs2069707)) have been previously
for prostate cancer [218]. identied [23, 61, 223]. This promoter variant is
Another multicenter study evaluated associa- adjacent to a HSF-binding motif. In addition,
tions of 308G>A (rs1800629) with NHL in there is a CA repeat microsatellite within the rst
7,999 cases and 8,452 controls from participating intron of the gene ranging from 12 to 15 repeats
studies from the InterLymph Consortium. [23, 223]. It was shown that allele 2 of the micro-
Carriers of 308A allele had increased risk for satellite and T allele in +874 T>A (rs2430561)
NHL, B-cell NHL, diffuse large B-cell lym- are in complete LD [23].
phoma (DLBCL), and other marginal zone lym- In vitro studies showed that T allele of +874
phoma. However, no signicant associations was T>A (rs2430561) is associated with higher IFN-
found between 308G>A (rs1800629) and production. EMSA studies showed that this allele
chronic small lymphocytic lymphoma CLL/SLL has higher afnity for NF-B which is in accor-
or T-cell NHL [219]. dance with the location of this SNP in the rst
Although this study also did not nd any sig- intron of the gene, a region related to binding of
nicant association between LTA +252 A>G NF-B [61, 223].
(rs909253) and NHL, carriers of G allele in this A meta-analysis of 17 studies with 1,929 can-
SNP had increased risk to DLBCL and mycosis cer cases and 2,830 controls showed a nonsigni-
fungoides [219]. cant increased risk of cancer in the presence of
In a meta-analysis of 33 studies with 14,435 AA genotype for +874 T>A (rs2430561).
cancer patients and 10,583 healthy controls, sta- However, this study showed signicant increased
tistically signicant increased risk of malignant susceptibility in individuals with AT genotype
transformation was found in carriers of G allele compared with TT genotype (Table 17.18) [223].
in +252 A>G (rs909253) which remained signi- Another meta-analysis with 32 studies and 4,524
cant in both Asian population and Caucasians cases and 5,684 controls did not nd a signicant
[220]. A recent study performed a meta-analysis association either [61]. Interestingly, in stratied
on 11 individual case-control studies with 2,270 meta-analysis for ethnicity, carriers of T allele
cases and 4,404 controls and found that G allele had signicant increased susceptibility to cancer
of +252 A>G (rs909253) is associated with a sig- in European and African population but not in
nicant increased risk of gastric cancer, but this Asian population [61]. This study also found that
risk was signicant only in Asians, but not TT genotype signicantly contributes to the risk
Caucasians [221]. An older study also showed of breast cancer in all ethnicities [61].
such a risk only in Asians especially those with
H. pylori infection [222].
17.7.10 Transforming Growth
Factor- (TGF-)
17.7.9 Interferon Gamma (IFN-)
Transforming growth factor- (TGF-) is a func-
Interferon gamma (IFN-) is a proinammatory tional mediator of epithelial and broblast cell
cytokine of Th1 subset with major roles in antitu- proliferation and a regulator of immune cell pop-
mor immune response. This cytokine enhances ulations [224]. In early stages of tumor progres-
differentiation of lymphocytes and their function sion, it acts as a tumor suppressor; however, in
and Ag presentation through inducing expression advanced cancers, TGF- induces many activities
Table 17.18 Signicant results from published meta-analysis of associations of (+874 T>A (rs2430561) in IFN-
gene with cancers
Total number Total number Population
Cancer site of cases of controls Analysis type OR 95 % CI included Reference
Cervical cancer 661 835 AT vs. TT 1.10 (1.021.19) India, South Mi et al. [223]
Africa
Breast cancer 527 715 TT vs. AA 1.58 (1.102.27) Iran, Italy, Liu et al. [61]
TT vs. AT/AA 1.53 (1.142.06) Turkey,
China, USA
Table 17.19 Genotype details for SNPS of TGF-

rs1800470 G = 0.444 UA +29 T>C Pro10Leu C allele:
rs1800471 G = 0.046 UA +74G>C Arg25Pro G allele:
rs1800469 T = 0.359 CEU
HCB
509 C>T NA T allele:
JPT
YRI
AVG
0 20 40 60 80 100
(C;C) (C;T) (T;T)
that lead to growth, invasion, and metastasis of of TGF-1 in in vitro studies [23, 228]. Arginine
cancer cells [224226]. in +915 G>C (rs1800471) was also associated
TGF- family consists of three isoforms with with higher levels of TGF-1 in in vivo stud-
pleiotropic roles in cancer immunity [227229]. ies [23]. In vitro studies showed that A allele in
TGF-1 as the most common isoform of this 1287 G>A (rs11466314), another variant of
family has enhancing effects on angiogenesis and this gene, is associated with higher expression of
its regulatory role in growth, differentiation, and TGF-1 [23]. EMSA studies showed that C allele
apoptosis of different cells [60, 133, 229]. It also in 387 C>T (rs11466315) had greater afnity
results in escape of malignant cells from immu- for Sp1 and Sp3 complexes [23].
nosurveillance by suppressing expression of Results of 40 case-control studies (includ-
HLA molecules [130, 133, 228, 229]. ing three studies with African population, 14 on
TGF-1 gene is located in the long arm Asian descendants, and 23 studies with European
of chromosome 19 (19q13.1). +869 T>C population) with 16,166 patients with various
(rs1800470; also called +29 T>C, or rs1982037) cancers and 19,126 controls were analyzed in a
is a common variant in the rst exonic region of systematic review. Although this meta-analysis
TGF-1 which results in substitution of leucine did not nd any signicant association with over-
to proline at codon 10 in signal sequence [227]. all risk of cancer, its result suggested that indi-
+915 G>C (also called +74 or rs1800471) is viduals with C allele in +869 T>C (rs1800470)
another exonic variant resulting in an arginine- have signicantly greater risk for prostate cancer.
to-proline substitution at codon 25. 509C>T In addition, in Asian populations, this allele was
(rs1800469) and 800G>A are two promoter signicantly associated with susceptibility to
variants in a proximal negative regulatory region cancers (Table 17.20) [229].
(Table 17.19) [230, 231]. In vivo studies showed A meta-analysis of 30 studies including
that T allele in 509 C>T (rs1800469) was asso- 20,401 patients with breast cancer and 27,416
ciated with higher levels of TGF-1 in plasma controls showed increased risk of breast cancer in
and also higher expression [23, 71]. Despite individuals with C allele in +869 T>C
some contrary results, C allele in +869 T>C (rs1800470). In stratied analysis, this association
(rs1800470) was associated with higher secretion remained signicant in Caucasian population and
332
Table 17.20 Signicant results from published meta-analysis of associations of SNPs of TGF- gene with cancers
rs1800470 Malignancy 5,183 6,524 CC vs. TT 1.26 (1.031.53) (Asian) Korea, China, Japan Wei et al. [229]
CT vs. TT 1.20 (1.011.43)
Prostate cancer 2,605 3,129 CT vs. TT 1.28 (1.011.61) USA, Germany, Brazil, Japan Wei et al. [229]
(CC + CT) vs. TT 1.24 (1.021.52)
Breast cancer 20,401 27,416 CT vs. TT 1.046 (1.0031.090) Mixed from Asian, Caucasian, and African Qiu et al. [60]
(CC + CT) vs. TT 1.052 (1.0121.095)
rs1800469 Gastric cancer 2,130 2,374 TT vs.(CC + CT) 1.35 (1.11.65) India, China Li et al. [71]
Colorectal cancer 994 2,335 CC vs. TT 1.62 (1.302.02) Iran, Germany, Korea, China Fang et al. [232]
(TC + CC) vs. TT 1.30 (1.081.58)
CC vs. (TC + TT) 1.48 (1.261.75)
4,440 6,785 (CC + CT) vs. TT 1.18 (1.061.32) USA, UK, Iran, China, Korea, Germany Wang et al. [233]
Colon cancer 1,760 2,454 (CC + CT) vs. TT 1.31 (1.051.63) UK, USA, China Wang et al. [233]
population-based studies [60, 234]. However, colorectal cancer [239]. Another systematic
older meta-analysis on breast cancer with almost review of 12 studies with 4,440 colorectal cancer
half of this sample could not nd such an associa- patients and 6,785 controls could nd such an
tion [234, 235]. Another recent meta-analysis of association only in colon cancer [233]. Regarding
20,022 cases and 24,423 controls could nd this HCC, a review of 11 studies including 2,577
increased risk for C allele just in Caucasians HCC cases and 4,107 controls revealed a signi-
[231]. This study also reviewed results of 8 stud- cant association between this SNP and the risk of
ies with 10,633 cases and 13,648 controls for HCC only in Caucasians [240].
509 C>T (rs1800469) and did not nd any sig-
nicant association between alleles of this poly-
morphism and risk of breast cancer in accordance 17.8 Concluding Remarks
with another meta-analysis (including 10,197
patients with breast cancer and 13,382 healthy In the recent decades, a great scientic effort has
controls) [231, 236, 237]. Some authors sug- uncovered the importance of immune polymor-
gested that the effect of TGF-1 is different phisms in cancers. However, this uncovered part,
according to expression of estrogen receptor and although is promising, only reminds us that there
progesterone receptor in breast cancer tumors is much more to reveal in this eld. There comes
[230]. a day that gathering immunogenetic data becomes
A systematic review analyzed results of 55 one main part of every clinical trial in cancer.
studies with a total number of 21,639 cancer This information will help understand more about
patients and 28,460 controls for associations of subgroups of patients, natural history of the can-
509 C>T (rs1800469) and susceptibility to dif- cers, responsiveness of cancer to treatment, or
ferent cancers. Although there was no a signi- toxicity of treatment, all in relation to immune
cant association between overall risk of cancer polymorphism [14]. One day, it might be possible
and genotypes of this SNP, this study found to assess the degree of predisposition to different
increased susceptibility of carriers of C allele to cancers for each individual and to employ preven-
colorectal cancer particularly in Caucasians tive measurement, and in case of suffering from
[238]. In addition, a meta-analysis of ve studies cancers, to efciently choose between treatment
with 994 colorectal cancer patients and 2,335 options and predict their clinical effectiveness
controls found increased risk of colorectal cancer [26]. Although it seems a vague dream in the far
for C allele of 509C>T (rs1800469) which future, it is becoming closer to reality everyday
remained signicant only in Asian population but considering the pace of scientic advancements.
not Caucasians in stratied analysis [232]. On the
other hand, a systematic review of seven original
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Primary Immunodeciencies
and Cancers 18
Mona Hedayat, Waleed Al-Herz,
Asghar Aghamohammadi, Kim E. Nichols,
and Nima Rezaei
Contents 18.5.2 Warts, Hypogammaglobulinemia,

Infections, and Myelokathexis Syndrome ....... 354
18.1 Introduction .............................................. 344
18.6 Diseases of Immune Dysregulation......... 354
18.2 Primary Antibody Deciencies ............... 344 18.6.1 X-Linked Lymphoproliferative Disease..... 354
18.2.1 Common Variable Immunodeciency ....... 344
18.2.2 X-Linked Agammaglobulinemia ............... 345 18.7 Syndromes with Autoimmunity .............. 355
18.2.3 Selective IgA Deciency ........................... 346 18.7.1 Autoimmune Lymphoproliferative
Syndrome ................................................... 355
18.3 Combined Immunodeciencies............... 346 18.7.2 Autoimmune Polyendocrinopathy
18.3.1 IL-2-Inducible T-Cell Kinase Deciency .. 346 with Candidiasis and Ectodermal
18.3.2 Purine Nucleoside Phosphorylase Dystrophy................................................... 356
Deciency .................................................. 347
18.3.3 Dedicator of Cytokinesis 8 Deciency ...... 348 18.8 Other Well-Dened
18.3.4 RHOH Deciency ...................................... 349 Immunodeciencies ................................. 356
18.3.5 MAGT1 Deciency.................................... 350 18.8.1 DNA Repair Defects .................................. 356
18.8.2 Signal Transducer and Activator
18.4 Phagocyte Defects .................................... 351 of Transcription 3 Deciency .................... 357
18.4.1 Severe Congenital Neutropenia 18.8.3 WiskottAldrich Syndrome ....................... 360
(Kostmann Syndrome) ............................... 351 18.8.4 Chromosome 22q11.2
18.4.2 ShwachmanDiamond Syndrome.............. 352 Deletion Syndrome .................................... 361
18.4.3 GATA2 Deciency ..................................... 352
18.9 Concluding Remarks ............................... 361
18.5 Defects in Innate Immunity .................... 353
18.5.1 Epidermodysplasia Verruciformis.............. 353 References ............................................................... 361
M. Hedayat, MD K.E. Nichols, MD

Division of Immunology, Boston Childrens Hospital, Department of Oncology, St. Jude Childrens
Harvard Medical School, Boston, MA, USA Research Hospital, Memphis, TN 38105, USA
e-mail: mona.hedayat@childrens.harvard.edu e-mail: Kim.Nichols@STJUDE.ORG
W. Al-Herz N. Rezaei, MD, MSc, PhD (*)
Department of Pediatrics, Faculty of Medicine, Research Center for Immunodeciencies,
Kuwait University, Safat, Kuwait Childrens Medical Center, Pediatrics Center of
e-mail: wemh@hotmail.com Excellence, Tehran University of Medical Sciences,
Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran
A. Aghamohammadi, MD, PhD
Research Center for Immunodeciencies, Department of Immunology, School of Medicine, and
Department of Allergy and Immunology, Molecular Immunology Research Center,
Childrens Medical Center, Pediatrics Center of Tehran University of Medical Sciences,
Excellence, Tehran University of Medical Sciences, Dr Qarib St, Keshavarz Blvd, Tehran 14194, Iran
Tehran 14194, Iran e-mail: rezaei_nima@tums.ac.ir
e-mail: aghamohammadi@tums.ac.ir

344 M. Hedayat et al.
18.1 Introduction 18.2 Primary Antibody

Deciencies
Immunodeciency disorders are classied as
either primary (genetic) or secondary (acquired). 18.2.1 Common Variable
Primary immunodeciencies (PIDs) are a hetero- Immunodeciency
geneous group of disorders that predispose to
frequent and severe infections, autoimmunity, Common variable immunodeciency (CVID) is the
and, in certain diseases, malignancies. According second most common PID (second to selective
to the updated classication of PIDs by the IgA deciency), which is estimated to affect as
International Union of Immunological Societies many as 1 in 25,000 individuals [12]. CVID is a
Expert Committee in 2011 [1], over 175 PIDs clinically and genetically heterogeneous group of
have been identied, with a total incidence of diseases characterized by hypogammaglobu-
1 in 10,000 births [2]. The overall risk for devel- linemia of two or more isotypes (IgG, IgA, or
oping malignancies in children with PIDs is IgM), impaired functional antibody responses,
425 % [3], with lymphomas representing up to and consequently increased susceptibility to
60 % of all cancer types [4]. Considering the chronic recurrent bacterial infections [13].
improved therapeutic options and increasing Furthermore, affected individuals are predisposed
life expectancy of PID patients, it is possible that to autoimmune and granulomatous diseases as
the incidence of malignancies may increase as well as hematological and certain solid malig-
patients live longer lives. Increasing evidence nancies in up to 15 % of subjects [13, 14]. Non-
suggests that defective immunosurveillance Hodgkin lymphomas (NHLs) represent the most
mechanisms, interacting with oncogenic viruses, common malignancies with up to a 259-fold
chronic antigen stimulation, defective DNA increase in risk compared to the general population
damage response, and genetic alterations of [1517]. NHLs in CVID are mostly extranodal, well
oncogenic and tumor suppressor genes, are the differentiated, and of B-cell origin [15]. In older
major factors driving the development of cancer studies, there was an increased risk of gastric
in patients with PIDs [59]. While further eluci- cancer (up to 47-fold) [16, 18], probably related
dation of the precise molecular pathogenesis of to the increased frequency of pernicious anemia
malignancies in the context of immunodeciency or Helicobacter pylori infection [19]. However, a
syndromes offers an exciting prospect for the 2010 study of 476 patients revealed that gastric
development of targeted cancer therapies, we cancer was diagnosed in only 0.6 % of patients,
report here the most recent clinical observations suggesting a potential downward trend. In this
on the incidence and types of malignancies, study, 6.7 % of patients developed NHL and
which should alert clinicians to the potential 0.8 % developed Hodgkin lymphoma (HL).
importance of more vigilant screening in immu- Other solid malignancies, including breast, colon,
nodecient patients. It should be noted, oral, and other cancers, collectively accounted
however, that surveillance protocols should for cancer in up to 4 % of patients [14].
be applied judiciously, without indiscriminate Defects in genes encoding the inducible
and frequent use of certain radiological proce- costimulator (ICOS; OMIM*604558) [20],
dures, due to increased risk of radiosensitivity tumor necrosis factor (TNF) receptor super-
in some syndromes [10]. Furthermore, early family members 13B (TNFRSF13B or TACI;
intervention with hematopoietic cell transplanta- OMIM*604907) [21, 22] and 13C (TNFRSF13C
tion, which is indicated in certain PIDs, may or BAFF-R; OMIM*606269) [23], CD19
decrease not only the infection but also the (OMIM*107265) [24], CD20 (OMIM*112210)
cancer risk [11]. [25], CD81 (OMIM*186845) [26], CD21 (CR2;
18 Primary Immunodeciencies and Cancers 345
OMIM*120650) [27], and LRBA (OMIM*606453) as evident by enhanced radiosensitivity, has been
[28] have thus far been identied in patients reported in patients with CVID [47, 48], with
with CVID. those having the highest rate of chromosomal
The immunologic defects in CVID are multi- aberration developing lymphoma [48].
faceted. Despite normal numbers of B cells in
the majority of affected individuals, their
inability to undergo terminal differentiation into 18.2.2 X-Linked
immunoglobulin-secreting plasma cells forms the Agammaglobulinemia
core common defect [15, 29]. T-cell abnormalities
are also frequently encountered in patients with X-linked agammaglobulinemia (XLA) is the
CVID, including impaired T-cell proliferative prototypic humoral immunodeciency arising
responses, partly due to defects in T-cell receptor from a defect in B-cell maturation, affecting the
signaling [30, 31]; decreased numbers of CD4+ T transition of B-cell progenitors into mature B
cells in conjunction with normal to increased lymphocytes and leading to the consequent failure
numbers of CD8+ T cells, giving rise to reversed of immunoglobulin production. It is estimated to
CD4:CD8 ratio [32, 33]; imbalanced T-helper cell afict three to six out of every million males of
responses, representing a shift toward a Th1 all racial and ethnic groups. As the maternally
phenotype [3436]; increased suppressor T-cell derived antibodies (Abs) are degraded, most
activity [34]; and diminished expression of the patients with XLA begin to experience recurrent
costimulatory molecule CD40 ligand [37]. infections by the end of the rst year of life
Moreover, the absolute and relative NK, invariant [18, 49]. Approximately 1015 % of individuals
NKT, and plasmacytoid dendritic cell numbers with XLA have higher concentrations of serum
are reported to be decreased in patients with immunoglobulin than expected or are not recog-
CVID [3840]. nized to have immunodeciency until after the
The complex derangement in numerical and age of 5 years. XLA is mainly characterized by
functional characteristics of B, T, NK, and den- recurrent bacterial infections, in particular with
dritic cells results in impaired humoral and extracellular encapsulated bacteria, most com-
cellular immune responses. As a result, patients monly localized in the respiratory tract. Diarrhea
often develop chronic inammatory and autoim- and skin infections are also frequently seen [18,
mune diseases, as well as recurrent bacterial 49, 50]. Despite general resistance to viral infec-
infections. These factors, along with persistent tions, affected individuals are susceptible to
antigenic stimulation, mainly from chronic severe and chronic enteroviral infections [51].
Helicobacter pylori [41, 42], human herpesvirus The gene defective in XLA, Brutons tyrosine
8 [43], and cytomegalovirus [44] infections, may kinase (BTK; OMIM*300300), encodes a cyto-
ultimately drive tumorigenesis; however, their plasmic tyrosine kinase of the Btk/Tec family [52].
relative contribution and the precise underlying The crucial role of BTK in B-cell growth and
mechanisms remain to be elucidated [13]. differentiation has been documented by a devel-
Furthermore, given the possible role of an auto- opmental block at the pro-B-cell to pre-B-cell
crine B-cell activating factor (BAFF) signaling transition with a reduction in mature B cells [50],
circuit in promoting tumor cell survival and pro- whereas T-lymphocyte subsets are normal and
liferation [45, 46], it is possible that aberrant may show a relative increase. In B cells, B-cell
BAFF-R signal transduction resulting from antigen receptor (BCR) cross-linking activates
CVID-related mutations might enhance malignant BTK downstream of the Src family kinases [53, 54],
transformation [13]. Finally, defective DNA repair, where it is a critical component in BCR-coupled
calcium signaling cascade [55, 56]. BTK also In IgAD, the common nding is a defect in
acts as a mediator of oxidative stress-induced the maturation of B cells producing IgA [73].
apoptosis of irradiated neoplastic B cells and The genetic basis of IgAD is complex and has
B-cell precursors [57], probably via the negative remained unclear. Autosomal recessive, autoso-
regulation of the antiapoptotic signal transducer mal dominant, and sporadic transmission pat-
and activator of transcription 3 (STAT3) function terns have all been observed. In view of the lack
[58]. BTK also interacts with and functions of an identied primary genetic defect and the
downstream of Toll-like receptor (TLR)-8 and variation in the inheritance patterns, it is likely
TLR9. These latter functions might explain the that IgAD represents a heterogeneous group of
susceptibility to enteroviral infections in XLA genetic abnormalities such as CVID. In support
patients [59, 60]. of this notion is the observation that mutations in
Although the overall chance of developing transmembrane activator and calcium modulator
malignancies in XLA is low, there are reports of and cyclophilin ligand interactor (TACI) gene
a 30-fold increased risk of colorectal cancer in (TNFRSF13B; OMIM*604907), which appear
patients with XLA [61, 62]. Aberrant immuno- to act as a disease-modifying mutation, have
logical function and/or persistent asymptomatic been found in IgA deciency and CVID [22].
inammation in the colon is generally thought to Moreover, a novel shared risk locus associated
contribute to the increased risk of colorectal with lower inducible costimulator (ICOS)
cancer. However, it has been shown that BTK and higher cytotoxic T-lymphocyte-associated
loss of function is associated with excessive protein-4 (CTLA4) expression has been recently
Wnt--catenin signaling [63], which is known as dened in both diseases [76]. The presence of
a major contributor to the development of specic major histocompatibility complex (MHC)
colorectal carcinoma [64]. In addition to colorectal haplotypes, in particular the ancestral HLA-A1,
cancer, cases of pituitary adenomas [18], gastric B8, DR3, and DQ2 (8.1), have been associated
adenocarcinoma [65], and squamous lung cancer with susceptibility to IgAD [77].
[66] have been reported. The association of malignancy, especially of
the lymphoreticular and gastrointestinal systems,
with IgAD has been documented mainly in adults
18.2.3 Selective IgA Deciency [78, 79] with an estimated two fold increased risk
compared to general population [80]. However,
Selective IgA deciency (IgAD) is the most in a combined Danish and Swedish study of 386
common PID with a prevalence that varies from patients with IgAD, the incidence of cancer was
1 in 143 to 1 in 18,550 in different ethnic groups not increased. Yet, the investigators in the same
[67, 68]. It is dened as occurring when serum study found that relatives of the same patients
IgA levels are equal to or below 0.07 g/L with had slightly elevated cancer rates. In contrast to
normal IgM and IgG levels in individuals 4 years adults, children with IgAD appear not to be at
of age or older in whom other causes of hypo- risk of malignancy [81, 82], which has only been
gammaglobulinemia have been excluded [69]. reported in case reports [8385].
As many as 8590 % of patients with IgAD are
asymptomatic, which could be explained by a
compensatory increase in IgM production and 18.3 Combined
subsequent increase in secretary IgM in the Immunodeciencies
mucosal lumen [70]. However, IgAD can present
with a broad spectrum of clinical manifestations, 18.3.1 IL-2-Inducible T-Cell Kinase
including recurrent sinopulmonary and gastroin- Deciency
testinal infections, allergic disorders, GI diseases
(especially celiac disease), progressive neurode- IL-2-inducible T-cell kinase (ITK) deciency is a
generative disorders, autoimmunity, and malignancy, novel PID characterized by severe EVB-associated
with gastric carcinomas and lymphomas being immune dysregulation, with a clinical picture
frequently associated with the disease [7075]. similar to that seen in X-linked lymphoproliferative
disease (XLP) [86, 87]. ITK deciency was NKT cells and impaired NK- and T-cell-mediated
originally described in 2009, where two ITK- cytotoxicity, plays contributory roles in the devel-
decient female siblings from a consanguineous opment of EBV-associated LPDs in ITK-decient
Turkish family developed uncontrolled Epstein patients [99].
Barr virus (EBV) infection resembling hemo-
phagocytic lymphohistiocytosis (HLH) with
eventual progression to HL [86]. In a report of 18.3.2 Purine Nucleoside
three cases from a single Arab family, the rst Phosphorylase Deciency
presentation was HL, whereas fulminant hemo-
phagocytosis and severe mononucleosis appeared Purine nucleoside phosphorylase (PNP) de-
after remission of lymphoma [87]. Adding to the ciency is a rare, autosomal recessive, combined
complexity of the disease, seven additional ITK- immunodeciency disorder, with an estimated
decient patients, of whom four developed HL, frequency of 4 % among patients with SCID [100].
were identied following the screen of patients The disease usually manifests during the rst
with autoimmune lymphoproliferative syndrome year of life; however, the onset of symptoms may
or suspicion of congenital forms of HLH [88, vary, with some patients having no apparent
89]. More recently, the clinical spectrum of ITK clinical immunodeciency until later in childhood
deciency has been further extended to include [101104]. Common clinical manifestations in
late-onset isolated involvement of the lungs and patients with PNP deciency include recurrent,
the mediastinal lymph nodes with a polyclonal bacterial, viral, and opportunistic infections;
proliferation of small B cells not suggestive of prolonged diarrhea; failure to thrive; neurologic
any malignant lymphoma [90]. abnormalities, including nonprogressive cerebral
In ITK deciency, germ line loss-of-function palsy, ataxia diplegia, or disequilibrium; and
mutations in the ITK gene (OMIM*186973) autoimmune disorders, including autoimmune
result in pronounced instability or truncation of hemolytic anemia, idiopathic thrombocytopenia,
the ITK protein [86, 87]. ITK, a member of the autoimmune neutropenia, lupus, and central
Tec family tyrosine kinases, is expressed in T as nervous system vasculitis [100102, 105108].
well as NK cells, invariant NKT cells, and mast Due to profound T-cell abnormalities, patients
cells [9193]. ITK plays a critical modulatory role are extremely susceptible to viral infections and
in the T-cell receptor (TCR) signaling cascade. may develop disseminated or even fatal disease
In mice, it functions in the positive/negative [100, 103]. A high frequency of malignancy is
selection of thymocyte development, as well as also noted, including pharyngeal tumors, lym-
regulation of conventional vs. innate-type CD8+ phoma, and lymphosarcoma [100, 109, 110]. In a
T-cell development [94, 95]. Moreover, Itk report of 33 patients with PNP deciency, four
CD8+ T cells fail to mount effective primary or had developed lymphoma or lymphosarcoma and
memory immune responses to a variety of viral one had a pharyngeal tumor [100]. Immunological
infections [9597]. Itk is also crucial for invariant evaluations of patients with PNP deciency
NKT-cell development and function in mice [93]. revealed marked T-cell lymphopenia, with
Similarly, a characteristic reduction in naive decreased T-cell proliferative responses and
CD45RA+ T cells and NKT cells has been abnormal humoral immunity in most cases, as
reported in ITK-decient patients [8688]. assessed by B-cell number, total immunoglobulin
Moreover, ITK has been shown to differentially levels, and specic antibody formation. NK numbers
regulate NK-cell-mediated cytotoxicity, which might may be variable.
be impaired in the absence of ITK protein [98]. Several disease-causing mutations have been
The development of LPDs in ITK-decient identied in the PNP gene (OMIM*164010),
patients almost always follows primary EBV producing proteins with differing degrees of
infection and is diagnosed as HL, as opposed to enzymatic activity that inversely correlate with
Burkitts lymphoma or other NHL seen in XLP. It clinical severity (i.e., more functional proteins
is speculated that perturbed innate and adaptive are associated with milder forms of disease,
antitumor immunosurveillance, including lack of while less functional proteins lead to severe
phenotypes) [104, 111, 112]. PNP is an enzyme Moreover, EBV and/or cytomegalovirus infections
in the purine salvage pathway that reversibly con- are documented in up to 40 % of patients
verts inosine to hypoxanthine and guanosine to [126, 127]. Increased frequencies of malignancies,
guanine. Of all accumulated PNP substrates, only including squamous cell carcinoma (SCC),
deoxyguanosine can be phosphorylated further cutaneous T-cell lymphoma/leukemia, Burkitts
in the mammalian cells. Thus, in PNP deciency, lymphoma, anaplastic B-cell lymphoma, as well
there is accumulation of abnormally high levels as adrenal leiomyoma and microcytic adnexal
of lymphotoxic dGTP [113, 114]. This, in turn, carcinoma, have been reported in up to 17 % of
inhibits ribonucleotidase reductase activity, DOCK8-decient patients [124, 125, 127, 128].
depletes dCTP, and inhibits DNA synthesis and The disease is due to biallelic mutations in the
repair [113, 114]. Moreover, mitochondrial dGTP DOCK8 gene (OMIM*611432), which encodes
is also likely to inhibit mitochondrial DNA repair DOCK8, a member of the DOCK180-related
and initiate the apoptotic protease cascade trig- family of atypical guanine nucleotide exchange
gered by cytochrome C release [115117]. The factors (GEFs) [129]. DOCK8 was shown to bind
most characteristic immune abnormality is thus a to the Rho GTPases Cdc42, Rac1, RHOJ, and
profound defect in T-cell number and function; RHOQ in a yeast two-hybrid system but not in
however, abnormal B-cell functions, including GST pulldown assay [130]. Following the gen-
defective Ab production, are common and in part eration of DOCK8-knockout mice, it has been
due to abnormal T-cell help [100, 118]. However, documented that DOCK8 is a Cdc42-specic
an intrinsic defect in B-cell function has not been GEF [131] and that DOCK8 exists in a macromo-
excluded. The T-cell specicity of PNP lies in the lecular complex with the WiskottAldrich syndrome
high deoxyguanosine phosphorylating activity in protein (WASP), an actin nucleation-promoting
the T lymphocytes, as compared with B lympho- factor activated by Cdc42, as well as with talin, a
cytes or other tissues [119, 120], and the inherent protein required for integrin-mediated adhesion
susceptibility of immature thymocytes to apoptosis [132]. These ndings further support the role of
during T-cell selection [121, 122]. DOCK8 in the regulation of actin dynamics and
formation of the immunologic synapse, which are
required for full T-cell activation, proliferation,
18.3.3 Dedicator of Cytokinesis 8 and acquisition of effector functions.
Deciency Immunological features of DOCK8 de-
ciency, besides high serum IgE levels and eosino-
Dedicator of cytokinesis 8 (DOCK8) deciency, philia, include lymphopenia (progressive with
initially described as a form of autosomal reces- age) affecting CD4+ and CD8+ T cells (especially
sive hyper-IgE syndrome [123], is now regarded the CD4+ T cells) and, to a lesser extent, NK and
as a combined immunodeciency disorder [1], B cells [124127], plus a virtual lack of circulat-
presenting early in life with: (1) recurrent sin- ing CD19+CD27+ memory B cells [133]. Studies
opulmonary infections; (2) cutaneous viral, bac- in DOCK8-decient patients have demonstrated
terial, and fungal infections; (3) severe atopy, decreased T-cell activation and proliferation in
asthma, and allergies; (4) immune-mediated response to mitogens [124127], but not to spe-
pathologies including autoimmune hemolytic cic antigens [126]; however, these functional
anemia and vasculitis; (5) neurological complica- studies are inconclusive due to the difculty in
tions; (6) malignancies; and (7) extremely high isolating naive T cells from the peripheral blood.
serum IgE levels and eosinophilia [123127]. In murine models of DOCK8 deciency, the
Cutaneous viral infections are the most distinctive defect has been localized to normal survival of
clinical feature and often identied as recalcitrant, CD8+ memory T cells [134]. DOCK8-decient
extensive lesions caused by herpes simplex (HS), humans and/or mice also exhibit abnormalities in
human papilloma (HP), molluscum contagiosum cytokine secretion associated with a T-helper
(MC), and varicella zoster (VZ) viruses [124127]. 2-biased immune response [124, 126, 134],
low serum IgM levels and impaired Ab responses spectrum of the disease is not restricted to
[133, 135], decreased CD4+ T-helper type 17 susceptibility to HPV [146].
cells, and impaired NK-cell cytotoxicity [124, RHOH deciency results from homozygous
125, 132, 136]. loss-of-expression mutations (Y38X) in the
Increased susceptibility to malignancy in RHOH gene (OMIM*602037) located on chro-
DOCK8-decient patients can be explained by mosome 4p13, which encodes an atypical Rho
failure of CD8+ T- and NK-cell-mediated tumor GTPase (RHOH) expressed predominantly in
immunosurveillance, as well as chronic antigenic hematopoietic cells. RHOH is GTPase decient
stimulation. Moreover, there is evidence that and remains constitutively in the active, GTP-
DOCK8 itself might have direct tumor sup- bound state, suggesting that its activity is likely
pressor activity [137140], and that loss of regulated by the level of the protein expressed in
DOCK8 expression might contribute to carcino- the cells rather than guanine nucleotide cycling
genesis [141]. Reduced DOCK8 expression has [147]. It has been shown to counteract Rac
been demonstrated in the vast majority of primary GTPase activities in lymphoid cell lines and
lung cancers, irrespective of the histological type, cytokine-stimulated hematopoietic progenitor
compared with normal lung tissue. Epigenetic cells, resulting in reduced proliferation, increased
mechanisms, including DNA methylation and apoptosis, and defective actin polymerization
histone deacetylation, were indicated to be [147150].
involved in DOCK8 downregulation in lung Immunologic evaluation of RHOH-decient
cancer cells [137], as with other candidate tumor patients revealed no major abnormality in the
suppressor genes, such as p16, RASSF1A, and frequencies of B-cell subsets, NK cells, NKT cells,
MYO18B [142145]. Moreover, homozygous monocytes, and polymorphonuclear cells and in
deletions of the DOCK8 gene has been shown in Ab production. Despite maintaining normal
breast and gastric cancer cell lines. These results T-cell counts, both patients displayed a restricted
suggest that genetic and epigenetic inactivation T-cell repertoire, lack of circulating naive T cells
of DOCK8 is involved in the development and/or consistent with the defect in thymic T-cell devel-
progression of lung cancers and other cancers by opment observed in Rhoh-/- mice [149], expan-
disturbing the regulatory functions of DOCK8 in sion of effector memory T cells (more likely to be
cell migration, morphology, adhesion, and growth consequences of chronic infection), altered
of cells [137]. expression of T-cell tissue-homing markers with
strikingly lower than normal proportion of
skin-homing 7+ T cells, and impaired T-cell
18.3.4 RHOH Deciency proliferative responses to anti-CD3 but variable
responses to mitogens and recall antigens (Ags)
Ras homolog family member H (RHOH) de- [146]. It is evident that on TCR stimulation,
ciency is a novel form of PID recently identied murine RHOH undergoes tyrosine phosphoryla-
by genome-wide linkage analysis in two young tion and mediates recruitment of ZAP70 and Lck
adult siblings born to consanguineous French to the TCR/linker of activation in T-cell (LAT)
parents [146]. Since childhood, both patients signalosome [151]. This nding has been con-
displayed a phenotype resembling epidermodys- rmed in RHOH-decient T cells of patients,
plasia verruciformis (EV), characterized by per- showing little or no ZAP70 phosphorylation in
sistent cutaneous infections with EV-specic the presence or absence of CD3 stimulation [146].
HPV (EV-HPV) genotypes. The older sibling had The combination of T-cell defects common to
also developed Burkitts lymphoma in childhood, both mice and humans, including impaired T-cell
granulomatous lung disease, and psoriatic-like responses, a lack of naive cells, and smaller than
lesions, whereas the younger sibling had mol- normal proportion of 7+ T cells, might explain
luscum contagiosum, psoriatic lesions, and the pathogenesis of susceptibility to cutaneous
gingivostomatitis, indicating that the phenotypic EV-HPVs.
The RhoH/TTF (translocation three four) gene role as a second messenger in intracellular
was rst identied by fusion to the BCL6/LAZ3 signaling has only begun to be unraveled [158,
oncogene resulting from t(3;4)(q27;p11) translo- 162165].
cation in an NHL cell line [152154]. Another Immunological investigations in patients with
chromosomal alteration involving the RhoH/TTF MAGT1 deciency revealed CD4 lymphopenia,
gene in a patient with multiple myeloma and leading to an inverted CD4:CD8 ratio and
t(4;14)(p13;q32) translocation has also been reduced number of recent thymic emigrant T
identied [154]. Moreover, aberrant somatic cells, indicating that impaired thymopoiesis may
hypermutations in RHOH gene have been contribute to CD4 lymphopenia. No major distur-
previously reported in various B-cell malignan- bance was observed in other lymphocyte popula-
cies, including diffuse large B-cell lymphomas tions. MAGT1-decient T cells showed impaired
[155], AIDS-related NHL [156], primary central proliferation and activation upon in vitro stimula-
nervous system lymphomas [157], and, rarely, tion with anti-CD3 Ab. In contrast, T-cell activa-
Burkitts lymphoma [155]. However, it remains tion in response to phorbol myristate acetate and
unclear whether these mutations translate into ionomycin was intact, showing that the patients
abnormal levels of RhoH expression in lympho- had a proximal TCR signaling defect prior to the
mas and what pathophysiological contribution induction of the Ca2+ ux. MAGT1-decint B
hypermutation in the RhoH gene plays in cells showed normal activation upon BCR
lymphomagenesis. stimulation [158]. Recapitulating the patients
phenotype by knocking down MGAT1 in normal
T cells, as well as rescuing patients T cells with
18.3.5 MAGT1 Deciency ectopic expression of MAGT1, established that
MAGT1 is required for TCR-stimulated Mg2+
A novel X-linked immunodeciency has been inux that transiently raises free [Mg2+]i in
recently identied in seven male patients (two of order to temporarily coordinate T-cell activation
which were siblings) with mutations in the mag- [158, 166].
nesium transporter 1 (MAGT1) gene [158, 159]. Individuals with genetic deciencies in
The clinical phenotype of MAGT1 deciency is MAGT1 have uncontrolled EBV infection and a
characterized by chronic viral infections, EBV in predisposition to lymphoma. This has been attrib-
particular, which led to the development of EBV- uted to a selective loss of NKG2D expression
related lymphomas or related lymphoprolifera- (posttranscriptional, accelerated protein turn-
tive disorders in four patients. Other clinical over) and the resultant impaired cytolytic
features include recurrent upper respiratory tract responses of NK and cytotoxic CD8+ T lympho-
infections, viral pneumonia, HSV-1 infections, cytes [159], which are essential for control of
recurrent shingles, molluscum contagiosum, and viral infections and tumor immunosurveillance
chronic diarrhea. [158, 159] [167]. Hence, MAGT1 not only mediates TCR-
MAGT1 deciency (OMIM*300715), named induced Mg2+ ux but also regulates the basal-
X-linked immunodeciency with Mg2+ defect, free [Mg2+]i homeostasis required for NKG2D
EBV infection, and neoplasia (XMEN) disease, cytolytic activity. This has been veried by culti-
has been reported to be caused by null mutations vation of NK and cytotoxic CD8+ T lymphocytes
in the MAGT1 gene [158]. MAGT1 encodes a from XMEN patients in Mg2+-supplemented
membrane-associated transporter that selectively medium, causing a dose-dependent increase in
conducts Mg2+ across the membrane, with almost free [Mg2+]i, which did recover the cytotoxicity
no permeability to other cations including Ca2+ defect partially in cytotoxic CD8+ T lymphocytes
[160, 161]. Despite the well-known essential and almost completely in NK cells [159]. Most
roles of Mg2+ as a cofactor for ATP, nucleic acids, notably, magnesium supplementation in vivo
and numerous metabolic enzymes, its critical concurrently reduced EBV-infected cells, which
may provide an adjunctive treatment to prevent in neutrophils and monocytes, and is stored in
early lymphoma development and mortality in the primary granules of neutrophils [174].
XMEN patients. Interestingly, mutations in the ELA2 gene are
also responsible for the clinical phenotype of
cyclic neutropenia. The pathophysiological
18.4 Phagocyte Defects mechanisms responsible for the development of
different phenotypes, congenital or cyclic neutro-
The underlying mechanism of cancer develop- penia, are not yet understood [175]. Most patients
ment in PIDs caused by defects of phagocytic with autosomal recessive disease, which comprises
cells is quite different from that observed in other approximately 30 % of SCN, have mutations in
immunodeciency disorders. Here the implicated the HS-1-associated protein X (HAX1) gene
genes are important for proper myeloid cell (OMIM*605998) [176]. HAX-1, a mitochondria-
development; thus cancers form due to dysregu- targeted protein containing Bcl-2 homology
lated myelopoiesis. This is distinct from cancers domains, is an apoptosis-regulating protein [176].
that occur in some other conditions including Mutations in the glucose-6-phosphatase catalytic
impaired immunosurveillance and presence of subunit 3 (G6PC3) gene (OMIM*611045) have
specic viruses. recently been identied in a group of autosomal
recessive SCN patients with additional syn-
dromic features including cardiac and urogenital
18.4.1 Severe Congenital anomalies and increased venous marking [177].
Neutropenia (Kostmann Patients with X-linked SCN harbor activating
Syndrome) mutations in WiskottAldrich syndrome (WAS)
gene (OMIM*300392), leading to a constitu-
Severe congenital neutropenia (SCN) is a rare tively active form of the WAS protein and unreg-
PID characterized by a maturation arrest of ulated actin polymerization [178]. Inactivating
myelopoiesis at the level of the promyelocyte/ mutations in the proto-oncogene growth factor-
myelocyte stage with peripheral blood absolute independent 1 (GFI1) gene (OMIM*600871) are
neutrophil counts (ANCs) below 0.5 109/L, in also associated with SCN [179]. In addition, SCN
addition to early-onset supercial and systemic without a maturation arrest has recently been
bacterial infections [168, 169]. The skin and associated with p14 protein deciency [180].
mucous membranes are usually affected by Finally, acquired nonsense mutations in colony-
ulceration, gingival hyperplasia, periodontitis, stimulating factor 3 receptor (CSF3R) gene
and abscess formation [170]. Patients may also (OMIM*138971) have also been found to affect
suffer from neurological disorders including 20 % of SCN patients [181].
developmental delay, mental retardation, epilepsy, SCN patients are at an increased risk of
and decreased cognitive function [171, 172]. myelodysplasia (MDS) and acute myeloid leuke-
SCN follows an autosomal dominant or reces- mia (AML) development with a cumulative inci-
sive pattern of inheritance or can occur sporadi- dence of leukemia of 22 % after 15 years of
cally. It is a genetically heterogeneous disorder G-CSF treatment [182, 183]. Independent of the
caused by a variety of mutations in several differ- genetic subtype, conversion to leukemia in
ent genes. Nonetheless, the different genetic patients with SCN is often associated with one or
forms of SCN share a rather similar clinical phe- more somatic cellular genetic abnormalities
notype. Mutations in the neutrophil elastase (e.g., monosomy 7, RAS mutations, trisomy 21, or
(ELA2) gene (OMIM*130130) are found in CSF3R mutations), which may be diagnostically
approximately 50 % of all cases, i.e., those with useful to identify subgroups of patients at high
dominant autosomal or sporadic SCN [170, 173]. risk of developing leukemia [175]. Other risk
ELA2 is a serine protease, exclusively expressed factors for progression to MDS and/or AML are
the severity of neutropenia, younger age at (OMIM*607444) [194], with the encoded protein
diagnosis, and prior exposure to G-CSF. [184] being essential for normal ribosome maturation,
Interestingly, marrow cells from nearly 80 % of though its precise molecular function remains
patients with SCN who transform to leukemia unclear [195, 196]. In addition to a stem-cell
show point mutations in CSF3R, suggesting that defect [197, 198], patients with SDS have also a
these mutations play an important role in leuke- serious, generalized marrow dysfunction with an
mogenesis [185]. abnormal bone marrow stroma in terms of its
Hematopoietic stem-cell transplantation (HSCT) ability to support and maintain hematopoiesis
is the only denitive treatment for patients with [196, 199].
bone marrow failure, MDS, or leukemia; how- Similar to other marrow failure syndromes,
ever, it seems that patients with SCN may be at patients with SDS have an increased risk for
increased risk of transplant-related mortality for MDS and AML [200], with an estimated risk of
unknown reasons. As a result, there is no clear 19 % at 20 years and 36 % at 30 years [184].
consensus on when a patient with SCN should There are also three reported cases of solid
undergo HSCT [186]. tumors in patients with SDS [201203]. The rea-
son behind this malignant predisposition is not
known. However, several theories have been
18.4.2 ShwachmanDiamond proposed, including chromosome instability
Syndrome [204, 205], accelerated apoptosis linked to
increased expression of the Fas Ag and to hyper-
ShwachmanDiamond syndrome (SDS) is a rare activation of the Fas signaling pathway [206],
autosomal recessive, systemic disease charac- and abnormal gene expression patterns as
terized by exocrine pancreatic insufciency, evident by upregulation of several oncogenes,
impaired hematopoiesis, and leukemia predis- including LARG, TAL1, and MLL, and down-
position [187]. Other clinical features include regulation of several tumor suppressor genes,
skeletal, immunologic, hepatic, and cardiac dis- including DLEU1, RUNX1, FANCD2, and
orders [186]. There is considerable phenotypic DKC1, which might result in continuous stimu-
variability between individuals, and making the lation favoring evolution or progression of
diagnosis can be challenging, particularly in malignant clones [207]. Accordingly, all
older patients in whom symptoms such as steat- patients with SDS should be monitored with
orrhea may have resolved [186] or may not be peripheral blood counts every 34 months and
present [188]. The most common hematologic marrow evaluation on a yearly basis, and if indi-
abnormality in patients with SDS is neutropenia, cated, HSCT should be done prior to the devel-
which can be chronic or intermittent. Anemia and opment of overt leukemia.
thrombocytopenia are also common manifestations.
Patients with SDS are susceptible to recurrent
infections [189] likely due to neutropenia. Other 18.4.3 GATA2 Deciency
immune defects have also been reported. These
include neutrophil chemotactic defects [190, A novel inherited immunodeciency clinically
191], decreased proportions of circulating B characterized by disseminated mycobacterial
cells, low immunoglobulin levels, decreased infections (typically Mycobacterium avium com-
in vitro B-cell proliferation, lack of specic plex [MAC]), opportunistic fungal infections,
antibodies or decreased total circulating T lym- disseminated HPV infections, and pulmonary
phocytes, as well as decreased proliferative alveolar proteinosis, with an increased risk of
responses [192, 193]. myelodysplasia, cytogenetic abnormalities, and
Around 90 % of patients with clinical features myeloid leukemias, has been recently described
of SDS have mutations in the Shwachman [208211]. This novel inherited immunode-
BodianDiamond syndrome ( SBDS ) gene ciency, termed monocytopenia and mycobacterial
infection (MonoMAC) syndrome, precedes the 18.5 Defects in Innate Immunity

development of overt MDS by many years, and
eventually leukemias. This form of immunode- 18.5.1 Epidermodysplasia
ciency occurs either as an autosomal dominant Verruciformis
form or sporadically [212].
Heterozygous mutations in the critical hema- Epidermodysplasia verruciformis (EV) is a
topoietic regulator of stem-cell integrity, GATA2 chronic, genetically inherited skin condition
gene (OMIM*137295), have been recently impli- characterized by increased susceptibility to cuta-
cated as the cause of the MonoMAC syndrome, neous infection with certain HPV genotypes,
suggesting dominant interference of gene func- referred to as EV-HPVs. [222, 223] EV begins
tion by either dominant negative effects or haplo- during infancy or early childhood, and the more
insufciency [209, 213, 214]. Mutations in the benign lesions manifest as at, wart-like,
same gene may result in two more different phe- hypopigmented, or hyperpigmented papules or
notypes: familial MDS/AML without other pityriasis versicolor-like plaques, whereas lesions
hematopoietic defects [215, 216], and Emberger with greater potential for malignant transforma-
syndrome, which is characterized by congenital tion present more variably as verrucous and seb-
deafness and primary lymphedema of the lower orrheic keratosis-like lesions, occurring mainly
limb with a predisposition to MDS or AML on sun-exposed areas [222224]. Approximately
[217]. The GATA family of transcription factors, 3060 % of individuals eventually develop skin
which contain zinc ngers in their DNA-binding malignancies, eventually in the fourth to fth
domain, have emerged as candidate regulators of decades, with Bowen carcinoma in situ being
gene expression in hematopoietic cells. GATA2 the most frequent tumor, followed by invasive
functions in the regulation of hematopoiesis and, SCC and, less frequently, basal-cell carcinoma
in particular, is required for maintenance and sur- [224227].
vival of the hematopoietic stem-cell pool [218, EV is inherited primarily in an autosomal
219]. GATA2 also functions in the formation of recessive pattern [228], although both X-linked
early blood and lymphatic vessels [220, 221]. recessive and autosomal dominant modes of
The role of GATA2 mutation in disease manifes- inheritance have been reported [229, 230].
tation is incompletely understood but likely com- Genome-wide linkage studies have identied two
plex and thought to be linked to the generation or EV susceptibility loci EV1 and EV2, on chromo-
maintenance of progenitors required for the somes 17 and 2, respectively [231]. Mutations
affected cell subsets [213]. in the EVER1 (OMIM*605828) and EVER2
Immunological characterization of patients (OMIM*605829) genes, which are part of the
with the MonoMAC syndrome revealed pro- EV1 locus, have been identied in approximately
foundly decreased or absent monocytes, NK 75 % of patients with EV [222].
cells, and B cells as well as a severe decrease in The EVER proteins, localized in the endo-
circulating and tissue dendritic cells (DCs). In plasmic reticulum of human keratinocytes
most cases, GATA2 deciency is accompanied by [232], interact with ZnT-1 [233], a zinc trans-
a severe reduction in peripheral blood NK cells, porter regulating cellular zinc homeostasis.
specically the CD56bright subset, with marked Loss of EVER zinc homeostasis enhances the
functional impairment [209], which predispose to expression of viral genes, specically the pro-
signicant HPV and other viral infections, as well oncogenic E6 and E7, contributing to HPV-
as HPV-associated SCC. Bone marrow failure mediated carcinogenesis. Besides keratinocytes,
resulting from loss of stem cells may underlie the EVER proteins are expressed in T and B lym-
multilineage cytopenias described in most phocytes, NK cells, endothelial cells, myeloid
patients; however, the underlying mechanisms for cells, and DCs. Zinc has been shown to con-
cytogenetic abnormalities or the leukemic trans- tribute to TCR signaling by increasing ZAP70
formation need to be further claried. phosphorylation [234]. Mutated, dysfunctional
EVER genes would disrupt zinc homeostasis chemokine receptor CXCR4 (OMIM*162643)
and consequently produce a defect in cell-medi- [247], a member of the G-protein-coupled recep-
ated immunity, which could compromise viral tor superfamily specic for the CXC chemokine
clearance and lead to malignant transformation stromal cell-derived factor 1 (SDF-1) [248], also
[222, 233]. Although the immunological pheno- known as CXCL12. All CXCR4 mutations
type of EV might be normal, it can also mani- reported to date disrupt receptor downregulation
fest with decreased total T-lymphocyte counts; leading to enhanced and prolonged chemotactic
reduced cell-mediated immunity, as measured responsiveness to SDF-1 [249, 250].
by reduced responsiveness to mitogens and Immunological and hematological abnormali-
Ags as well as cutaneous anergy to recall Ags ties in WHIM syndrome include peripheral
[235, 236]; and defective cell-mediated immu- neutropenia, B lymphopenia with a particular
nity toward EV-HPVs or infected keratinocytes reduction in the number of switched memory B
[237, 238]. cells (CD27+ IgD), T lymphopenia with
decreased number of nave T cells, and a relative
expansion of memory T cells with a restricted
18.5.2 Warts, Hypogammaglobulinemia, repertoire, deciency of plasmacytoid DCS, and
Infections, and Myelokathexis hypogammaglobulinemia [251254]. The mech-
Syndrome anisms by which dysregulated CXCR4 signaling
affects leukocyte homeostasis and predisposes
Warts, hypogammaglobulinemia, infections, and to a selective susceptibility to HPV infection and
myelokathexis (WHIM) syndrome is a rare, carcinogenesis are still unknown. It remains pos-
dominantly inherited PID characterized by warts, sible that defective trafcking of effector cells (T
hypogammaglobulinemia, infections, and myelo- cells and NK cells) and Ag-presenting cells might
kathexis, which refers to neutropenia resulting contribute to defective cutaneous immunity,
from abnormal retention of mature neutrophils explaining the abnormal susceptibility to viruses
and increased neutrophils apoptosis in the bone affecting the skin [99].
marrow [239241]. The incidence of WHIM
syndrome has been estimated to be 0.23 cases per
million births [242]. The clinical onset usually 18.6 Diseases of Immune
occurs during infancy or early childhood with Dysregulation
recurrent gastrointestinal, respiratory, and cuta-
neous bacterial infections and increased suscep- 18.6.1 X-Linked Lymphoproliferative
tibility to HPV infection, causing numerous, Disease
recalcitrant skin and genital warts [240, 241].
Genital warts (condylomata acuminata) may X-linked lymphoproliferative disease (XLP),
undergo dysplastic changes conferring to an formerly known as Duncan disease, is a rare and
increased risk of malignancy [239241]. Contrary often fatal inherited immunodeciency disorder,
to the long-held belief, HPV is not the only initially described by Purtilo et al. [255], with an
unique viral susceptibility in WHIM syndrome; estimated incidence of one to three per million
more recently, EBV-associated lymphoproliferative male births [256]. It is characterized by severe
disease [243, 244] as well as herpes zoster [245], immune dysregulation in males with a variable
herpes simplex virus [245, 246], and molluscum clinical presentation, often following EBV
contagiosum [243] infections have been infection, manifesting as fulminant infectious
reported, indicating a generalized susceptibility mononucleosis and/or acquired hemophagocytic
to Herpesviridae viruses. lymphohistiocytosis (HLH), dysgammaglobu-
WHIM syndrome is primarily caused by gain- linemia, and malignant lymphoma [257260].
of-function mutations in the gene encoding the Other, albeit less common, clinical features of
XLP include aplastic anemia, lymphocytic 18.7 Syndromes

vasculitis, pulmonary lymphoid granulomatosis, with Autoimmunity
arthritis, colitis, and psoriasis [260262].
Most cases of XLP are caused by germ- 18.7.1 Autoimmune
line mutations in the Src homology 2 domain- Lymphoproliferative
containing gene 1A (SH2D1A; OMIM*300490) Syndrome
encoding the 128 amino acid signaling lympho-
cytic activation molecule (SLAM)-associated Autoimmune lymphoproliferative syndrome
protein (SAP) [263265]. A second XLP-like dis- (ALPS) is a rare disease characterized by defec-
order caused by mutations in the X-linked inhibi- tive Fas-mediated apoptosis [276]. The incidence
tor of apoptosis protein (XIAP; OMIM*300079) and prevalence of ALPS are unknown. Estimated
was described in 2006 [266]. Although XIAP cases of ALPS exceed 500 cases worldwide;
deciency is predominantly associated with however, it is not reliably conrmed. Classically,
recurrent EBV-associated HLH, no lymphoma patients present with autoimmunity, lymphade-
occurrence has been reported in affected patients nopathy, and/or splenomegaly along with elevation
till now [141, 260, 266]. In humans, SAP is in TCR /+ B220+CD4+CD8+ double-negative T
expressed predominantly in NK, NKT, and T (DNT) cells (a constant feature of the disease
cells [267269]. It has been shown to serve as with undetermined origin) and defective in vitro
an adaptor molecule downstream of several Fas-mediated lymphocyte apoptosis [277].
SLAM immunomodulatory receptors family Furthermore, certain biomarkers may be useful to
[270]. The SLAMSAP association potentiates aid in diagnosis [278]. These include elevated
the development of NKT cells, TB-cell conju- circulating levels of sFASL, IL-10, vitamin B12,
gation required for the development of germinal and IL-18. Patients who do not fulll the ALPS
centers and immunoglobulin production, and diagnostic criteria are now classied as having
EBV-directed cytotoxicity by T and NK cells. In ALPS-related conditions caused by germ-line
addition, it is required for normal T-cell homeo- mutations in CASP8, NRAS, and SH2D1A [277].
stasis mediated by reactivation-induced cell XLP, a genetic immunodeciency caused by
death (RICD) [271, 272]. mutations or deletions in the SH2D1A gene, can
SAP-decient patients are at increased risk of be included in the spectrum of ALPS-like disor-
lymphoma development, as well as other lym- ders, since these patients frequently display
phoproliferative diseases. Approximately 30 % defective apoptosis in response to TCR restimu-
of patients develop lymphoma at a mean age of lation [279, 280]. Mutations in the ALPS and
15 years at diagnosis [260, 273]. Expectedly, the ALPS-related genes often manifest with variable
majority are of B-cell origin, arising in extrano- penetrance [281]. Thus, patients with ALPS often
dal sites, most commonly localized in the ileoce- have family members with the same genetic
cal region, with Burkitts lymphoma comprising mutation with no clinical phenotype or very mild
approximately 5060 % of total lymphomas symptoms. The penetrance of the mutation is not
[260, 274, 275]. Notably, not all cases of lympho- related to the type of mutation but probably
mas arise due to malignant transformation of depends on unknown genetic and environmental
EBV-infected B cells, as up to one-third of modiers. Hence, the clinical signicance of
patients with lymphoma are EBV seronegative isolated detection of a heterozygous Fas mutation
[260, 273, 275], indicating that the genetic defect in a healthy relative of a patient with ALPS is
per se can result in lymphoma. It is likely that not yet clear.
defective antitumor immunosurveillance due to Autoimmunity, affecting over 70 % of patients,
poor CD8+ T- and NK-cell cytotoxic responses is mainly directed against blood cells [282].
and lack of NKT cells contributes to lymphoma- Other autoimmune manifestations are rare and
genesis [99]. include autoimmune nephritis, hepatitis, arthritis,
uveitis, iridocyclitis, and vasculitis [283]. (3) Addisons disease. Additional autoimmune
Autoantibodies are more common than obvious components may appear throughout life and
clinical disease and present in up to 92 % of include gonadal failure, type 1 diabetes, hypothy-
patients [284]. Elevation of TCR /+ DNT cells roidism, pernicious anemia, hepatitis, alopecia,
in the peripheral blood and lymphoid tissues is a vitiligo, and/or ectodermal dystrophies.
hallmark of ALPS, but it is not pathognomonic as The disease is characterized by loss of toler-
patients with other autoimmune diseases such as ance against self-antigens [294, 295], which is
SLE and ITP may also have mild elevations in caused by mutations in the autoimmune regulator
these cells [285]. (AIRE) gene (OMIM*607358) [296, 297].
ALPS is caused by germ-line or somatic Although the endocrine features are clearly
mutations in FAS gene (TNFRSF6, or CD95; autoimmune, the underlying immunodeciency
OMIM*134637), or germ-line mutations in the predisposing to CMC has been a long-standing
FAS ligand (FASL) (TNFSF6, or CD95L; puzzle. Recently, autoantibodies against the
OMIM*134638) or CASP10 (OMIM*601762) Th17-related cytokines IL-22, IL-17A, and
genes. IL-17F, which are implicated in protection against
Apoptosis is critical in tumor scrutiny as FAS, fungi at epithelial surfaces, were discovered in
a putative tumor suppressor, is silenced in many the sera of APS-1 patients [298, 299], suggesting
tumors [286288]. As anticipated, patients with that the underlying immunodeciency in patients
ALPS who harbor germ-line mutations in the with APECED has an autoimmune basis.
ALPS-related genes have an increased risk of Several cases of oral and esophageal SCC
developing malignancy [289], with the risk of have been reported in APECED patients with
NHL and HL, respectively, being 14 and 51 times CMC [300303]. In a cohort of 92 Finnish
greater than expected [290]. An increased risk of patients, six had developed oral or esophageal
cancer has also been observed in unaffected fam- SCC by the mean age of 37, representing 10 % of
ily members who may inherit the same mutation patients older than 25 years [300]. The partial
but fail to develop an overt ALPS phenotype T-cell defect of APECED seems to favor the
[290]. Sporadic NHL harbors somatic mutations growth of Candida albicans and predispose to
of the FAS gene in 11 % [291] of cases and in chronic mucositis and the development of
the CASP10 gene in 14.5 % of cases [292]. SCC. Besides chronic inammation and increased
Furthermore, in HL, somatic FAS gene mutations cell turnover, Candida albicans biotypes are
are found in ReedSternberg cells in 1020 % of capable of producing the carcinogenic nitrosa-
cases [286, 293]. mine N-nitrosobenzylmethylamine [304, 305],
and can also act to promote oral carcinogenesis in
rats when a known carcinogen, 4-nitroquinoline-
18.7.2 Autoimmune 1-oxide, is repeatedly applied [306].
Polyendocrinopathy
with Candidiasis
and Ectodermal Dystrophy 18.8 Other Well-Dened
Immunodeciencies
Autoimmune polyendocrinopathy with candidi-
asis and ectodermal dystrophy (APECED), also 18.8.1 DNA Repair Defects
called autoimmune polyendocrine syndrome
type I (APS-1), is a rare autosomal recessive B- and T-lymphocyte development depends
disease, most commonly seen in Iranian Jews, largely on multiplex genetic rearrangements,
Sardinians, and Finns. The diagnosis of APECED i.e., V(D)J recombination, class switch recombi-
is reached if patients manifest at least two of the nation, and somatic hypermutation, carried out
following conditions: (1) chronic mucocutaneous by multiple DNA repair and damage response
candidiasis (CMC), (2) hypoparathyroidism, or protein complexes [307]. Variations in the DNA
repair genes might compromise the delicate bal- the usual local or systemic features of inamma-
ance between the generation of genetic variation tion, forming so-called cold abscesses [328].
and replication delity of DNA [308, 309]. PIDs Recurrent sinopulmonary infections, resulting in
associated with defects in DNA repair, collec- bronchiectasis and pneumatocele formation fre-
tively termed genomic instability syndromes, are quently superimposed with bacterial and fungal
generally associated with cellular radiosensitiv- infections, are the major causes of morbidity and
ity, developmental defects, and predisposition to mortality in patients with HIES [329]. Despite
cancer [309311]. Syndromes known to be asso- having extremely high serum IgE levels and
ciated with malignancies, including ataxiatel- eosinophilia, patients with HIES are usually free
angiectasia, Nijmegen syndrome, Bloom from other allergic manifestations, recognized as
syndrome, DNA ligase IV deciency, Artemis a marked difference from DOCK8 deciency
deciency, cartilage hair hypoplasia, and PMS2 [325, 327]. In patients with HIES, serum IgG,
deciency, are summarized in Table 18.1. IgM, and IgA levels are usually normal; however,
Although these defects are associated with an most have impaired antigen-specic Ab response
increased risk of lymphoid malignancies, mainly to immunization [330]. Diminished circulating
NHL, nonlymphoid tumors affecting the brain, memory B cells and defects in the differentiation
skin, breast, and gastrointestinal tract have also of Th17 cells have also been demonstrated
been reported [311, 312, 314, 316, 319321]. [330332]. The multisystem nature of the disease
This is partly due to the fact that diverse DNA extends beyond the immune system and
repair processes are not specic to Ag receptor accounts for the characteristics craniofacial,
diversication. DNA double-strand breaks, aris- musculoskeletal, dental, and vascular abnormalities
ing from multiple sources, including exposure to [333336].
ionizing radiation, can potentially lead to repli- Dominant negative mutations in STAT3
cation errors, loss or rearrangements of genomic (OMIM*102582) have been identied as the
material, and eventually cell death or carcino- major molecular etiology of autosomal dominant
genesis. The DNA damage response pathway, and sporadic cases of HIES [337, 338]. STAT3,
responsible for sensing and repairing the dam- one of the seven STAT proteins in the human, is a
aged DNA, comprises the most powerful tumor transcription factor and plays a critical role in
surveillance mechanism [320]. The observation responses to many cytokines and growth factors
of an increased risk of cancer development in through the shared signal-transducing molecule
heterozygote carriers provides additional insight gp130 [326, 327]. It is crucial for cell prolifera-
into their tumorigenic potential [321324]. tion, survival, migration, apoptosis, and inam-
Additionally, defects in immunosurveillance mation in various tissues [339], probably
mechanisms per se, similar to certain PIDs not explaining the diverse clinical ndings in patients
associated with DNA repair defects, contribute with HIES.
to cancer development. STAT3 deciency is associated with an
increased risk of LPDs, most notably HL and
NHL (relative risk: 259), with the majority of
18.8.2 Signal Transducer B-cell origin and aggressive histology [340342].
and Activator of Transcription Other cancers described in patients with HIES
3 Deciency include leukemia and cancers of the vulva, liver,
and lung [343]. The underlying mechanisms,
Hyper-IgE syndrome (HIES) is a complex PID however, remain unclear. The higher risk of
characterized by recurrent staphylococcal infec- tumor formation has been attributed to defective
tions beginning early in infancy, predominantly immunosurveillance and chronic B-cell stimula-
involving the skin and lungs, chronic eczema, tion, resulting in an increased turnover of B cells
and markedly high serum IgE concentrations and accumulating genetic aberrations, giving rise
[325327]. Skin infections due to S. aureus lack to malignant B-cell clones [99].
358
Table 18.1 Clinical and Immunological Features of DNA Repair Defects Associated with Cancers
Gene Disease Inheritance Clinical features Pathogenesis Immune defects Associated cancers
ATM (OMIM*607585) Ataxia-telangiectasia AR Cerebellar ataxia, Disorder of cell cycle Often decreased Lymphomas,
oculocutaneous checkpoint and IgA, IgE, and IgG lymphoid leukemias
telangiectasia, DSB repair, role in subclasses, increased (mainly T cells),
chromosomal instability, V(D)J, CSR IgM, antibodies epithelial tumors,
radiosensitivity, thymic variably decreased, gastric carcinoma
aplasia, recurrent progressive T-cell [311313]
sinopulmonary infections, lymphopenia, normal
cancer predisposition B-cell count
(up 40 %)
NBN (OMIM*602667) Nijmegen breakage AR Severe microcephaly, Disorder of cell cycle Often decreased Hodgkin and
syndrome bird-like face, mental checkpoint and DSB IgA, IgE, and IgG non-Hodgkin
and growth retardation, repair, role in V(D)J, subclasses, increased lymphomas,
chromosomal instability, CSR, SHM IgM, antibodies leukemias (mainly
radiosensitivity, recurrent variably decreased, B cells), brain
sinopulmonary infections, decreased B- and tumors [314]
strong predisposition to T-cell counts
lymphoid malignancy
BLM (OMIM*210900) Bloom syndrome AR Short stature, bird-like Role as a RecQ-like Low IgM and IgA, Leukemias,
face, sun-sensitive helicase normal B- and lymphomas,
erythema, erythema, T-cell counts carcinomas [315]
marrow failure,
chromosomal instability,
cancer predisposition
LIG4 (OMIM*601837) DNA ligase IV AR Microcephaly, facial Impaired NHEJ, Decreased serum Igs, EBV-positive B-cell
deciency dysmorphisms, radiation role in V(D)J, CSR decreased B- and lymphomas, T-cell
sensitivity, may present T-cell counts ALL [316, 317]
with RS-SCID, Omenn
syndrome, or with a
delayed clinical onset
M. Hedayat et al.
18
DCLRE1C (OMIM*602450) Artemis deciency AR Radiation sensitivity, may Role in V(D)J, CSR Decreased serum Igs, EBV-positive B-cell
present with RS-SCID or markedly decreased lymphomas [318]
Omenn syndrome B- and T-cell counts
RMRP (OMIM*250250) Cartilage hair AR Short-limbed dwarsm Role in ribosomal Normal or reduced Non-Hodgkin
hypoplasia with metaphyseal RNA processing, serum Igs, variably lymphomas,
dysostosis, sparse hair, mitochondrial DNA decreased antibodies, basal-cell carcinoma
bone marrow failure, replication, and cell normal B-cell count, [319]
autoimmunity, cycle control decreased or normal
predisposition to cancers, T-cell count,
impaired spermatogenesis, impaired lymphocyte
neuronal dysplasia proliferation
of the intestine
PMS2 (OMIM*600259) PMS2 deciency AR Recurrent infections, Defective CSR- Low IgG and IgA, Leukemias,
(class switch caf au lait spots, cancer induced DSBs in Ig elevated IgM, lymphomas,
recombination predisposition switch regions abnormal antibody colorectal
deciency due to responses, normal carcinoma, brain
Primary Immunodeciencies and Cancers
impaired mismatch B-cell count, tumors [320]

repair) decreased, switched,
and non-switched
B-cell counts
359
18.8.3 WiskottAldrich Syndrome 154 patients with WAS, 21 (13 %) developed

malignancies, mostly of lymphoreticular origin,
WiskottAldrich syndrome (WAS) is a rare with the average age at onset of 9.5 years [345].
X-linked immunodeciency with highly variable Nonlymphoid malignancies, including glioma,
manifestations characterized by thrombocytope- acoustic neuroma, testicular carcinoma, and
nia with small platelets, eczema, and humoral Kaposi sarcoma, have infrequently been reported
and cellular immunodeciency with increased [345, 355]. The development of hematological
susceptibility to pyogenic and opportunistic malignancies in WAS patients is at least partly
infections. Patients with WAS may also manifest due to NK cell and cytotoxic T-lymphocyte
with an increased incidence of autoimmunity and dysfunction [356358], absent of invariant NKT
malignancies [344349]. cells [359, 360], and chronic stimulation of auto-
The disease is caused by mutations in the WAS reactive cells and ineffective clearance of virally
gene (OMIM*300392), which is expressed infected cells [361, 362]. It has been reported
exclusively in hematopoietic cells. Around 300 that despite normal expression levels of lytic
unique mutations spanning the WAS gene have molecules, the cytotoxic CD8+ T cells from WAS
been described. The effect of a given mutation on patients failed to effectively kill B-cell lymphoma
WASp expression correlates with the disease target cells due to inefcient polarization of cyto-
severity: mutations that cause decreased WASp toxic granules toward the target tumor cells
levels result in the mild variant X-linked throm- [356]. Recently, activating mutations in WASp
bocytopenia (XLT), characterized mainly by (which give rise to XLN) have been found to lead
thrombocytopenia [350, 351], whereas mutations to genetic instability through dysregulation of
that abolish WASp expression or result in the actin polymerization. Enhanced and delocalized
expression of a truncated protein are associated actin polymerization throughout the cell was
with classic WAS. In addition, a third disorder shown to inhibit myelopoiesis through defective
termed X-linked neutropenia (XLN), character- mitosis and cytokinesis, with micronuclei forma-
ized by neutropenia and variable myelodysplasia, tion indicative of genomic instability [363].
has been attributed to activating mutations in the Despite lack of direct evidence, genomic instability
GTPase-binding domain of WASp [178, 352, 353]. might contribute to the development of malig-
The WAS protein (WASp) is a multifaceted nancies in WAS patients [347].
protein which exists in complex with several Early HSCT is the treatment of choice for
partners involved in relaying signals from cell patients with classic WAS, preferably from a
surface receptors to the actin cytoskeleton; lack matched related donor [364]. Furthermore,
of WASp results in cytoskeletal defects that immune reconstitution in WAS patients following
compromise multiple aspects of normal cellular HSCT leads to a decrease in cancer risk [364].
activity including proliferation, phagocytosis, Gene therapy is an alternative to HSCT in the
immune synapse formation, adhesion, and directed treatment of WAS [365]; however, the long-term
migration [347]. It is therefore not surprising that outcome needs to be further monitored. This
lack of WASp results in a wide range of defects in could be explained by the fact that the viruses
cellular function involving all hematopoietic cell used for therapy integrate in the host genome,
lineages [347]. with preferential insertion at transcription start
Malignancies are relatively common in older sites, promoter and enhancer regions of active genes,
patients (adolescent and young adults), especially and at conserved noncoding DNA, resulting in a
in those with autoimmune manifestations, and high rate of transformations and the development
are frequently associated with a poor prognosis of secondary malignancies [366, 367]. Acute
[345, 348, 354]. The most frequent malignancy T-cell leukemia due to vector insertion in the
reported is B-cell lymphoma, which often occurs vicinity of the T-cell oncogene LMO2 has been
in EBV-positive patients [345, 349]. In a report of reported in one patient [368, 369].
18.8.4 Chromosome 22q11.2 leukemia, SCC, astrocytoma, neuroblastoma,

Deletion Syndrome hepatoblastoma, renal cell carcinoma, and
rhabdoid tumors [381, 386390].
Chromosome 22q11.2 deletion syndrome is
relatively common (estimated in 1 in 4,000
births) [370], and about 6 % of newly diagnosed 18.9 Concluding Remarks
cases are familial [371]. The presenting symp-
toms of chromosome 22q11.2 deletion syndrome The expanded life expectancy of patients with
vary depending on the patients age. While devel- PIDs has increased the overall risk for developing
opmental delay and speech issues are the usual cancers. However, the management of cancers in
presenting symptoms in older children and adults, such patients remains challenging, in part due to
cardiac anomalies, hypocalcemia, and infection the rarity, the increased risk for infection and
are the major manifestations in infants. Cardiac other complications, as well as the rather slow
defects are seen in approximately 80 % of pace of scientic advancement related to these
patients; on the other hand, tetralogy of Fallot conditions. Continued progress in understanding
and interrupted aortic arch type B have a strong the interplay between chronic Ag stimulation,
positive predictive value for chromosome oncogenic viruses, genetic factors, and impaired
22q11.2 deletion syndrome [372, 373]. Palatal host immunity during tumor formation in various
dysfunction, feeding problems, facial dysmor- PIDs will facilitate renement of current and
phism, renal anomalies, and gastrointestinal emerging therapeutic approaches.
manifestations also are seen in most of these
patients [374]. Patients are also at an increased
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Immunosenescence, Oxidative
Stress, and Cancers 19
Tamas Fulop, Graham Pawelec, Gilles Dupuis,
Rami Kotb, Bertrand Friguet, and Anis Larbi
Contents
19.1 Introduction
19.1 Introduction ................................................ 377
19.2 Immune System and Cancer ..................... 378 The most important risk factor for cancer devel-
19.2.1 Immunosenescence or Immune Aging ........ 378 opment is age [1]. With increasing age, numerous
19.2.2 Innate Immune System ................................ 379
19.2.3 Adaptive Immune System ............................ 383 alterations at multiple levels including molecular,
19.2.4 Interaction Between Innate and Adaptive cellular, organ, and systemic levels are observed.
Immune Responses: Effect of Aging ........... 384 On the one hand, cellular senescence seems to be
19.3 Inammation Aging an anticancer mechanism related to aging due to
and Oxidative Stress .................................. 385 the combined effects of proliferation and envi-
19.4 Immunosenescence and Cancer................ 387 ronmental factors such as oxidative stress or
DNA damage and telomere shortening [2]; on the
19.5 Modulation ................................................. 388
other hand, there are various interactions among
19.6 Concluding Remarks ................................. 388 physiological systems which can favor the devel-
References ................................................................. 389 opment and progression of cancers with aging
where cellular senescence is also a contributor,
together with hormonal changes [2]. One of the
B. Friguet, PhD
T. Fulop, MD, PhD (*) Biological Adaptation and Ageing
Geriatrics Division, Department of Medicine, UMR UPMC-CNRS 8256 ERL INSERM U1164,
Research Center on Aging, University of Sherbrooke, Unit de vieillissement stress, inammation UR 4,
3001, 12th Avenue North, Sherbrooke, QC, Canada Universite Pierre et Marie Curie-Paris 6, Jussieu,
e-mail: tamas.fulop@usherbrooke.ca 75005 Paris, France
e-mail: bertrand.friguet@upmc.fr
G. Pawelec, MA, PhD
Tbingen Ageing and Tumour Immunology Group, A. Larbi, PhD
Second Department of Internal Medicine, Center for Singapore Immunology Network (SIgN), Biopolis,
Medical Research, University of Tuebingen, Agency for Science Technology and Research
Tuebingen, Germany (A*STAR), Singapore, Singapore
e-mail: graham.pawelec@uni-tuebingen.de e-mail: anis_larbi@immunol.a-star.edu.sg
R. Kotb, MD, FRCPC G. Dupuis, PhD
Department of Medicine, BCCA Victoria, Biochemistry Department and Graduate Program
British Columbia Cancer Center in Immunology, Faculty of Medicine and
and The University of British Columbia, Health Sciences, University of Sherbrooke, 3001,
2410 Lee Ave, V8R 6V5 Victoria, BC, Canada 12th Avenue North, Sherbrooke, QC, J1H 5N4, Canada
e-mail: rami.kotb@bccancer.bc.ca e-mail: gilles.dupuis@usherbrooke.ca

378 T. Fulop et al.
physiological systems involved is the immune inhibited or exhausted, and resistant cancer cells
system. After several years of debate, it is now will survive and proliferate as explained by the
clear that the immune system plays a major role decit of the built-in tumor suppressor mecha-
in the control of the emergence of cancerous cells nisms such as cell senescence, DNA damage-
[3, 4]. With aging there are changes in the induced apoptosis, etc. Eventually, the tumor
immune system collectively called immunose- escapes from immune surveillance and becomes
nescence which might adversely affect the anti- clinically apparent. At this stage the tumor is
cancer activity [5, 6]. One of the most important orchestrating the behavior of the immune system
characteristics of immunosenescence is the by actively suppressing the immune response
occurrence of inammaging [79], indicating through the production of various inhibitory sub-
that aging is accompanied by a state of low-grade stances, such as NO, IDO, PGE2, and via other
inammation which can also contribute to the pathways. At the same time, immune suppressor
increase in cancer incidence, and, more effec- cells including Tregs and MDSCs may become
tively, combat the emergence of tumor cells. dominant, hence inhibiting the tumor-eliminating
Experimental data implicating immune aging at activity of the immune system. Thus, to eliminate
various stages of cancer development are accu- the nascent tumor cells, organisms need a com-
mulating, but there remains much to discover. pletely and fully functioning immune system. As
Here, we describe changes in innate and adaptive we age there are several physiological alterations
immune systems with age in relation to age- in the immune system ultimately contributing to
related increased cancer development. the appearance of cancers with higher incidence
in the elderly.
19.2 Immune System and Cancer

19.2.1 Immunosenescence or
It took some time to understand how the immune Immune Aging
system may interact with the cancer at various
stages of its development [1012]. Currently, this It is currently well established that the immune
synthesis of ideas developed over the decades response is profoundly altered with aging [13].
following the original suggestion of immunosur- Most changes concern the adaptive immune sys-
veillance against tumors, known as immunoed- tem, but it is now accepted that the innate immune
iting that describes all facets of the interaction system is also affected [1417]. Collectively, it is
between the immune system and cancer. very difcult to establish whether the changes are
Immunity plays an important role in the host only detrimental or are at least partly an adaptation
defense against tumor development. Despite the to sustain decreasing immune responses by chang-
fact that cancer originates from self cells and as ing the threshold for immune activation. The pres-
such may be only weakly antigenic. This phase ence of low-grade inammation can be part of this
of the interaction is called the elimination stage adaptation process. This phenomenon can over-
or true immunosurveillance. At this level the come the decreased immune reserve with aging.
immune system involves many different immune Nevertheless, as the immune response is impli-
cells and is efcient at eliminating cancer cells. cated in cancer immunosurveillance, it can be
However, this action can result in the emergence hypothesized that even if the changes in the aging
of tumor variants and the establishment of a tem- immune system may be adaptive in respect to the
porary equilibrium between the transformed cells pathogenic environment, they can still contribute
and the efcient immune defense. At this stage, to the increased incidence of cancers [1821]. The
the cancer remains clinically insignicant. As the age-related changes in the innate and adaptive
equilibrium shifts and the continuously growing immune system in view of their implication in
genetically unstable malignant cells generate putative cancer development and progression will
variants, the immune response can become be discussed here.
19 Immunosenescence, Oxidative Stress, and Cancers 379
19.2.2 Innate Immune System with aging [3032] suggesting altered foreign anti-
gen (Ag) processing. Recently, it has been shown
The innate immune system plays an essential role that the inammasome is a complex of molecules
in cancer immunosurveillance by directly elimi- activated by specic PRRs (NLRs and AIM2)
nating the tumor cells and maintaining them in a responding specically to challenge via the activa-
quiescent state but may also favor the develop- tion of inammatory caspases such as caspase-1
ment and progression of cancers in some ways. It and caspase-5. This ultimately results in the pro-
should be stressed that interactions between the duction of a wide range of cytokines, particularly
innate and adaptive immune system are recog- IL-1 [33], playing a role in inammation. There
nized as essential for an efcient adaptive immune are currently no data on how these inammasomes
response. These functions are mediated by vari- are affected by aging. After the alterations observed
ous innate cells including neutrophils, monocyte/ in neutrophil functions, it can only be suggested
macrophages, NK cells, and ILL. It is now recog- that their assembly and function may be altered.
nized that most phenotypes and functions of the The causes of these dysregulated effector
cells of the innate immune system are altered with functions remain unknown, but changes in the
aging, as briey summarized in the following. inammatory environment and in the signaling
pathways may contribute. Neutrophils can also
19.2.2.1 Neutrophils be stimulated via their pattern recognition receptors
Neutrophils are the most abundant innate immune by Ags that may be present in higher amounts in
cells. They are the rst to arrive at the site of any the periphery of aged individuals, such as DNA
aggression but are markedly altered with aging [17, degradation products, altered proteins, latent/
22]. It is interesting to note that not all their func- chronic viral antigen, and/or tumor-derived Ags.
tions are changed with aging. Thus, the number of Recently, one of the most important discoveries
neutrophils and their capacity to adhere at inam- was of PRRs on the surface of many immune
matory sites is not altered with aging [23, 24]. It is cells including neutrophils recognizing pathogen-
also of note that while most of the effector func- associated molecular patterns (PAMPs) [34]. The
tions are increased with aging at the basal level, ever-growing family of the PRRs now includes
they cannot be further modulated [2528]. The three main types: the TLRs, the retinoic
most important functions increased at quiescent acid-inducible gene 1 protein (RIG-1)-like heli-
state are the production of free radicals and the pro- cases (RLRs), and the nucleotide-binding domain
duction of proteases [25, 26] which can be impor- and leucine-rich-repeat-containing proteins
tant for tumor ghting/development. Nonetheless, (NLRs) [35]. It is now recognized that they play
this can also contribute to the low-grade inamma- an essential role in many cell functions, including
tion observed with aging, which can be detrimen- neutrophil biology, allowing immune cells to dis-
tal. In contrast, an acute stimulation of neutrophils criminate between self and nonself and acting as
in the elderly reveals that they are unable to per- danger-sensing receptors to alert the organism to
form correctly by increasing chemotaxis, phagocy- the presence of microorganisms, transformed
tosis, and intra- and extracellular killing and to stay cells, or damaged cells.
viable and active for a longer functional period There are currently 13 TLRs described with
[27]. These functions are mediated through the different recognition specicities and signaling
activation of specic receptors such as pattern rec- pathways leading to well-characterized cellular
ognition receptors (PRRs), Fc, and complement responses [34]. Bacterial products are recognized
receptors. Another important function recently rec- by TLR2 and TLR4, while TLR3 and TLR7 rec-
ognized for the elimination of foreign invaders is ognize intracellular pathogens. Signaling is medi-
autophagy. Engagement of different Toll-like ated either by the MyD88 pathway [36] or by the
receptors (TLRs) such as TLR4 and TLR7 has TRIF pathway [37, 38]. Activation of these TLRs
been implicated in the activation of macroautoph- results in the activation of NF-B, a transcription
agy [29], which has been shown to be defective factor furthering strong cytokine production [39].
380 T. Fulop et al.
Neutrophils from aged individuals display altera- age-related dysfunction [4446]. These altera-
tions in the signaling of these TLRs leading to tions include a decrease of cell surface TLR
their altered functionality [14, 27]. While the num- expression (TLR1 and TLR4), although this nd-
ber of these receptors is not signicantly changed ing is controversial [31, 47, 48]. Other receptors
with age, there is a signicant alteration in the also show an altered expression, such as the
trafcking of signaling molecules in and out of expression of the important T-cell CD80/CD86
lipid rafts. There is a need for further studies in co-stimulatory receptors which is decreased on
order to truly appreciate the role of TLR in the monocytes upon TLR stimulation [49]. In vitro
altered functions of neutrophils with age [27]. studies in humans demonstrated a higher proin-
Taken together, all available experimental evi- ammatory cytokine prole, especially for IL-6
dence indicate that neutrophils participate in and IL-8 production by resting monocytes [9],
inammaging but can no longer effectively coun- despite the nding that cytokine production fol-
teract pathological challenges and as such may lowing stimulation with LPS is reduced.
contribute to the inammatory process becoming Consistent with this, another recent study found
more chronic. Neutrophils also interact with other that the four monocyte subsets had lower IL-6
cells of the immune system, in addition to the production upon TLR1/TLR2 stimulation, con-
adaptive arm such as B cells for antibody produc- rming earlier studies on TLR stimulation [50,
tion and T cells for efcient effector functions [40, 51], which indicates that monocytes are not a
41]. They also participate in the recruitment of homogeneous population and react differently
monocyte/macrophages to the challenge site which depending on the nature of the stimuli.
take over their functions for a longer time period. Many years ago, it was shown that several
surface receptors such as Fc and FMLP had
19.2.2.2 Monocyte/Macrophages altered signal transduction upon appropriate
Monocyte/macrophages have been relatively stimulation, resulting in altered function [25, 26].
poorly studied in human aging. However, cur- Recent data further suggest that in addition to the
rently available data indicate that there are pheno- decrease in some TLR expression, the TLR sig-
typic changes associated with altered effector naling pathways show age-related alterations
functions in older individuals. Recent studies [27] linked to altered chemotaxis as evident by
characterizing monocytes showed the existence of the reduced number of inltrating macrophages
two distinct subpopulations: CD14++(high) CD16- in wounds of elderly humans. Alteration in the
and CD14+(low) CD16+ [42]. These subpopula- MAPK signaling pathways including p38 MAPK
tions are very distinct in their surface protein and ERK1/2 MAPKs has been reported in human
expression and their functions. The rst monocytes with aging.
CD14++(high) CD16 subpopulation expresses Macrophages from elderly people produce
CD62L, CD64, and CCR2 with low levels of more prostaglandin E2, which suppresses T-cell
CXCR1. The second CD14+(low) CD16+ lacks all activation via decreased IL-12 production [52].
these surface markers but expresses high levels of Furthermore, it was demonstrated that phagocy-
CX3CR1. These latter cells are considered to be tosis, free radical production, and chemotaxis
mainly proinammatory as they produce high lev- were reduced in monocytes/macrophages from
els of TNF- in response to TLR2 and TLR4 liga- healthy aged subjects [53]. No data seem to exist
tion. By analyzing the four subpopulations of regarding age-related changes in the clearance of
human monocytes, it was found the CD14+ (low) apoptotic cells, known as an important macro-
CD16+ and the CD14++(high) CD16+ populations phage function. We can only speculate that con-
were increased with aging, whereas the propor- sidering the functional changes described above
tion and number of CD14+ (low) CD16- were and the inammaging, the clearance of apop-
decreased compared to the young [43]. totic cells may be impaired with aging. Decrease
The few existing data suggest that monocyte/ in some receptors, as well as altered signaling
macrophages from aged individuals display leading to changes in chemotaxis and phagocytosis,
supports the hypothesis that apoptotic cells are clear virus-infected cells [39]. They can also
not cleared efciently. This could lead to their activate NK cells, which directly eliminate tumor
persistence in becoming proinammatory and cells. In addition to presenting Ag, they also provide
sustaining the quiescent state stimulation of co-stimulatory signals and cytokines for optimal
monocyte/macrophages, nally contributing to T cell priming, differentiation, and proliferation
the process of inammaging. Furthermore, [58]. Whether the numbers of DCs change dur-
these data conrm that, like neutrophils, mono- ing aging is still controversial.
cytes are to some degree activated at the basal There are several studies demonstrating alter-
state, but cannot be further stimulated through ations in pDC function in aged humans including
their surface receptors. This baseline activation reduced type I interferon production following
state may be very important to maintain their TLR stimulation, e.g., via TLR7 and TLR9. It has
functions for combating/constraining constant been suggested that the increased basal oxidative
and chronic challenges but insufcient for elimi- stress related to aging could be the underlying
nating new infections. Therefore, it seems that cause of the decreased upregulation of the inter-
neutrophils and monocytes are probably both feron regulatory factors by TLRs [59, 60]. In con-
contributing to the low-grade inammation with trast, mDCs from aged humans showed increased
aging which not only impairs the immune envi- expression of CD86 signaling, another sign of
ronment but also creates a vicious circle which activation even in the quiescent state. However,
maintains their functioning at an adequate level these ndings have not been corroborated by
whereas impairing their contribution to combat- in vitro studies. Nonetheless, they do seem to
ing new invaders, including tumor cells. Taken retain the capacity to produce proinammatory
together, all the experimental data available sug- cytokines and to activate CD8+ T cells [61], as
gest that with aging, most monocyte/macrophage well as to induce IL-17 production, which is
functions are changed with age, leading to altered known to recruit neutrophils [62]. DCs have also
tumor cell and pathogen clearing and altered reg- been reported to have a decreased ability in nave
ulation of the adaptive immune response and the CD4+ T cell activation via Ag presentation [63,
inammatory process resulting in chronic low- 64], attributed to decreased PI3K activity, a major
grade inammation and ultimately to increased pathway mediating cell function. Reduced PI3K
age-related diseases such as infections, cardio- was implicated in both age-related reduced DC
vascular disease, and cancers. migration and also as a negative regulator of TLR
signaling. Thus, the global result of this decreased
19.2.2.3 Dendritic Cells PI3K activation is a higher stimulation of the
Dendritic cells (DCs) are the most potent NF-kB pathway further contributing to inam-
antigen-presenting cells (APC) that can prime maging due to greater production of proinam-
specic T cells. There are several types of DCs matory cytokines such as IL-6 and TNF- in the
[54]: Plasmacytoid dendritic cells (pDCs) are basal state [63]. DCs have reduced Ag processing
important in host defense as they are one of the capacity concomitant with the altered expression
rst cells to produce type I interferon, hence ini- and function of their co-stimulatory molecules.
tiating several other responses, including NK Natural killer (NK) cells are one of the most
cell activation which amplies host response important antitumor players in the innate immune
[5557]. The second type of DC is the conven- system [10]. The NK cell population is now also
tional or myeloid-derived dendritic cell (mDC), divided into different subpopulations; those with
regarded as the most important APC for T-cell a CD16-CD56+ or CD16+CD56++ phenotype pro-
activation. They express TLRs and C-type lec- duce high amounts of IFN- and are among the
tins for the detection of Ags and subsequently most cytotoxic subtypes [65]. Subset distribution
produce IL-12, IL-15, and IL-18. IL-12 is essen- changes with aging, and the number of CD56dim
tial for induction of Th1 cell responses which NK cells increases, while CD56bright cells decrease
will induce cytotoxic T lymphocyte responses to [66, 67]. Furthermore, the expression of CD57 is
382 T. Fulop et al.
increased on CD56dim NK cells from elderly that decreased NK cell functions with aging were
subjects, representing a highly differentiated associated with a higher incidence of infectious
subset of NK cells. These observations were diseases [71]. IL-2-induced NK cell proliferation
recently extended by the nding that CD94 is decreased with aging and many cytokines and
(member of the C-type lectin family) and KLRG1 chemokines produced by NK cells, such as IL-2,
expression on NK cells was signicantly IL-8, are also decreased but with maintenance of
decreased in elderly subjects. Although the exact IFN- production [72]. This decreased production
consequence of this decrease is not known, it was of cytokines contributes to the altered activation
hypothesized that the decreased expression of of macrophages with aging, resulting in decreased
these surface markers induces unregulated cell microbicidal and tumoricidal activities. Thus,
lysis contributing to chronic inammatory condi- NK cells of elderly people show decreased prolif-
tions. Moreover, the same study revealed the erative responses to cytokines; higher total cyto-
presence of a greater proportion of IFN--positive toxic capacity when stimulated with certain
CD3-CD56bright NK cells with aging. This may cytokines including IL-2, IL-12, or IFN-; and a
suggest a shift to a more cytotoxic, cytokine-pro- greater sensitivity to stimulation via CD16. The
ducing and potentially immunomodulatory NK cytotoxic activity of NK cells depends on whether
cell phenotype occurring as a mechanism to com- the whole NK cell population or activity per cell
pensate for the decreased proportion of CD56bright is considered. On a per cell basis it is decreased,
NK cells. Aging also inuences the dynamics of which might be important for protection against
NK cells [65]. NK cells from the elderly have a developing cancer cells.
signicantly decreased proliferation and produc- Furthermore, other receptors involved in the
tion rate, and there is an increased proportion of cytotoxic activity of NK cells including members
long-lived NK cells which can be related to the of the natural cytotoxicity receptor family,
increased proportion of CD56dim NK cells. The namely, NKp30 and NKp46, decrease with aging
increased expression of CD57 may also suggest [73]. NKp30 has also been shown to be important
that the NK cells of elderly people are late-stage in the regulation of the cross-talk between NK
or terminally differentiated, like many of their cells and DCs. By this interaction the NK cells
CD8+ T cells [68]. Taken together, the data indi- can activate the DCs to more efciently prime
cate that although the number of NK cells often T cells. DCs release Th1 cytokines which further
increases with age, there is a profound redistribu- enhance NK activation. Thus, NK cells can mod-
tion of NK cell subsets with altered receptor ulate the adaptive immune response against
expression, explaining the functional alterations virus-infected or tumor cells via this interaction
leading either to decreased direct defense against with DCs.
virus-infected and tumor cells and/or decreased NKT cells are innate T lymphocyte population
regulatory activity for other components of the that recognize lipid Ags presented in the context
innate immune response, ultimately resulting in of the CD1d molecule found on monocytes,
decreased modulation of the adaptive immune macrophages, and DCs [74]. They can increase
response. Recently, it has been shown that NK the functions of NK cells. NKT cells are rapidly
cell activity is also under the control of IL15R/ recruited from the circulation during acute
IL15, released by nonimmune cells such as inammation and interact with various APCs
muscle cells, which, by its decrease with aging expressing the CD1d molecule. Recently, it has
can also contribute to these NK cell functional been shown that NKT cells are able to recruit
alterations [69]. neutrophils and activate them via their IFN-
Studies in very healthy elderly populations secretion [75]. Thus, NKT cells may play an
revealed that the total NK cell number tends to important regulatory role in the acute phase of a
increase with age, while their cytotoxicity is not microbial and/or tumor cell challenge by inter-
signicantly affected [70]. However, other stud- acting with various APCs via CD1d lipid anti-
ies in unselected elderly populations revealed genic presentation and secretion of different
cytokines. There are only a few reports on NKT suggested reason for this accumulation is a chronic
cell functioning in the elderly [72]. However, it antigenic stimulation especially caused by chronic
can be hypothesized that the altered activation of viral infections (predominantly CMV); however,
APCs via their TLR receptors will create an unfa- other chronic inammatory stimulations related to
vorable milieu for NKT activation either directly specic diseases may also contribute (including
or by their cytokine secretion. diabetes mellitus type 2, atherosclerosis, and pos-
IL-17 is mainly secreted by T cells, Th-17, sibly Alzheimer disease) [7881]. Interestingly,
and NKT cells [76]. This cytokine acts indirectly there are some reports showing that these cells
on neutrophil survival through stimulation of the also accumulate in cancer, such as at the early
secretion of G-CSF. IL-17 is also released by stage of breast cancer [82] and in renal carcinoma
neutrophils themselves and reinforces their [83]. Furthermore, they also express the character-
survival and recruitment [77]. It can also promote istic inhibitory surface receptors of exhausted and/
tumor vascularization by angiogenic factors. or senescent cells like KLRG1, CD57, PD-1, and
These immune cells as well as IL-17 itself may CTLA-4, as well as having reduced replicative
have pro- and antitumor activities; currently it is capacity and decreased survival after TCR activa-
not known what determines this dual effect on tion [84]. The role of these cells in cancer develop-
cancer. However, their differentiation in various ment is still questionable. Whether they are
subtypes, expression of specic receptors, and metabolically inert as senescent cells with short
production of various cytokines is likely to be telomeres and decreased telomerase activity or
determined by and in turn inuence the tumor they are metabolically active and able to secrete
microenvironment [75]. How aging affects various proinammatory cytokines and contribute
T cells has not been well investigated to date. to cancer development is a matter which is yet to
be elucidated. The cause of this exhaustion is not
known with certainty, but could either be due to
19.2.3 Adaptive Immune System direct antigenic stimulation by viral Ags such as
CMV or they could be innocent bystanders
Although there are changes in the innate immune affected by the chronic low-grade inammatory
response with aging as described above, it is still environment induced by such chronic antigenic
thought that the most important and relevant stimulation caused by constant basal proinam-
changes occur in the adaptive immune response. matory cytokines such as TNF- produced by the
Among the cells composing the adaptive immune innate immune system [85]. It was shown that p38
response, the T cells are thought to be the most has a role in cell activation, proliferation, and cell
affected; in addition, more and more data are cycle progression [86, 87]. TNF- can further acti-
emerging showing that B cells are also changed vate p38, thus contributing to immunosenescence
with aging. Nonetheless, it is well recognized that [85]. Interestingly, p38 is constitutively phosphor-
some of the most marked immune alterations ylated in EM and EMRA T cells, contributing to
associated with aging concern T lymphocyte sub- their reduced telomerase activity. Thus, the proin-
populations and functions [13]. The most recog- ammatory environment causing hyperphosphor-
nized model for T-cell subpopulations identies ylation of signaling molecules, such as p38, may
nave (CD45RA+ CCR7+), central memory TCM inuence the development of T-cell subpopula-
(CD45RA CCR7+), effector memory TEM tions as found in aging and inammatory diseases.
(CD45RA- CCR7-), and TEMRA (CD45RA+ CCR7) Together, these changes may be well tumorigenic
cells. Among these subpopulations, the highly dif- by altering adequate tumor-specic immune
ferentiated populations of EM (effector memory: response; they may be good targets for therapeutic
CCR7-, CD28-, CD27-, CD45RA-) and EMRA- modulation, as recently demonstrated so encour-
like CD4 and CD8 T cells (T effector memory agingly for PD-1/PDL-1 [8890]. Considering
cells re-expressing CD45RA) have been shown to these changes, it is reasonable to assume that an
accumulate in older humans [13]. Currently, the alteration in T lymphocyte activation is a central
384 T. Fulop et al.
issue in the age-related modications of the broblast senescence [85]. Nearly all of the
immune response. Currently, the most important signaling pathways associated with TCR activa-
paradigm underlying these changes is the repetitive tion or IL-2 receptor responses are found to be
antigenic stimulation over the life span that could altered with aging [105, 106]. There is an alteration
lead to partial unresponsiveness (immune exhaus- in the early steps of T-cell activation including
tion) and accumulation of memory cells. This has protein tyrosine phosphorylation, calcium mobi-
been shown for both CD4+ and CD8+ T cells with lization, and the translocation of PKC to the
distinct senescent status, surface molecule expres- plasma membrane. In addition, subsequent steps
sion, telomere length, and functionality. This was of the signaling pathways including the Raf-Ras-
further supported by a longitudinal study, the MAP kinase pathway are impaired. Decline in
OCTA/NONA study, resulting in the development proximal and intermediate events of transmem-
of the Immune Risk Prole integrating several of brane signaling leads to the decreased activity of
these parameters [9194]. It is of note that as transcription factors, especially NF-kB and
appealing as the CMV paradigm may appear, it is NF-AT. Not only activation signaling but also the
not yet proven [9597]. It is likely that other fac- negative regulatory network is altered with aging
tors could also contribute to causing the changes in [106]. This altered signaling followed by
the T cell compartment of the immune system decreased activation may be caused by a differen-
with aging including the slight but detectable tial inammatory state and subsequent T cell
amounts of the proinammatory cytokines con- phenotypic and functional change.
comitant with increased reactive oxygen species There are also age-related changes in the B
found in this basal proinammatory state. cell compartment [107111]. Production of B
Moreover, the intracellular T cell redox environ- cells is altered with aging at different levels,
ment inuences T cell function in aging [98, 99] resulting in decreased naive B cells. In addition,
which will be discussed later. Concomitant with an age-dependent loss of diversity of B cell
these phenotypic changes, the functions of T cells receptors is also observed which has been corre-
are also altered, and there is increasing evidence to lated to poor health and may reect expanded
implicate altered activation in the decreased T cell clones of memory B cells. These changes may
functions with increasing age. also lead to a shift in antibody specicity and the
Studies of elderly humans and animals have increase of autoantibodies. These alterations in
revealed that one function of T cells most notice- the B cell compartment may also favor the emer-
ably altered is the production of interleukin-2 gence of cancers related to aging.
(IL-2) compared to younger counterparts [100]. Taken together, aging is associated with an
It can be hypothesized that defects or alterations exhaustion of the adaptive immune response,
in the proximal events during T cell activation especially by rendering T cells dysfunctional and
will strongly affect the efciency of immune unable to appropriately respond to receptor
responses [100]. Thus, appropriate signal trans- ligation. This, together with B cell alterations,
duction cascades trigger an appropriate T-cell contributes to the establishment of a chronic
response, whereas alterations in the early events inammatory state, leading to higher susceptibility
of T cell signaling will result in less effective, to diseases such as cancer and increased mortal-
altered overall responses [101104]. The most ity predicted by the Immune Risk Prole [91].
important changes occur in CD4+ T cells result-
ing in decreased production of IL-2 and clonal
expansion. Although there are no changes in 19.2.4 Interaction Between Innate
TCR number at the cell surface, the number of and Adaptive Immune
CD28 co-stimulatory molecules decreases with Responses: Effect of Aging
aging, especially on CD8+ T cells. One of the
most important driving forces to decrease surface It is evident that if any component of the immune
CD28 expression is TNF-. This cytokine can response is not functioning, the outcome cannot
also activate p38 which plays an essential role in be optimal. Thus, the rst line of defense of the
organism, the innate immune response, is not the key to aging and age-related pathologies
only a powerful eradicator of foreign invaders but such as cancer.
is also responsible for the activation of the adaptive Alterations in the T-cell compartment can also
immune system for long-lasting and highly spe- trigger changes in the innate immune system
cic immunity by Ag specic, clonally expanded because the accumulation of memory and termi-
B and T lymphocytes. The reduced functioning nally differentiated/exhausted T cells secreting
of both monocytes/macrophages and DCs with more proinammatory cytokines and chemo-
aging will lead to reduced Ag presentation and kines will chronically stimulate and attract the
activation of T cell immune responses by these innate immune cells. The increased susceptibility
APCs. In addition, neutrophils secrete many mol- to apoptosis of certain T-cell subsets like CD4+
ecules such as HMG-B1 and alarmins which can naive T cells may also chronically contribute to
directly induce DC maturation or the activation the stimulation of innate cells.
of both the innate and the adaptive immune All these data demonstrate that with aging,
response. It is possible that the reduced neutro- alterations in both arms of the immune system, as
phil function with aging will also affect this well as in their efcient cooperation, contribute
aspect of their role in immune response. to altered protection against different challenges
A very efcient network exists among the dif- and participate in the development and mainte-
ferent cells participating in the innate immune nance of age-related low-grade inammation and
response aiming to eradicate invaders, restore increased susceptibility to diseases such as can-
homeostasis by resolving acute inammation, cer [9]. The same interaction between the innate
and ultimately to efciently activate the adaptive and adaptive immune response may either favor
immune response [16]. The individual function- the eradication or the progression of cancers
ing of the innate immune cells was shown to be depending on their state of activation, the pheno-
dysregulated with aging either because of type repartition, and the microenvironment.
receptor-driven signaling pathway alterations or
because of an age-related proinammatory
milieu sustained by cytokines and oxidative 19.3 Inammation Aging
stress [22]. These alterations will induce a dis- and Oxidative Stress
ruption in their functioning and in their mutually
supporting network resulting in inefcient eradi- The relationship between chronic low-grade
cation of the challenge, contribution in chronic inammation related to immunosenescence and
antigenic stimulation, and a chronic low-grade age-associated diseases, such as cancer, remains
inammation. On the other hand, they ultimately to be elucidated. It is of note that alterations of
lead to the altered and inadequate activation of certain proinammatory (IL-6, TNF, IL-1) as
the adaptive immune response. well as anti-inammatory cytokines (IL-10, IL-4)
One of the important central players of the are observed at greater frequencies in age-
cooperation of the innate and adaptive immune associated diseases compared to healthy aging [9].
response is TNF-. This factor is at center stage Thus, age-related immune dysregulation manifested
of the cytokines secreted by various cells of the essentially by a basic chronic low-grade inam-
innate immune system, such as monocytes stim- mation and a suppression of the adaptive response
ulated by many external or internal agents lead- may eventually lead to the development of
ing to modulation of the T-cell response either clinically signicant pathological conditions
to enhance it or dampen it via downregulation of including cardiovascular disease, dementia, dia-
CD28 or exhaustion of T cells [112]. TNF- betes mellitus, osteoporosis, and cancer [8]. Age-
production is increased in oxidative stress, related low-grade inammatory process seems to
chronic antigenic stimulation, CMV infection, accelerate the progression of chronic diseases, as
and visceral adiposity [113115]. Thus, the reg- well as having an immunosuppressive effect on
ulation and control of this vital molecule to cellular immune responses by contributing to
maintain it under a benecial threshold may be their exhaustion. The question arises as to
386 T. Fulop et al.
whether this proinammatory activity is the cancer development [125]. Recently, it was
primum movens for disease development or just a recognized that local inammatory processes
secondary reaction following latent chronic such as in the intestine and stomach may lead to
inammatory diseases. Moreover, this low-grade the development of cancers. However, the rela-
inammation may also represent an adaptive tionship between oxidative stress and inam-
mechanism to maintain an acceptable level of maging is less well established. When innate
response against cells including nascent tumor immune cells are chronically activated, they
cells. However, when increasing over a certain continuously release free radicals which can con-
level, it could become predominantly detrimental tribute to tumorigenesis directly as well as via the
by favoring their proliferation and the clinical alterations they cause to the adaptive immune
appearance of cancer. system, as already mentioned [126]. It is of note
What are the molecular events underlying that free radicals can create a vicious circle by
inammaging? It seems that NF-kB is at the cen- maintaining (through TLRs and inammasome
ter stage of metabolic pathways, as it controls the activation) the production of free radicals by
secretion of proinammatory molecules, such as other innate immune cells such as neutrophils,
cytokines, chemokines, MMPs, COX2, and DCs, and monocyte/macrophages which in turn
iNOS [116, 117]. NF-kB is also activated by reactivate them. Thus, free radicals directly and
many of these molecules via various pathways indirectly via oxidatively modied proteins or
such as the MAPK and the IP3/Akt pathway. As lipids activate NF-kB leading to proinammatory
might be expected from knowledge of the path- cytokine production. Similarly these free radicals
ways leading to their development, NF-kB activ- and lipid peroxides also activate the Nalp3
ity is highest in CD8+ TEMRA cells [118]. inammasome. These events lead to low level of
Moreover, the FOXO family of transcription fac- activation of innate cells at the basal level and
tors plays a role in longevity, cell survival, and participate in its maintenance.
proliferation via the modulation of NF-kB by Oxidatively modied proteins are also con-
free radical production [119]. Thus, NF-kB mod- tinuously produced as a result of the low-grade
ulating pathways are heavily implicated in the inammation [127, 128], accumulating in
occurrence, as well as in the perpetuation of this immune cells, especially in T cells, which interfere
low-grade inammation. with their functioning. Many proteins including
Thus, what is the relation between inammag- TCR, CD45, and enzymes are targeted by free
ing and free radicals which have been shown to radicals and become carbonylated or glycoxy-
increase with aging as a result of increased oxida- dated. This accumulation is further enhanced by
tive stress [120]? The degree of oxidative stress is decreased proteasome activity to eliminate these
the result of the disequilibrium between the pro- altered proteins [129, 130]. Thus, the free radicals
duction of ROS and endogenous antioxidant spe- create an altered cellular environment favoring
cies. Free radicals are produced as by-products of the activation of innate cells and decreased
aerobic respiration [121]. They are benecial for functioning of adaptive immune cells.
signaling, enzyme activation, and microbial elim- Furthermore, these free radicals will affect the
ination, but over a certain threshold, they may surrounding cells in inltrating tissues by inducing
become detrimental by causing mutations in cell proliferation, evasion of apoptosis, tissue
DNA and oxidation of macromolecules [122]. invasion, angiogenesis, autophagy, and altera-
The role of free radicals became the basis of one tions in macromolecule functions either by gain
of the leading theories of aging and consequently of functions or by loss of functions. All these
has been related to many age-associated diseases activities may contribute to some extent of
including cancer [123, 124]. In this context, it has tumorigenesis. Free radicals mediate these func-
been known for many years that age-related tions by stimulating different molecular pathways
increased ROS production due to mitochondrial including the Ras, MAPK, PI3K, mTOR, and
dysfunction may cause DNA damage and favor NF-kB pathways. Consequently, ROS also alter
Nrf2 activity which is considered to be the master phages via TLRs, thus contributing to a sustained
regulator of the antioxidant response [126]. Nrf2 proinammatory state which is measurable in
modulates a large number of genes that control some circumstances via increased circulating
several processes including immune and inam- levels of IL-6, IL-1, or TNF-. Thus, aging is
matory responses [131]. We have shown that with accompanied by a chronic low-grade inamma-
T cell aging, the Nrf2 is altered [22], which is tory process and by many other changes, some
also hypothesized in innate immune cells, and related to inammaging, some independent
further contributes to the inammatory process thereof. Hence, this may be the price that has to
and consequently to carcinogenesis. Thus, the be paid for maintaining immunosurveillance
immunosenescence associated inammaging against persistent pathogens or endogenous
contributes to cancer development by many stressors such as cancer cells. All these changes
pathways, especially by t increased basal free contribute to a decreasingly effective immune
radical production, which in turn further activates environment, probably unable to appropriately
these cells by propagating inammatory signal respond either to new Ags such as represented by
by free radicals. the continuous risk of exposure to new pathogens,
or to chronic persisting Ags such as those from
CMV or tumor cells during the life span.
19.4 Immunosenescence Therefore, inammaging related to immunose-
and Cancer nescence is likely to be one of the most important
general driving forces for cancer development. It
A causative connection between inammation is of note that every individual alteration at all
and some cancers is well established [132]. cellular and molecular levels also contributes to
Inammation in its uncontrolled state highly increased tumorigenesis. The most important ele-
favors tumorigenesis by increasing genomic ments for immunosenescence are the decreased
instability via the production of free radicals, neutrophil, macrophage, and DC functions but
persistence of proinammatory cytokines and maintaining uncontrolled proinammatory cyto-
chemokines and the subversion of Treg, T cell, kine production, as well as the decreased specic
and MDSC functions, as well as through angio- adaptive immune response by T cells to tumor
genesis [133]. The apparent disequilibrium Ags. TNF- seems to play a particularly impor-
between the retention of a reactive innate immune tant role as it is secreted mainly by immune cells,
response at basal state and the more severely in contrast to IL-6. It is the consequence of and
altered adaptive immune response with aging the support for inammaging via NF-kB and
leads to the presence of the low-grade inammatory AP-1 signaling.
status commonly present in the elderly, termed as Furthermore, an important aspect of the
inammaging as discussed above. Although inammatory response is the production of free
the cause of this increased basal inammatory radicals which leads to the activation of various
state is certainly multifactorial, it is likely that signaling cascades resulting in effector functions
one of the most important causes is chronic and apoptosis as well as in the further production
antigenic stimulation concomitant with increased of proinammatory cytokines. They also increase
free radical production related to oxidative stress. the possibility of genomic instability and epigenetic
The Ag source can be exogenous, as with persistent deregulation leading to enhanced mutations [134].
viral infections such as CMV [95] and subclini- These proinammatory cytokines secreted by the
cal bacterial infections, or endogenous like the cells of the innate immune system are also able to
various posttranslationally modied macromole- induce the production of free radicals. Thus, the
cules such as DNA or proteins which can be deregulation of innate immune responses strongly
modied by oxidation, acylation, or glycosyl- contributes to age-related chronic inammatory
ation. Such altered molecules can stimulate the processes and associated pathologies, as well as a
innate immune response, particularly macro- functionally neutral consequence of the aging
388 T. Fulop et al.
process. As a result, its modulation could be gen to cytotoxic T lymphocytes in order to elicit
benecial in the treatment of these diseases. an immune response. This process, termed
Moreover, the deregulated immune response cross-presentation, is crucial for the generation
with aging also produces directly pro-tumor mol- of immune response to viruses and tumors and
ecules as well as inducing the accumulation of in autoimmune disease. The ability of DCs to
immunosuppressive immune cells either systemi- cross-present exogenous Ag to CTLs makes
cally or in the tumor microenvironment. Data them an attractive target for exploitation in
suggest that pro-tumor molecules such as NO, immunotherapy. In recent years, signicant
indoleamine-2,3-dioxygenase (IDO), TGF, advances have been made in understanding the
IL-10, VEGF, PD-1 are increasing with age, as mechanism of cross-presentation and the DC
well as MDSCs (CD11b+, CD33+, CD34+, CD14- subsets involved. The recent discovery of human
HLADR-) under the high proinammatory cyto- cross-presenting DC has given this eld a new
kine micro- and macroenvironment, and Tregs lease of life relative to cancer immunotherapy
which suppress the antitumor activities of T cells, [136]. Such an example is the injection of mono-
NK, and NKT cells [18, 21]. These changes com- clonal antibodies (mAbs) which not only
pletely alter tumor-immune interactions neces- directly eliminate tumor cells but also result in
sary for cancer eradication or at least for the the release of new tumor antigens by killing
maintenance of the equilibrium stage. tumor cells. These can then participate in cross-
Finally, altered immune network functioning presentation to T and B cells, thus amplifying
also favors tumorigenesis. The altered presenta- the primary treatment [137].
tion of antigens by DC and macrophages decreases Modern immunotherapy clearly needs to con-
the activation of T cells, the functions of which are sider many aspects of tumor biology and associ-
further altered by oxidative stress and proinam- ated immune reactions. The heterogeneity of
matory cytokines produced by innate immune tumors and their microenvironment combined
cells. In contrast, the altered T cell phenotype and with the diversity of immune cells/molecules
functions are further increasing the innate cell will need complex approaches to immunother-
functions. Thus, a vicious circle is created leading apy. The new paradigm is to use autologous
to the appearance of tumor cells. tumor cells for vaccine and/or in combination
with personalized peptide vaccination which
would lead to eradication of tumors or at least to
19.5 Modulation the retardation of their development and metas-
tasis formation [21]. In an aging/geriatric envi-
Due to our increased understanding of tumor- ronment, certain characteristics specic to
immune interactions now, the patients immune elderly subjects, such as functional status and
system, even in nonimmunological treatments, comorbidities, should denitely be further
like radiotherapy, should be taken into consid- considered.
eration [12, 135], in order to achieve long-term
tumor control or complete tumor elimination.
Thus, the patients immune system needs to 19.6 Concluding Remarks
become integral to cancer therapy. It is also
clear that immunotherapies are mostly used in There is no doubt that aging is the main risk fac-
late-stage cancers when the immune system is tor for the development of many diseases includ-
already subverted. Thus, immunotherapy ing cancers, type 2 diabetes, and cardiovascular
should be initiated when the immune system is and neurodegenerative diseases. Understanding
still able to react. the mechanisms regulating aging is the most
Dendritic cells (DCs) possess the specialized important for the comprehension of the occur-
potential to present exogenously derived anti- rence of these different diseases. The low-grade
inammation seen with aging can be a common References

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Nutrition, Immunity, and Cancers
20
Hassan Abolhassani,
Niyaz Mohammadzadeh Honarvar,
Terezie T. Mosby, and Maryam Mahmoudi
Contents 20.3 Aging as a Confounder of the Triangle

of Nutrition, Immunity, and Cancer ....... 398
20.1 Introduction .............................................. 395
20.4 Role of Cancer in Predisposition
20.2 Role of Nutrition in Predisposition to Malnutrition from
of Cancer from an Immunologic View...... 396 an Immunologic View ............................... 398
20.2.1 Protein-Calorie Balance ............................. 396
20.2.2 Essential Fatty Acids .................................. 397 20.5 Role of Nutritional Support in Immune
20.2.3 Antioxidants (Selenium, Vitamin E, Restoration of Cancer Patients ............... 399
and Vitamin C) ........................................... 397 20.5.1 Arginine ...................................................... 400
20.2.4 Vitamin D ................................................... 397 20.5.2 Glutamine ................................................... 400
20.2.5 Vitamin B6 ................................................. 397 20.5.3 Branched Chain Amino Acids.................... 400
20.2.6 Folate .......................................................... 397 20.5.4 Nucleotides, Long-Chain Omega-3
20.2.7 Calcium ...................................................... 397 Polyunsaturated Fatty Acids,
and Eicosapentaenoic Acid ........................ 400
20.5.5 Fructooligosaccharides ............................... 400
20.5.6 Bioactive Compounds ................................ 400
H. Abolhassani, MD, MPH 20.5.7 Vitamins C and E........................................ 400
Research Center for Immunodeciencies, 20.5.8 Vitamins A.................................................. 401
Childrens Medical Center Hospital,
Tehran University of Medical Sciences, 20.6 Concluding Remarks................................ 401
62 Qarib St., Keshavarz Blvd, Tehran 14194, Iran References ............................................................... 401
Division of Clinical Immunology,
Department of Laboratory Medicine,
Karolinska Institutet at Karolinska University
Hospital Huddinge, Stockholm, Sweden
e-mail: abolhassanih@yahoo.com
20.1 Introduction
N.M. Honarvar, PhD M. Mahmoudi, MD, PhD (*)
Changes in immunologic pathways have a lead-
School of Nutrition and Dietetics,
Tehran University of Medical Sciences, ing role in all stages of cancer. Proinammatory
Poursina St., Keshavarz Blvd., Tehran 14155, Iran cytokines such as tumor necrosis factor- (TNF-
e-mail: honarvar@sina.tums.ac.ir; ), interferon- (IFN-), and interleukins 1 and 6
m-mahmoudi@sina.tums.ac.ir
(IL-1 and IL-6) are important mediators of cancer
T.T. Mosby, MS, RD, LDN complications such as cachexia [1]. A tumor can
Department of Nutrition, St. Jude Childrens
trigger the release of cytokines such as IL-6 [2],
Research Hospital, 262 Danny Thomas Place,
Memphis, TN 38105-3678, USA which is associated with an increase in lipolysis
e-mail: terezie.mosby@stjude.org and proteolysis, which in turn affect the appetite

396 H. Abolhassani et al.
and host neuroendocrine axis and induce anorexia may affect the regulation of carbohydrate absorp-
and cachexia [2, 3]. Several neuropeptides such tion and short chain fatty acid formation, which
as neuropeptide Y (NPY) and adipokines such as affects the metabolism of carcinogens [12]. This
leptin have been implicated in the pathogenesis of process is linked to colon cancer and its progres-
cancer cachexia syndrome [3, 4]. NPY receptors sion [13]; apparently, a decrease in ber intake may
appear to be resistant to NPY, and production of allow more time for exposure of colon cells and the
NPY appears to be decreased in cancer cachexia. immune system to the potential carcinogens, affect-
This hypoleptinemia may play a role in increased ing intestinal transit [14]. Moreover, based on the
insulin resistance seen in cancer patients [5, 6]. evidence used to draw conclusions about a gluten-
Nuclear factor-kappa B (NF-B) plays an impor- free diet in patients with celiac disease leading to
tant role in cancer development and may be inu- cancer protection, it seems reasonable to consider
enced by proinammatory chemokines to activate gluten as a booster for cancer in celiac patients [15].
inammatory response genes and regulate cell Meat consumption is a risk factor for some
proliferation and apoptosis [7]. NF-B activa- cancers, especially colon, rectum, and prostate.
tion also promotes the cyclooxygenase-2 (COX2) Red meat consumption increases the risk of colon
cascade, leading to increased oxygen free radical cancer by causing increased production of het-
synthesis and cell damage [8, 9]. Thus, an imbal- erocyclic amines [16, 17].
ance of cytokine production and neuropeptide and On the other hand, a change in the normal diet
adipokine dysfunction may be a major cause of and deciency of vitamins or minerals may affect
the nutritional consequences of cancer. the adequacy of either innate immunity (phago-
cytic activity, chemotaxis of neutrophils, or
release of cytokines from monocytes) or adaptive
20.2 Role of Nutrition immunity (immunoglobulin production of B cells
in Predisposition of Cancer or cell-mediated immunity) [18, 19]. Many of the
from an Immunologic View consequences of malnutrition in the regulation of
signal transduction and immunoregulatory gene
One of the known risk factors for cancer is obesity, expression were rst recognized in the early
especially with the modern lifestyle and low physi- 1800s as nutrigenomics [20, 21]. The majority of
cal activity [1]. Dietary patterns have a signicant these changes are reversible after administration
effect on the cytokine prole; for instance, the high of adequate nutrition supplements [2224].
intake of saturated fats, especially in obese people, The following list of specic dietary factors
leads to inltration of adipose tissue by macro- has been studied in relation to the immune aspects
phages producing IL-1b, IL-6, and macrophage of cancer.
inhibitory factor (MIF) [24]. Moreover, a decrease
in the secretion of anti-inammatory adipokines
such as adiponectin may maintain proinammatory 20.2.1 Protein-Calorie Balance
signals and activate the production of C-reactive
protein (CRP) by the liver [5, 6]. Based on previous The formation of lymphocytes, eosinophils, and
studies, this chronic inammatory process is related vital immune system proteins such as thymic
to an increased susceptibility to various types of hormones, antibody (Ab) responses to T-cell-
cancer, including cancers of the gastrointestinal, dependent antigens (Ags), and Ab afnity are
respiratory, and genitourinary systems [7, 10]. affected by protein-calorie imbalance [25]. It has
Inuenced by this important effect of nutrition long been recognized that caloric restriction with
on the immune system, characteristics of the human a well-balanced diet avoiding certain nutrient
diet can directly stimulate gastrointestinal malig- deciencies can increase longevity and has can-
nancies [11]. A diet low in ber and vegetables cer preventive effects in mammals [26].
20 Nutrition, Immunity, and Cancers 397
20.2.2 Essential Fatty Acids clear how vitamin B6 mediates this effect, but it
has been reported that high dietary vitamin B6
Essential fatty acids in our body can regulate the attenuates and low dietary vitamin B6 increases
production of prostaglandins, prostacyclins, the risk of cancer [37].
thromboxanes, and leukotrienes, causing a sig-
nicant effect on the host immune system and
regulation of inammation [27]. 20.2.6 Folate
Folate is important for DNA methylation, repair,

20.2.3 Antioxidants (Selenium, and synthesis [3841]. Epidemiologic studies
Vitamin E, and Vitamin C) have shown that low folic acid intake is associ-
ated with a higher risk of various cancers, most
These nutrients have strong antioxidative effects notably colon [42], breast [43], and probably
and may reduce the risk of cancer by neutralizing cervical cancer [43]. The fact that methylenetet-
reactive oxygen species or free radicals that can rahydrofolate reductase, an enzyme predicted to
damage DNA [2830]. reduce the risk of colon cancer, is associated with
folate status supports the role of folate in cancers
20.2.3.1 Vitamin A [42, 44].
Vitamin A plays an important role in protection
against measles, white blood cell (WBC) func-
tion, resistance to carcinogens, and skin and 20.2.7 Calcium
mucous membrane defenses. Vitamin A precur-
sor carotenoids, such as lycopene, have a poten- Many studies show that calcium may reduce the
tial effect on cancer prevention [31, 32]. risk of colorectal cancer via direct and indirect
effects. Calcium has a direct effect on prolifera-
tion, stimulating differentiation, and apoptosis in
20.2.4 Vitamin D the colonic mucosa [45, 46]. Its indirect effect is
binding to toxic secondary bile acids and ionized
Vitamin D has been of interest based on eco- fatty acids to form insoluble soaps in the lumen
logic studies on populations with greater expo- of the colon [47, 48].
sure to ultraviolet light who had a lower risk of In addition to deciency, an overdose of some
breast cancer [33], colon cancer [34], and micronutrients can also have an immunosup-
prostate cancer [35]. This vitamin regulates pressive effect, especially megadoses of vitamin
humoral Ab response and supports a Th2- E [49]. High doses of certain minerals such as
mediated anti-inammatory prole of cyto- chromium, copper, iron, manganese, and zinc
kines; therefore, its anticancer properties are also may induce cancer and immune dysfunction
strongly suggested [36]. [5052].
In summary, attenuated innate and adaptive
immunity as a result of an inadequate diet
20.2.5 Vitamin B6 leads to a higher risk of cancer and lower
homeostasis for cancerous antigens, thus
Pyridoxine induces WBC responses, Th1 cyto- reducing nutrient intake, increasing losses, and
kine-mediated immune responses, and shrinkage interfering with utilization due to altering met-
of the thymus [36]. Epidemiologic studies and abolic pathways. Thus, nutrition may have a
laboratory animal models have shown that vita- significant role in prevention and treatment of
min B6 modulates the risk of cancer. It is not cancer [40].
20.3 Aging as a Confounder

of the Triangle of Nutrition,
Immunity, and Cancer
Aging may be a confounder of the triangle of

nutrition, immunity, and cancer; however, neither
the relationships nor the mechanisms of interac-
tion are known. Unfortunately, only a few studies
have considered that nutrition and immune func-
tion simultaneously decrease in elderly individu-
als [53]. It is known that increased age adversely
affects the function of the immune system as well
as nutrient intake habits [54]. Therefore, both
immunosuppression (decreased effectiveness of Fig. 20.1 Schematic overview of complex network of
diet-immunity-cancer
T and natural killer cells) and nutritional de-
ciencies (as dened by the 1989 recommended
dietary allowances) in the elderly may have inde- deaths may be linked to improper diet [6365].
pendent correlations with increased risk of infec- Moreover, a proportion of patients with malig-
tion and neoplasia development [55, 56]. One of nancy develop cachexia, a progressive invol-
the probable mechanisms that may affect both untary weight loss status that is attributed to
immunity and nutrition in old people is turnover clinicopathologic factors of the tumor (origin,
uctuations of cellular components in lysosomes metastasis, and size), host immunity, and anti-
or autophagy. Advanced age leads to a reduction tumor treatment (Fig. 20.2) [65, 66]. During
in the autophagy of loading viral Ags and cross- the development of cancer-associated cachexia,
presentation of tumor Ags into MHC class I mol- several Th2-dominant condition mediators such
ecules, as well as pathogen killing [57, 58]. as IL-2 and TNF- (prognostic markers) are
Similarly, the capability of autophagy for ener- implicated in appetite loss and metabolic dis-
getic balance recycling of amino acids to main- turbances, as well as leptin, IL-1, IL-6, IFN-,
tain protein synthesis under starvation conditions leukemia inhibitory factor, NPY, and proteogly-
and the capacity of intracellular lipid stores or can 24K [67, 68]. These immunologic and meta-
glycogen mobilization are disturbed [59, 60]. bolic changes induce cancer cachexia syndrome,
However, only minimal information has been which is characterized by patient tissue wasting,
produced concerning human cancer initiation as anorexia, appetite loss, prolonged fatigue and
a direct result of a specic dietary etiology in the lethargy, insulin resistance, microcytic anemia,
elderly. hyperlipidemia, and hypoalbuminemia [69, 70].
Metabolic features of this syndrome include
increases in heterogeneity of energy require-
20.4 Role of Cancer ment, substrate cycling and turnover, Cori cycle
in Predisposition activity, and hepatic protein synthesis, as well as
to Malnutrition decreases in peripheral muscle protein synthesis,
from an Immunologic View serum protein lipase activity, and plasma concen-
tration of branched chain amino acids [71, 72]. In
Despite the role of nutrition in either prevent- general, the severity of malnutrition and cachexia
ing or causing cancer in humans, malnutrition in digestive neoplasias is in highest percentages
is a common problem (global percentage of (from 79 % in esophageal cancer to 40 % in rec-
56.5 %) [61], and weight loss is often predictive tum cancers) due to the involvement of all areas
of shortened survival in these patients [62]. In described in Fig. 20.2 during the development of
advanced stages of cancer, up to 35 % of related cancer and in chemotherapy or tumor resection.
Fig. 20.2 The casual

pathways of cachexia
occurrence after malignancy
It should be noted that antitumor agents with and activity of (NK) cells are the most common
their side effect on cells with high turnover may changes in the defense system of patients with
exacerbate malnutrition. This could be explained cancer cachexia, leading to more infectious com-
by the competition between cancerous regions plications and poor prognosis [81]. Neutrophil
and normal cells of the gastrointestinal system to chemotaxis, monocyte phagocytosis and killing,
use nutrients to repair the adverse effects of anti- number of T cells, and proliferation of lympho-
tumor drugs (hypermetabolic state). Briey, cytes are also defective in patients with lung can-
impaired caloric intake, side effects of therapy, cer [82]. Phagocytic and bactericidal activities of
changes in taste and mood, pain and other adverse neutrophils were low in hepatocellular carcinoma
consequences of eating, obstruction, stula, and patients [83]. In addition, surgical stress in can-
malabsorption all promote malnutrition in cancer cer patients enhances Th2 and compromises the
patients [7377]; therefore, well-nourished Th1/Th2 balance and expression of HLA-DR on
patients with intact gastrointestinal integrity have monocytes, which is considered to be a central
lower morbidity and mortality than others [78]. marker of immune paralysis after surgical trauma
It should be noted that cachexia after can- [84]. Most of these immune parameters are also
cer differs from cachexia following starvation. reduced during radiotherapy and chemotherapy
Increased protein and glucose turnover, high because of their side effects on bone marrow.
whole body synthesis and catabolism, accelerated However, these factors are reversible after nutri-
hepatic protein production (especially acute phase tion improvement [85].
agents), increased serum free fatty acid levels, and
depletion of fat stores were reported only in can-
cer patients. However, metabolic abnormalities 20.5 Role of Nutritional Support
and, paradoxically, impaired immune response in Immune Restoration
are probable consequences of cancer cachexia, of Cancer Patients
as explained in the previous section [79, 80].
Increased levels of immunosuppressive mediators Adjuvant therapy of cancer patients by different
(e.g., TGF-), decreased C3 and delayed hyper- nutritional support strategies (dietary counsel-
sensitivity response, and diminished numbers ing, oral nutritional supplements, enteral tube
feeding, and parenteral tube feeding) is the of studies that used parenteral glutamine postop-
mainstream recommendation to increase their eratively showed it was associated with a shorter
quality of life and to obviate the risks associ- hospital stay and a lower incidence of infectious
ated with gastrointestinal complications and complications [95].
reverse malnutrition [86]. However, there is no
comprehensive approach based on the needs
of cancer patients with cachexia or those with 20.5.3 Branched Chain Amino Acids
increased nutrient requirements [87]. Several
studies have shown the effectiveness of nutri- L-valine, L-leucine, and L-isoleucine can
tional supply in groups of patients with malig- improve the immune response and maintain
nancy that resulted in weight gain, increased serum albumin level in the course of hepatocel-
appetite, increased energy and protein intake, lular carcinoma recurrence [96].
reduced gastrointestinal toxicity, and enhanced
immune function [8890]. In the clinical set-
ting with standard treatment protocols, it turns 20.5.4 Nucleotides, Long-Chain
out that the implementation of nutrition support Omega-3 Polyunsaturated
in patients with cancer is most effective when Fatty Acids, and
it is limited to special, well-described circum- Eicosapentaenoic Acid
stances. Nonetheless, the potential advantages
of some specic nutrients have been described These lipid agents have anti-inammatory, anti-
and are outlined below. cachectic, immunomodulating, and antitumor
effects [97100].
20.5.1 Arginine
20.5.5 Fructooligosaccharides
Arginine is a semi-essential amino acid with
immunomodulatory potential such as stimulated This group of functional bers associated with
thymic growth and mononuclear cell response to increased lactic acid bacteria acts as an immuno-
mitogens, which enhances lymphokine-activated modulator by stimulating IgA synthesis, promot-
killer cell generation via a nitric oxide-mediated ing mucin production, modulating inammatory
mechanism and stimulates the release of poly- cytokines, and decreasing Ag absorption [101].
amines by the small intestine. In one randomized
trial of malnourished patients with head and neck
cancer, follow-up at 10 years indicated better sur- 20.5.6 Bioactive Compounds
vival in those who received supplemental argi-
nine preoperatively [91, 92]. Agaricaceae fungus consisting of ergosterol,
oleic acid, and triterpenes may inhibit neovascu-
larization induced by tumors and therefore atten-
20.5.2 Glutamine uate cancer progression [102].
Glutamine is the most abundant amino acid in the

human body and the preferential fuel of rapidly 20.5.7 Vitamins C and E
dividing cells such as lymphocytes and macro-
phages [93]. However, supplementing glutamine Since chemotherapy may induce mucositis and
in the diets of patients with cancer may be coun- bleomycin in particular induces chromosomal
terproductive because glutamine (which is essen- damage in lymphocytes, the administration of
tial for fast growing cells in culture) may promote vitamins C and E may reduce the side effects of
accelerated tumor growth [94]. A meta-analysis therapy [103105].
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Allergies and Cancers
21
Delia Rittmeyer and Axel Lorentz
21.1 Introduction .............................................. 407 Worldwide, especially in industrialized countries,

allergies and cancer cause high morbidity, mortal-
21.2 Molecular Mechanisms of Allergy.......... 408
ity, and nancial burden to healthcare systems. A
21.3 Types of Allergic Diseases ....................... 409 total of 12.7 million people were diagnosed with
21.4 Molecular Basics of Carcinogenesis ....... 409 cancer, and 7.6 million died from cancer in 2008,
21.5 Types of Cancers ...................................... 410
whereby incidences in industrialized countries are
nearly twice as high as in developing countries
21.6 Antitumor Immunity ............................... 410 [1]. In developed countries, for instance, in
21.7 Relationship Between Allergies Germany and in the USA, cancer is the second
and Cancers in General ........................... 411 leading cause of death after cardiovascular
21.7.1 Cancers Positively Correlated
with Allergies ............................................. 411
diseases [2, 3]. Cancer rates are rising due to an
21.7.2 Tumor-Promoting Effects of Allergies....... 412 increasingly aging population and changes in life-
21.7.3 Cancers Negatively Correlated style [1]. Allergies are more prevalent, but mortal-
with Allergies ............................................. 413 ity is much lower. In Germany, about 40 % of all
21.8 Tumor-Protecting Effects adults have experienced some type of allergy
of Allergies ................................................ 414 during their life time, and about 300 million peo-
21.9 Concluding Remarks ............................... 415 ple are suffering from asthma worldwide [4, 5].
References ............................................................... 416
Interest in possible relationships between
these prevalent diseases arose in the 1950s.
Following studies revealed a decreased preva-
lence of allergies among cancer patients [6].
Since then, much research has been done, but still
no generally accepted statement about the corre-
lation has been established. As the immune system
is involved in both allergic and neoplastic diseases,
a connection might be obvious; nonetheless, the
D. Rittmeyer, MSc A. Lorentz, PhD (*) nature of this connection is dichotomous. On the
Department of Nutritional Medicine, one hand, allergies are regarded as a hyperactive
University of Hohenheim, Fruwirthstrae 12,
state of the immune system which leads to better
70593 Stuttgart, Germany
e-mail: delia.rittmeyer@googlemail.com; detection and destruction of tumor cells. On the
lorentz@uni-hohenheim.de other hand, allergic reactions go along with

408 D. Rittmeyer and A. Lorentz
inammatory processes which may initiate and production of mainly IL-4 and IL-5. On the
support tumor growth [7]. Hence, there are dif- contrary, Th1 cells which develop under the
ferent hypotheses on the relationship which inuence of IL-12 from the same precursor cells
appears to be complex and not universally appli- predominantly produce interferon (IFN-) and
cable for every type of cancer or allergy. This IL-2. Further factors determining polarization are
chapter will give an overview about studies the Ags route of entry, Ag dose, and the way of
examining these relationships and describes Ag presentation [13, 14]. Th2 cells organize
possible mechanisms which could explain them. the further allergic response toward the specic
allergen, as shown in Fig. 21.1. Secretion of IL-4
or IL-13 by Th2 cells causes the isotype switch to
21.2 Molecular Mechanisms IgE in B cells. Additionally, a costimulatory
of Allergy signal, namely, the engagement of CD40 on the
surface of B cells and CD40 ligand on the surface
By denition, allergy is an immunologic reaction of Th2 cells, is required for the stimulation of
to normally innocuous environmental antigens the B cell [15]. As a result, sensitized B cells
(Ags), so-called allergens, and it is mostly differentiate into plasma cells and produce
equated with type I hypersensitivity (immediate- allergen-specic IgE. Moreover, B cells them-
type hypersensitivity) according to the classica- selves are also able to take up soluble Ags via
tion by Coombs and Gell. This type is mediated specic B cell receptors and present them to
by immunoglobulin (Ig) E in response to T helper nave CD4+ T cells inducing Th2 differentiation
cell type 2 (Th2) polarization of CD4+ T cells [8]. [9]. IL-5, IL-6, and IL-9 may enhance IgE pro-
Usually IgE is associated with defense against duction, whereas Th1 cytokines like IFN- and
helminthic infections [9]. Atopy is the hereditary IL-12 act as inhibitors [14].
tendency to immediate-type reactions and increased Most of the IgE engage to the high-afnity
production of IgE; however, not every allergic receptor FcRI on the surface of mast cells even
disease has to be atopic [10]. There are different in absence of Ag. If allergens bind to specic
genes associated with atopy, but environmental IgE, FcRI is cross-linked, followed by an inam-
factors are of great importance as well. During matory reaction [15]. Mast cell mediators such as
fetal and postnatal periods, the immune system is histamine, lipid mediators, and cytokines are
rather Th2 polarized which shifts toward Th1 released during the effector phase of an allergic
during the rst years of life [9]. According to the reaction and induce typical allergic symptoms.
hygiene hypothesis, infectious diseases in childhood FcRI is also expressed on basophils which are
are important for Th1 bias. This corresponds with also able to release allergic mediators being
an increasing incidence of allergic diseases in stored in granules [16]. As basophils are able to
industrialized countries where excessive hygiene produce IL-4 as well, they can amplify IgE pro-
leads to an inadequate Th1/Th2 balance [11]. duction [17]. When specic IgE was once built,
Allergic reactions are induced by low doses of further exposition to the corresponding allergen
allergens. Allergens are proteins, many of which elicits an allergic reaction without renewed sensi-
are enzymes, and their allergenicity is determined tization [9].
by protease activity, surface features, or glycosyl- Production of IL-5 by Th2 cells and mast cells
ation patterns. Soluble allergens enter the body, activates eosinophils to secrete inammatory
orally or by inhalation, where they are taken up mediators as well as highly toxic proteins and
by antigen-presenting cells (APCs) such as free radicals from their granules [8, 9]. Hours
dendritic cells (DCs) which present them to nave after the early phase of the reaction, the late phase
CD4+ T cells via major histocompatibility may take place which is characterized by inltra-
complex (MHC) class II [12]. In the presence of tion of further inammatory leukocytes and
interleukin (IL)-4, nave CD4+ T cells differenti- eventually a chronic inammation may be estab-
ate into Th2 cells which are characterized by the lished [18]. The cells involved in allergic
21 Allergies and Cancers 409
Allergen IL-4
TCR
Th2
DC MHCII
Sensitization phase IL-4 IL-5
B
Eo
IgE
IL-5
Inflammatory mediators:
Effector phase MC Histamine, Leukotrienes,
Proteases, Toxic proteins,
ROS, Cytokines etc.
Fig. 21.1 Type I allergic reaction. B B cell, DC dendritic cell, eo eosinophilic granulocyte, IgE immune globulin E, IL
interleukin, MC mast cell, Th2 T helper cell type 2, ROS reactive oxygen species. For further explanation, see text
reactions reside predominantly in tissues close to tract and others [8]. Anaphylaxis is a systemic
the body surface as their actual function is to reaction against an allergen with life-threatening
defend against multicellular parasites which symptoms like hypotension or airway constric-
invade primarily into skin and mucosa-associ- tion [20].
ated lymphoid tissue. Therefore, these cells are
specialized to evoke Th2 immune responses [8].
21.4 Molecular Basics
of Carcinogenesis
21.3 Types of Allergic Diseases
Cancer is a genetic disease in consequence of a
Allergic asthma is a chronic inammatory disease number of mutations in somatic cells. Unlimited
of the airways caused by inhaled allergens. growth of the mutated cells leads to formation of
Symptoms are breathlessness, wheezing and neoplasms. Tumor cells are capable of invading
coughing due to bronchial constriction, and into tissues and eventually of disseminating and
increased mucus secretion. It is often accompa- building metastases in distant regions of the
nied by hyperreactivity of the airways to other body. The clinical phenotype is varying as well as
stimuli [10, 19]. Allergic rhinitis or hay fever is the implications, depending on the type of cancer
an allergic inammation of the nasal mucosa and the affected patient. Although the incidence
which results in sneezing, itching, and runny or of cancer increases with age, tumors occur in
blocked nose and is often combined with allergic every age group [22].
conjunctivitis [20]. Atopic dermatitis or eczema The development of cancer, carcinogenesis, is a
is a manifestation of atopy which occurs predom- multistep process which requires progressive
inantly among children, showing symptoms like alterations in the genome of normal cells.
itching, red rashes, and small vesicles on the skin Mutations can occur spontaneously or can be
[20, 21]. Food allergies mostly cause diarrhea or generated by so-called carcinogens [23]. A car-
vomiting, but they may also affect the respiratory cinogen is an environmental factor like a chemical
compound, a biological substance, a virus, or common type is adenocarcinoma which affects

radiation that is able to interact with DNA and the exocrine component of the pancreas, but other
cause damages or alterations in the genome. components of the pancreas may also be affected.
Usually cells have several mechanisms to repair Main causes are smoking, diabetes mellitus, and
DNA damages. During the process of repair, the chronic pancreatitis [22]. Lung cancer is the third
cell cycle is stalled, preventing that this mutation is leading type of cancer among men and women
multiplied. If no repair is possible, the cell is and the leading cause of death from cancer among
destroyed by apoptosis [24]. An abolition of these men. More than two thirds of the cases are caused
mechanisms is a precondition for oncogenesis. by cigarette smoke [31]. Cancers of the colon and
Therefore, mutations have to occur in genes which rectum represent the second most common type
are responsible for the control of cell proliferation, of cancer. Besides the hereditary component,
differentiation, or apoptosis [25]. Such critical dietary habits are a major risk factor [3, 31]. Skin
genes can be divided into two groups: oncogenes cancer includes malignant melanoma, basal cell
and tumor suppressor genes [26]. Products of carcinoma, squamous cell carcinoma, and some
oncogenes are, e.g., transcription factors, growth fac- others [22]. The rst one causes more deaths;
tors, or their receptors. Tumor cells are character- however, the others are more prevalent, yet with
ized by gain-of-function mutations in oncogenes, higher curing rates [31]. Meningioma and glioma
resulting in overexpression of oncogene proteins are the two most common types of brain cancer,
and subsequent increased growth [27]. Tumor sup- whereby the causes are largely unknown [32].
pressor genes, or rather their products, have a Lymphatic and hematopoietic cancers are, e.g.,
repressive effect on cell growth. Loss-of-function leukemia, Hodgkin lymphoma, or non-Hodgkin
mutations in tumor suppressor genes result in unim- lymphoma. Leukemia is characterized by an
peded proliferation or evasion of apoptosis [25]. abnormal proliferation of leukocytes and can be
However, one single mutation is not sufcient for classied into acute or chronic and myelogenous
the formation of a cancer cell. Carcinogenesis is a or lymphocytic forms [22]. Acute lymphocytic
multistep process involving several events that inca- leukemia is the most common tumor disease in
pacitate control of the cell cycle, thereby creating a childhood, whereas the etiology is still not identi-
cell with growth advantages [28]. The initiation pro- ed [31]. Among reproductive cancer, prostate
cess of carcinogenesis, characterized by somatic cancer in men and breast cancer in women are
changes, is followed by the process of promotion. the leading types of cancer. Furthermore, breast
Different promoters like chemical irritants, hor- cancer is the most frequent cancer-induced cause
mones, or inammation induce proliferation of the of death among women. Other common reproduc-
damaged cells and further mutations, as the genome tive tumors are tumors of the uterus, cervix, and
of cancer cells is very unstable [25, 29]. The next ovaries [31].
step is tumor progression. By means of alteration of
cell adhesion molecules and protease activity, can-
cer cells are capable of leaving the primary tumor 21.6 Antitumor Immunity
and inltrating into tissues. Subsequently, tumor
cells spread through blood or lymphoid vessels and In 1970, Burnet and Thomas established the
build metastases in distant parts of the body while hypothesis of cancer immunosurveillance. It
they are displacing healthy tissue [30]. states that, to a certain degree, the immune sys-
tem is able to detect and destroy tumor cells
before they can arise to clinically detectable
21.5 Types of Cancers malignancies. Meanwhile this hypothesis has
been expanded to the theory of immunoediting
Pancreatic cancer is one of the cancer types with which is comprised of three phases: the elimina-
the poorest prognosis, as mortality rates almost tion phase, the equilibrium phase, and nally the
correspond to incidence rates [31]. The most escape phase [33].
The elimination phase complies with the Altogether the immunosurveillance hypothesis
process of immunosurveillance. Immune cells of describes that the immune system is in fact able
innate and adaptive immune response identify to ght tumor cells, but also promotes carcinogen-
tumor cells by so-called tumor Ags [34]. If these esis by sculpting poorly immunogenic mutants.
are presented to an activated CD8+ T cell, the
tumor cell is directly destroyed by the release of
cytotoxic proteins. Moreover, antigen-specic B 21.7 Relationship Between
cells produce specic antibodies which can Allergies and Cancers
opsonize tumor cells and lead to either antibody- in General
dependent cellular cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC) [35]. The rst studies relating to possible associations
Besides this adaptive immune reaction, there between allergies and cancer date back to more
are cells of the innate immune system involved in than half a century [39, 40]. Anyway until now
immunosurveillance which execute antigen- the results have not been consistent, despite
independent immune responses. Among them are various researches in this regard [41].
natural killer (NK) cells and NK T cells which Regarding cancer in general, there seems to be
are able to recognize and directly kill tumor cells a balance between studies reporting positive and
[25]. In addition, these two cell types produce negative correlations with different types of aller-
IFN- which is probably the most important gies. While analyses of the Cancer Prevention
cytokine in antitumor immunity [33]. It acts Study II indicate a slightly decreased risk for
indirectly by modulating the immune response, people suffering from hay fever or asthma [42],
e.g., by activation of macrophages or augmenta- data from the First National Health and Nutrition
tion of T cell response and NK cell activity, and it Examination Survey (NHANES I) show an up to
is able to increase immunogenicity of tumor 50 % increased risk of developing any type of
cells. Moreover IFN- itself has anti-proliferative, cancer [43]. Together with several other studies
apoptotic, and angiostatic capacities which [19, 21, 39, 4455], no conclusion can be drawn
directly affect tumor cells [36, 37]. However, which identies the role of allergies in cancer
cancer cells are capable of defending against epidemiology. As the term cancer includes
these immune mechanisms. Either they lack cer- diseases of diverse etiologies and a variety of
tain MHC peptides, making them unrecognizable affected tissues, it is necessary to distinguish
to T cells, or they do not express costimulating between different cancer sites as well as specic
signals which lead to T cell tolerance [38]. Hence, types of allergy. In the following, those associa-
if the immune system is not able to kill the entire tions which are supported by the majority of
tumor cells, the process of immunoediting studies are presented.
reaches the equilibrium phase, characterized by
dynamic dying and generation of further mutated
cancer cells [34]. Following Darwins rules, those 21.7.1 Cancers Positively Correlated
cells, which show surviving advantages through with Allergies
reduced immunogenicity, resist the immune
attacks. Thus tumor cells also get shaped and Without exception, all of the evaluated studies
sculpted by immune cells, leading to cell popula- suggest a positive association between a history
tions that are capable of evading any immune of asthma and lung cancer. Without controlling
reactions [33]. In this case, surviving tumor cells for smoking, a study of 78,000 asthmatic patients
enter the escape phase. Besides the absent immu- found an increased risk for women as well as for
nogenicity, tumor cells are also able to suppress men [49]. Another study observed a positive
immune reactivity so that they can proliferate association with asthma, yet no associations with
continuously and eventually develop a malignant hay fever only, both asthma and hay fever, and
tumor [38]. any of these conditions [42]. A further survey
calculated a lower, but still elevated, risk for Since inammatory responses are supposed to
asthma when controlling for smoking. An additional remove the causes as well as to rebuild damaged
analysis of the effect of respiratory drugs taken tissues, an environment rich in growth promoting,
for the treatment of asthma showed no connection but also rich in damage causing, factors is required.
to cancer development [19]. In a Taiwanese Consequently, the conditions for carcinogenesis
study, asthma was the only type of allergy associ- are established.
ated with risk of lung cancer [48]. Reactive oxygen species (ROS) released by
The prevalence of skin cancer was predomi- macrophages are capable of causing DNA dam-
nantly examined among subjects suffering from ages, thus promoting tumor initiation. Permanent
atopic dermatitis, for other types of allergy there is cell regeneration raises the probability of carci-
only little evidence available. Atopic dermatitis nogenic mutations [29]. Cancer promotion is
was associated with a clearly increased risk of supported by growth factors like TGF, IL-1, IL-6,
keratinocyte carcinoma which made up half of all or IL-8. Furthermore, several inammatory media-
observed excess cancers in this study. Among tors have angiogenic properties or stimulate the
6,275 hospitalized patients with atopic dermatitis, production of angiogenic factors. For dissemina-
not a single case of malignant melanoma was tion, cancer cells exploit the mechanisms that
found [50]. Another study involving patients with leukocytes utilize for extravasation into inamed
atopic dermatitis found an increased risk of mela- tissues. These are activation of selectin molecules,
noma as well as of nonmelanoma skin cancer [51]. interactions between integrins and adhesion
molecules of the immunoglobulin superfamily,
and secretion of proteinases [29].
21.7.2 Tumor-Promoting Effects Apparently, an inammatory microenviron-
of Allergies ment is essential for tumor progression, but vice
versa, tumors themselves also secrete inamma-
The positive association between specic types of tory mediators which recruit leukocytes and
cancer and allergies is mainly explained by exem- mediate inammation [38, 60]. Accordingly,
plary description of the relationship between Dvorak described tumors as wounds that do
asthma and lung cancer. Increased susceptibility not heal [61], indicating that pathogen-induced
to inhaled carcinogens due to impaired mucociliary inammation is usually self-limiting, while
clearance and pulmonary obstruction and, above cancer-related inammation is triggered perma-
all, inammatory processes are regarded to be nently [29]. Oncogenic mutations that initiate
responsible for the increased prevalence of lung carcinogenesis may also lead to the establishment
cancer among asthmatic patients [49, 5658]. As of an inammatory environment. The activation
described before, allergic reactions go along with of the Ras oncogene by mutation, for instance,
chronic or subchronic inammation. There is leads to the expression of proteins that induce the
evidence that tumors predominantly arise at the production of inammatory mediators [38, 59].
sites of inammation and that inammatory cells The main mediator cells of tumor-induced inam-
and mediators are found in all tumors [59]. mation are tumor-associated macrophages (TAM).
Inammatory reactions are usually triggered They are able to release almost all of the cytokines
by infections. Macrophages, which have detected and chemokines required for tumor progression,
infectious agents, release chemokines that attract and their abundance has been shown to correlate
other inammatory leukocytes, such as neutrophils with a poor prognosis [29, 62].
and further macrophages. Additionally they release One of the key molecules in the connec-
cytokines which increase vascular permeability tion between inammation and carcinogen-
to facilitate migration of attracted cells into esis is the transcription factor nuclear factor
aficted tissues. Leukocyte recruitment is mediated (TNF)-B. TNF-B is an endogenous tumor pro-
by adhesion molecules and extracellular prote- moter as it is activated immoderately by carcino-
ases which relieve movement into the tissue [29]. genic mutations. In addition, it is a coordinator of
inammation by regulating expression of several statistically signicant [67]. Another case-control

proinammatory and survival factors [59, 62]. study obtained a protective effect of any allergy
on cancer development. Self-reported allergy
was inversely associated with both colon and rec-
21.7.3 Cancers Negatively Correlated tum cancer [68]. The risk of colorectal cancer
with Allergies calculated by Turner et al. was reduced by more
than 20 % among patients suffering from both
The association between a history of allergy and asthma and hay fever, and less reduction was
pancreatic cancer seems to be quite denite. Five observed among patients suffering from hay
surveys could demonstrate an inverse association. fever only [42]. A prospective study from Iowa
Holly et al. reported a decreased prevalence of involving only women noted an inverse correlation
any self-reported allergy among pancreatic cancer for allergy in general which was the strongest in
patients. This correlation was available for patients with skin allergies. Moreover, the risk
multiple allergens like house dust, plants, molds, was decreasing with an increasing number of
animals, and food. Furthermore, with increasing allergies [69]. Allergic rhinitis was negatively
numbers of allergies and increasing severity of associated with rectum cancer among Taiwanese
symptoms, the risk of cancer development patients, and the association was stronger for
decreased. It should be noted that even after males than for females [48]. Combining the
receiving a hyposensitization therapy, allergic cohorts from the Cancer Prevention Study (CPS)
patients still showed a reduced risk [63]. Hay I and II, Jacobs et al. calculated a relative risk of
fever was correlated with a reduced risk of pan- 0.83 for colorectal cancer mortality when having
creatic cancer in Turners prospective study [42]. both asthma and hay fever [70].
Eppel et al. found a risk of pancreatic cancer in Most studies agree about a decreased risk of
allergic patients that was scaled down by more tumors of the brain, specically glioma, being
than 50 %, but not for asthma patients. For males associated with atopic diseases. In a hospital-based
separately, the risk was even lower [64]. Another case-control study, the prevalence of glioma was
study that additionally investigated a possible reduced in combination with physician-diag-
association between variants in IL-4 and IL-4 nosed history of any allergy and asthma as well
receptor genes and cancer prevalence found a as with self-reported allergy to chemicals.
negative correlation for any allergy, hay fever, Meningioma risk was not associated with any
and reaction to animals. But variants in the type of allergy. In addition, the risk of acoustic
abovementioned genes were not correlated to neuroma was positively associated with hay
cancer [65]. A more recent study detected a fever, allergy to food, and allergy to other
signicantly increased survival of non-resected substances [71]. One further case-control study
pancreatic cancer patients with self-reported found hospitalized glioma cases to be less likely
allergies. In the cohort that has undergone a to suffer from asthma, as well as hay fever, atopic
resection, results were nonsignicant [66]. dermatitis, or allergy in general. Moreover, there
Cancers of the colon and rectum are less prev- was a stronger risk reduction in conjunction with
alent among individuals that show a history of use of any allergic medication like nasal spray
allergy. Several studies identied allergies to be or antihistamines [72]. Wigertz et al. contrasted
inversely associated with colorectal cancer. The the prevalence of allergy among glioma and
probability of developing colorectal cancer with meningioma cases with noncancerous individu-
any self-reported allergy in an Italian study was als. They showed a decreased risk of glioma
lowered, whereas the association was stronger among subjects with asthma, atopic dermatitis,
when allergy was diagnosed at age 35 or older. and hay fever. Treatment of hay fever with nasal
Regarding colon and rectum cancer separately, spray or eye drops was associated with lower risks
the risk of rectum cancer development was lower than non-treated disease. Meningioma risk was
than colon cancer, whereas the latter was not only reduced among atopic dermatitis patients [73].
In children having asthma, a 45 % risk reduction as eosinophils are important effector cells in
could be observed [74]. One case-control study allergic reactions [82]. A role for eosinophils in
used IgE levels for the measurement of allergy immunosurveillance of tumors was considered
besides a self-reported history of allergy. As IgE since they were observed in different tumor inl-
levels did not signicantly conrm self-reported trates. Indeed, higher numbers of tissue or blood
allergies, odds ratios for the risk of glioma devel- eosinophils correlated with better prognosis, e.g.,
opment varied but both implicated a decreased improved survival rates in lung and colon cancer
risk [75]. A few years later the same research [83, 84]. Although eosinophils might contribute
group reported similar risks for meningioma to tumor growth by release of VEGF, thereby ini-
development [32]. A more recent study con- tiating angiogenesis, in vitro and in vivo studies
rmed this with an odds ratio of 0.46 for aller- substantiated rather antitumor activities [6, 85].
gen-specic IgE levels and glioma [76]. Besides Eosinophils are recruited by secretion of IL-5
glioma and meningioma, data from the from Th2 cells and eotaxin-1, a specic chemo-
INTERPHONE study also indicate allergies to kine. Particularly IL-5 induces differentiation
protect from acoustic neuroma [77]. from CD34+ precursor cells, stimulates synthesis
of granule proteins, and activates eosinophil
effector functions [86, 87]. These effector func-
21.8 Tumor-Protecting Effects tions are mainly mediated by the release of their
of Allergies granule proteins which are highly toxic toward
pathogens, as well as toward tumor cells. In vitro
The majority of the presented studies attribute studies could prove the direct cytotoxicity of
negative associations between allergies and cancers eosinophil cationic protein (ECP) [83, 84, 87].
to an enhanced immunosurveillance among allergic ECP causes lysis of tumor cells by creating pores
patients due to a hypersensitive and hyperactive in the cell membrane [88]. Further granule
immune system. This implies that immune cells proteins like major basic protein or eosinophil
of allergic subjects are more effective in detecting peroxidase have indirect antitumor properties in
and destroying cancer cells [48, 53]. The pivotal terms of triggering the release of histamine from
cells of immunosurveillance are NK cells by virtue mast cells. Besides the IL-5 dependent activation,
of their capacity to carry out ADCC and to produce eosinophils are also responsive to specic IgE. As
IFN- [37]. There is evidence for increased numbers they express IgE receptors on their surface, binding
and activity of NK cells in subjects suffering from of IgE leads to tumor-specic antibody-dependent
asthma or allergic rhinitis [7880]. Additionally, cellular phagocytosis (ADCP) [6].
it could be proved that there is a negative correla- A study which involved lung cancer patients
tion between cancer incidence and natural cyto- investigated antitumor activities of eosinophils
toxicity which would further explain an improved in vitro. For this purpose, eosinophilia was induced
potential for immunosurveillance among allergic by IL-2 treatment in cancer patients. Eosinophils
individuals [81]. were then puried from blood samples and added
Besides the classical cells of immunosurveil- to tumor cells. ADCC and direct lysis by eosinophils
lance, other immune cells may be antitumor effec- from IL-2 treated patients were highly increased
tors as well. Below, critical cells and mediators of compared to those of non-treated patients or
allergic reactions and their possible antitumor healthy donors, which did not harm tumor cells at
activities are given. While in nonallergic individu- all [83]. This suggests that in fact there are differences
als their activity may be negligible due to low in cytotoxic potentials between allergic and nonal-
occurrences, their actions may be increased among lergic individuals. The inuence of IL-2 was to
allergic subjects, explaining a negative correlation ascribe to secondary cytokine production because
between allergies and cancer incidence. IL-2 has no direct effect on eosinophils, but stimu-
Allergic disorders are marked by increased lev- lates lymphocytes. Thus, eosinophil activation was
els of eosinophils, a condition named eosinophilia, most likely mediated by IL-5.
Another study conrmed the involvement of ator of antitumor cytotoxicity as well as phagocy-
eosinophils in antitumor immunity in methyl- tosis of tumor cells.
cholanthrene-induced brosarcoma models. Among Typical Th2 cytokines are IL-4, IL-5, IL-13,
IL-5 transgenic mice, which show increased levels and IL-10. The role of IL-5 in recruiting and acti-
of eosinophils, tumor growth and incidence were vating eosinophils has already been described.
reduced, whereas among eotaxin-decient mice, IL-10 and IL-13 exhibit rather tumor-promoting
incidence was increased. An even greater increase than antitumor activities [85, 94]. IL-4 is known
of incidence was observed in eosinophil-decient as Th2 differentiation factor and mediator of IgE
mice. This provides evidence that, at least, chemi- isotype switch in B cells [95]. However, IL-4 also
cally induced cancers may be effectively fought and shows antitumor activities. First, IL-4 induces the
inhibited in growth by eosinophils [86]. inltration of macrophages and eosinophils
IgE is the key mediator of allergic reactions. which mediate cytotoxicity toward tumor cells [96].
Binding of IgE to the high-afnity receptor FcRI Second, IL-4 is one of the most potent inhibitors
on the surface of mast cells and basophils leads to of angiogenesis by blocking migration of endothe-
ADCC, whereas binding to the low-afnity lial cells. The resulting restricted tumor growth
receptor CD23 on the surface of macrophages or could be proved for local as well as for systemic
eosinophils leads to ADCP [6]. Usually IgE is application of IL-4 in vivo [97]. Moreover IL-4
predominantly present in tissues bound to its receptor has been shown to be expressed on dif-
receptors, but in allergic patients, serum IgE lev- ferent human tumors, and immunogenicity of
els are up to ten fold higher than normal [89]. In melanoma cells could be increased by IL-4 by
addition to defense against helminths and hyper- means of enhanced MHC class II expression [98].
sensitivity toward allergens, IgE antibodies may As described, many crucial components of
also be directed against tumor Ags, thereby allergic reactions were separately shown to have
mediating antitumor activities. In vitro studies antitumor activities, but only little research has
could demonstrate IgE-mediated effector activi- been done yet to evaluate the combined effects
ties against human ovarian carcinoma cells [89, of these cells. One study evaluated growth of
90]. Furthermore, treatment of mice with IgE tar- inoculated tumor cells in mice that were sensi-
geted on tumor cells resulted in decreased growth tized against ovalbumin. Tumor cells in allergic
of induced cancer. The effect was signicantly mice showed the same proliferation rate like
stronger for IgE than for treatment with IgG. those in nonallergic mice, whereas apoptosis was
Besides the curative potential of IgE, a protective increased [99]. Consequently, tumor progression
long-term immunity against the specic tumor was decreased in allergic mice which might sup-
cells were observed as well [91]. The incidence port the relationship between allergy and some
of survival was monitored within a case-control types of cancer in humans.
study among glioma patients. Those who had
elevated levels of IgE were observed to survive
on average 9 months longer compared to patients 21.9 Concluding Remarks
with moderate or borderline IgE levels.
Additionally, elevated IgE levels were more com- Even despite extensive research, the relationship
mon among control subjects than in patients between allergies and cancer remains poorly
which might support the assumption of an antitu- understood. As there are studies which show neg-
mor capacity of IgE [92]. Among pancreatic can- ative as well as positive correlations, one has to
cer patients, IgE levels were detected to be ve take a closer look at the specic type of cancer
fold higher than in control groups, whereas levels and the location it arises. Allergies are accompa-
of other Igs were similar. Tumor-specic IgE was nied by inammatory reactions which constitute
found to mediate ADCC against tumor cells, an optimal environment for carcinogenesis, thus
whereas IgE isolated from healthy controls did promoting the development of tumors at this spe-
not [93]. Recapitulating, IgE is an effective medi- cic site. Additionally, systemic effects in terms
of enhanced immunosurveillance can likewise be 13. Romagnani S. The role of lymphocytes in allergic
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Cancer Immunology
of Transmissible Cancers 22
Katrina Marie Morris and Katherine Belov
Contents
22.1 Introduction
22.1 Introduction .............................................. 419
22.2 Canine Transmissible Venereal Cancer, an afiction primarily of vertebrate
Tumor ........................................................ 420 animals, is a disease characterised by
22.2.1 Prevalence and Transmission ..................... 420
22.2.2 Histology and Clonality ............................. 420 uncontrolled cell proliferation which frequently
22.2.3 Disease Progression ................................... 421 results in the death of the host. Cancer clones are
22.2.4 Immunology ............................................... 421 under natural selection to avoid host immune
22.3 Devil Facial Tumor Disease ..................... 422 response and resist treatment, resulting in the
22.3.1 Prevalence and Appearance ....................... 422 generation of increasingly aggressive subclones.
22.3.2 Transmission .............................................. 422 Cancers nearly always originate and spread
22.3.3 Immunology ............................................... 422
22.3.4 Do Devils Have an Impaired
within a single individual, ending either with the
Immune System?........................................ 423 elimination of the tumor or the death of its host.
22.3.5 Devils Have Low MHC Diversity .............. 423 Cancers may be triggered by contagious
22.3.6 Expression of Immunosuppressive pathogens, most commonly viruses, such as
Cytokines ................................................... 423
22.3.7 Regulation of Cell Surface MHC............... 423
human papillomavirus which can cause cervical
cancer in humans or the Jaagsiekte sheep
22.4 Comparison of DFTD and CTVT........... 424 retrovirus which causes pulmonary tumors in
22.5 Evolution of Transmissible Cancers ....... 424 sheep. However, cases in which cancer cells
22.6 Transmissible Tumors as a Cancer themselves form a pathogenic agent do occur,
Model......................................................... 425 although they are extremely rare. There are only
22.7 Concluding Remarks ............................... 426 two naturally occurring cancers able to spread
between individuals. These are canine
References ............................................................... 426
transmissible venereal tumor (CTVT) found in
dogs and devil facial tumor disease (DFTD) of
Tasmanian devils. These cancers act as a para-
site transmitting from one host to the next. While
all tumor must adapt in order to avoid their
K.M. Morris, BAnVetBioSc, PhD hosts immune response, these two tumors have
K. Belov, BSc, PhD (*) evolved to avoid immune destruction not only
Faculty of Veterinary Science, University of Sydney, from their original host but also from the
Rm 505, RMC Gunn Building (B19), Regimental Dr.
Camperdown, Sydney 2006, NSW, Australia
immunologically disparate hosts that they are
e-mail: katrina.morris@sydney.edu.au; transmitted to. These two diseases give two
kathy.belov@sydney.edu.au different perspectives on transmissible cancers

420 K.M. Morris and K. Belov
Table 22.1 Comparison of DFTD and CTVT can affect both sexes of any breed of dog [3].
Disease DFTD CTVT Metastases are rare and are most commonly
Species affected Tasmanian devil Dog, wolf, found in the lymph nodes [2]. CTVT in domestic
coyote, jackals, dogs can be successfully treated with chemother-
foxes
apy [3]. CTVT can also be induced in adult
Distribution Tasmania Worldwide
immunocompetent dogs by inoculation with liv-
Age of origin About At least
18 years ago 6,000 years ago ing tumor cells [5].
Likely cell of Schwann cell Macrophage
origin
Site of primary Face, External genitalia 22.2.2 Histology and Clonality
infection mouth or neck
Mode of Biting Coitus Histologically, CTVT is described as an undiffer-
transmission
entiated round-cell neoplasm of histiocytic origin
Frequency of 65 % 7%
metastases [6]. Cytologically, CTVT cells do not have many
Mortality 100 % Rare distinctive ultrastructural features [7]. CTVT has
Effect on host Decline of host Little or no effect been proposed to be of macrophage lineage based
population population of on its expression of several macrophage charac-
80 %, extinction teristic proteins [8] and its ability to be parasit-
in wild likely
within 30 years
ised by Leishmania infantum [9], a parasite
Treatment Surgical excision Chemotherapy usually infecting macrophages. The transmissi-
if treated early bility of CTVT cells has been demonstrated by
studies which found that the disease can be
induced by transplanting live cells, but not killed
cells or cell ltrates [3]. The chromosome num-
and give us unique insights into the immunology ber of CTVT (5759 chromosomes) is consistent
of cancers. These cancers provide an ideal model across geographically dispersed samples and is
for studying the battle between tumor and host. different to the normal number of dogs (76 chro-
For a comparison of CTVT and DFTD, see mosomes) [1012]. CTVT genomes share chro-
Table 22.1. mosomal duplications and deletions which are
not found in the dog genome [13]. Transmissibility
has been further supported by the presence of a
22.2 Canine Transmissible LINE insertion near the c-myc gene which is
Venereal Tumor found in all CTVT tumor studied to date but is
not found in the normal dog genome [14].
22.2.1 Prevalence and Transmission Recently clonal transmission of CTVT has been
conrmed by molecular genetic studies. Rebbeck
Canine transmissible venereal tumor is a et al. [13] and Murgia et al. [15] found that the
contagious neoplasm found in domestic dogs [1]. pattern of microsatellite polymorphism strongly
The disease is found worldwide, but is mostly suggests a monophyletic origin. The most recent
prevalent in stray dog populations [2]. While it is common origin of CTVT may have been rela-
most commonly found in dogs, it can be trans- tively recent, predicted to be between 47 and
mitted to a wide range of canine species includ- 470 years [13] or between 250 and 2,500 years
ing wolves, foxes, jackals and coyotes [3]. In [15]. However, the date of CTVT origin is
some regions, such as Japan, it is the most com- ancient, predicted to have occurred at least
mon tumor found in dogs [4]. Transmission 6,000 years ago in either the dog or wolf and may
occurs by transplantation of viable tumor cells have predated the domestication of dogs [13].
during coitus [3]. The tumor establishes on the This makes CTVT the oldest known malignant
external genital mucosa of the infected dog and cell line.
22 Cancer Immunology of Transmissible Cancers 421
22.2.3 Disease Progression have selective mechanisms for downregulating

MHC class I molecules to escape recognition by
In experimental transplantation, the disease has a CD8+ cells [20]. CTVT cells express none, or
predictable growth pattern featuring three distinct very few, MHC class I and II molecules during
stages of progressive growth, stable growth and the progressive phase [21, 22]. Additionally they
then regression [3]. The initial progressive phase do not express 2M, a component of MHC class I,
lasts several weeks and is characterised by rapid on the cell surface [23].
tumor growth with a doubling time of about The initial lack of cell surface MHC should
47 days [16]. During the stable growth phase, result in cell destruction by natural killer (NK)
tumor expansion slows [16]. During regression, cells [24]. However, migration of NK cells to the
the tumor shrinks and eventually disappears [16]. tumor is impaired due to tumor expression of
Spontaneous regression is associated with an TGF-1 [25]. TGF-1 is a potent immunosup-
intense local lymphocytic inltration [17]. The pressive cytokine which commonly plays a role
number and size of tumor-inltrating lymphocyte in immune avoidance in cancers [26]. TGF-1 is
subpopulations vary with CTVT growth phase expressed in high concentrations in CTVT
[17]. In natural transmission, the disease will usu- tumors where it suppresses the killing activity of
ally regress after 69 months of growth, unless tumor-inltrating lymphocytes (TIL) [25].
treated earlier [2]. Experimental transplantation Natural killer cells, which migrate to the tumor
to immunosuppressed dogs results in tumors that due to the lack of cell surface MHC expression,
do not regress [18]. Additionally, the host is are impaired by TGF-1 [25]. In addition, the
immune to subsequent reinfection after remis- function of dendritic cells (DCs) is impaired with
sion, and offspring of infected mothers are par- inhibited antigen uptake and presentation,
tially protected from infection [19]. These impaired differentiation and apoptosis of mono-
ndings demonstrate that host immune response cytes and DCs [27].
is involved in tumor regression and protection Host expression of interleukin-6 (IL-6)
from subsequent reinfection. appears to be critical in forcing the tumor into
regression. At the onset of regression, expression
of IL-6 by TILs is increased, antagonising the
22.2.4 Immunology activity of tumor TGF-1 [25]. By downregulat-
ing TGF-1, the ability of interferon (IFN-) to
CTVT is initially capable of downregulating host promote MHC class I and II expressions is
immune response, but in the majority of cases, it restored [28]. IFN- and IL-6 work synergisti-
is eventually overcome by the host defences [2]. cally to enhance MHC expression [28]. It has
Regression of CTVT involves both humoral and been postulated that IFN- induces expression of
cell-mediated immunity. Rejection of foreign tis- an MHC class II transactivator, resulting in an
sue is initiated by the presence of major histo- increased MHC class II expression [28]. This
compatibility complex (MHC) antigens (Ags) on results in the attraction of CD4+ cells which pro-
the surface of foreign cells. MHC Ags present mote the generation of antibodies (Abs) against
peptides to T cells. There are two classes of MHC CTVT, driving tumor rejection and the subse-
antigen. Class I peptides are recognised by CD8+ quent immunity against it. Host IL-6 may also
T cells, while class II peptides are recognised by enhance T cell cytotoxcity when MHC molecules
CD4+ T cells. Cells without MHC, mutated or are expressed [25]. Additionally, during regres-
foreign MHC, or MHC presenting abnormal sion TILs secrete a heat-sensitive factor, enhanc-
peptides can trigger an immune response; there- ing MHC class I and II expressions [29]. At
fore, regulation of MHC is important for cancers commencement of regression, 3040 % of cells
to escape host immunosurveillance. CTVT has express both class I and II MHCs [21, 22]. DC
the additional distinction in that it is capable of activity is substantially recovered during
transmitting across MHC barriers. Many tumors regression [27]. Expression of IL-6 and the
re-establishment of DCs are believed to be the may occur due to starvation or complications from
critical factors in initiating tumor regression. metastases [37]. The tumors are undifferentiated
There is evidence that humoral immunity is soft tissue neoplasm, believed to be derived from
also involved in regression. Treating CTVT- Schwann cell originator cells [38]. Metastases
infected dogs with the sera of post-regressive occur in around 65 % of cases [36].
dogs caused regression, while dogs simultaneously
given CTVT and immune serum did not develop
the disease [18]. Antibodies to CTVT have been 22.3.2 Transmission
found in dogs after CTVT regression [30]. B lym-
phocytes and plasma cells appear in higher con- DFTD was discovered to be a transmissible
centration in regressive than progressive tumors allograft from karyotypes of the tumors and hosts
[17]. Additionally, Liao et al. [31] detected a [39]. Similar to CTVT, DFTD samples have a
CTVT-secreted factor which was specically conserved karyotype which is distinct from the
cytotoxic to B cells. normal devil karyotype [39]. The DFTD karyo-
type is highly rearranged with the absence of
both copies of chromosome 1, one copy of chro-
22.3 Devil Facial Tumor Disease mosome 5 and both sex chromosomes [39, 40].
Clonality has been conrmed by genotyping
22.3.1 Prevalence and Appearance which has shown that DFTD specimens taken
from different individuals are identical to each
Tasmanian devils are the worlds largest marsu- other, but usually different to their hosts, at
pial carnivore since the extinction of the several microsatellite markers as well as MHC
Tasmanian tiger in the early twentieth century. genes [41]. Further support for clonality comes
Devils were once widespread on mainland from next-generation sequencing [42]. The
Australia, but today are restricted to the island of disease is spread by biting. Devils bite one
Tasmania. Although several population crashes another frequently when ghting over food and
have been reported over the last two centuries, territory or during mating [43]. DFTD cells are
Tasmanian devils were classied as a species of transferred when a devil bites into the tumor of a
least concern prior to the outbreak of DFTD with diseased devil [44]. DFTD cells can then establish
a population of around 150,000 animals. DFTD into a tumor around wounds in the mouth or face
was rst witnessed in 1996 by a wildlife photog- of the new host [39, 45].
rapher in Mount William National Park in the far
north-east of Tasmania [32]. Since then, the dis-
ease has spread rapidly across the state, with the 22.3.3 Immunology
disease found in 85 % of the devil distribution as
of 2012 [33]. The disease is projected to have While much is known about the immunology and
spread to the entirety of devil distribution by pathology of CTVT, very little is known about
2016 [34]. Since its emergence, DFTD has wiped the same aspects of DFTD. Unlike CTVT, DFTD
out over 80 % of the devil population [34], and cells pass from one animal to another without
unless acted upon, the devil is expected to be provoking an immune response [46]. Both tumor
extinct within 30 years [33]. This had led to and host characteristics have been hypothesised
devils being listed as endangered by the IUCN as to be responsible for the ability of this cancer to
well as national and state authorities [35]. spread and go undetected by the host immune
DFTD appears as tumors mostly around the system. It has been suggested that an impaired
head and neck of the devil [36]. After appearance immune system and a lack of genetic diversity, in
of the rst lesions, death usually occurs within particular at MHC genes, may make the devil
6 months [36], and there have been no veried population susceptible to the spread of DFTD
cases of devils having survived the disease. Death [47]. However, it is likely that the tumor itself
actively avoids immune detection. Both down- spread has been questioned by recent studies.
regulation of cell surface MHC and the expres- Kriess et al. [53] conducted skin grafts in devils
sion of immunosuppressive factors have been and found that even MHC similar hosts were
investigated. Each of these host and tumor char- capable of rejection. This suggests that minor
acteristics will be discussed below. histocompatibility Ags may play a role in allograft
rejection. Most recently Lane et al. [54] found no
link between MHC diversity and susceptibility to
22.3.4 Do Devils Have an Impaired DFTD, suggesting that MHC is not critical to the
Immune System? disease spread. However, the lack of genetic
diversity in devils may still be responsible for the
Soon after the emergence of DFTD, it was spread of this disease. There is likely to be low
hypothesised that the spread of this disease may diversity at other key immune genes such as
be enabled by an impaired devil immune system minor histocompatibility Ags and genes of the
[47]. Tasmanian devils are known to be highly innate immune system, which may have a role in
susceptible to neoplasms [48]. However, research disease susceptibility.
over the last decade has demonstrated that devils
have a robust immune system functionally
equivalent to other marsupial and eutherian 22.3.6 Expression
immune systems. Their immune tissue architec- of Immunosuppressive
ture and immune cell distribution are similar to Cytokines
that seen in eutherians; their lymphocytes prolif-
erate in response to mitogen stimulation, and Expression of immunosuppressive cytokines is
subcutaneous injection of a cellular Ag produces found in many cancers and allows the cancers to
a strong antibody response [46, 49]. Additionally, avoid host detection and destruction of tumor cells
NK-cell responses have been demonstrated [50]. by suppressing the host immune response. As dis-
Therefore, with a robust immune response, it is cussed, the expression of TGF1 by CTVT cells is
unlikely that the absence of immune response to involved in preventing NK-cell response [25].
DFTD is due to a lack of functionality in the However, it has been recently shown that TGF1
devils immune system. as well as three other cytokines commonly
expressed by cancers to downregulate immune
detection are not over-expressed in DTFD tumors
22.3.5 Devils Have Low MHC compared to control tissues [55]. This includes
Diversity VEGF-A, IL-6 and IL-10. It therefore appears that
in DFTD, unlike CTVT, suppression of the
Devils lack genetic diversity across the genome immune system by release of immunosuppressive
[42, 51] as well as at MHC class I and class II cytokines does not play a key role in the pathology
genes [41, 52]. Having an important role in the of DFTD [56].
recognition of both cancerous and foreign cells,
MHC class I presentation should be critical in the
recognition of DFTD cells by the devils immune 22.3.7 Regulation of Cell
system. However, this recognition may be Surface MHC
impaired if MHC diversity is so low that it impairs
the host immune system from recognising these Like CTVT, regulation of cell surface MHC may
cells as foreign. Siddle et al. [41] found that dev- allow DFTD to avoid rejection. Recent research
ils have critically low MHC diversity and sug- has shown that DFTD cells express functional
gested that this was the rst link between a lack of MHC class I and class IIB RNA transcripts, but
MHC diversity and the spread of disease. little or no transcripts for genes involved in Ag
However, the role of MHC diversity in DFTD processing including B2M, TAP, MHC class IIA
and DMB [56]. This study found only trace cell surface [57]. However, TGF1 is not upregu-
amounts of MHC I proteins at the surface of lated in DFTD cells [55]. Therefore, it is yet to be
DFTD cells both in vivo and in vitro [56]. These seen how DFTD cells avoid NK-mediated
ndings may explain how DFTD cells evade rec- destruction.
ognition by T cells, though further work is needed With only two naturally occurring transmissi-
to build a full picture of how DFTD cells avoid ble cancers worldwide, it is somewhat surprising
immune recognition. that these diseases do not occur more frequently.
There are several commonalities between CTVT
and DFTD which appear to be signicant factors
22.4 Comparison of DFTD making their host species susceptible to this form
and CTVT of disease. Firstly, for such a disease to occur, a
route of transmission must be present. In both
DFTD and CTVT are the only naturally occur- species, transmission of cancer cells appears to
ring clonally transmissible cancers. Over the last occur at the site of tissue damage. Devils biting
decade, our understanding of the pathology and behavior [43] and the extended, rough copulation
immunology of these diseases has greatly devel- that occurs in dogs [57] provide routes of trans-
oped, but much is still unknown. Further research mission for the transfer of tumor cells. The prob-
into their origin, evolution and immunology will able low genetic diversity in both of the original
not only provide insights into transmissible can- host populations is another commonality.
cers, but may also have medical applications to Tasmanian devils have low genetic diversity, par-
human cancers. These two diseases differ in ticularly at MHC genes [41, 51], while it has
many aspects of their pathology and immunol- been hypothesised the CTVT arose in an inbred
ogy. However, they also share features in com- wolf population due to homozygosity at a num-
mon which may help reveal circumstances ber of loci in CTVT [15]. Additionally, a sponta-
favoring the generation of such diseases. neously arising sarcoma was found to be
While CTVT is an ancient disease having transmissible among a colony of laboratory
been around for at least 6000 years, DFTD is a Syrian hamsters which also had low MHC diver-
very new disease less than 20 years old. This sity [58]. A further trait which may predispose
allows us to compare a transmissible cancer populations to disease is susceptibility to neo-
which has been through thousands of years of plasms. Devils are naturally highly susceptible to
co-evolution with its host species to one which neoplasms [48]; however, this is not a trait shared
has recently emerged. CTVT, as a successful par- by dogs or wolves. If the presence of all these
asite, has likely undergone selection to become a factors is indeed required for the development of
benign tumor which does not kill its host popula- transmissible cancers, this may explain the rarity
tion, resulting in the low rate of metastases and of such diseases. However, with wildlife species
the characteristic regression. On the other hand, increasingly losing diversity due to anthropo-
the newly emerged DFTD results in 100 % mor- genic effects, the chance of seeing similar disease
tality and may drive its host population to extinc- occurring in wild vertebrate species may be
tion. Immunologically there are both similarities increasing.
and differences in how these diseases avoid host
immune rejection. CTVT initially has low or no
expression of MHC classes I and II to avoid 22.5 Evolution of Transmissible
rejection by T cells, and the expression of host Cancers
TGF1 appears to be important in preventing
NK-mediated destruction of tumor cells [25]. A further intriguing aspect of DFTD is that it
Similarly, recent evidence has shown that DFTD allows us to observe the evolution of a transmis-
has low or no expression of MHC class I on the sible cancer in real time. DFTD, as a clonal cell
line, has already gone through two stages of 22.6 Transmissible Tumors
adaptation: one in order to establish as a tumor in as a Cancer Model
its initial host and a second stage of adaptation in
order to be capable of transmission to other hosts. CTVT and DFTD provide in vivo models for
The disease is now going through further evolu- studying the battle between tumor and host
tion as it spreads through the population. A num- immune response. CTVT has been used as a
ber of strains have been identied based on model for human cancer since at least 1980 [63,
karyotype [59]. Although the functional signi- 64]. As these cancers have been propagated
cance of these changes is unknown, when cul- through many hosts over many years, they have
tured, the strains display different morphology had a long exposure to host immunosurveillance,
and growth rates [59]. These changes provide thus providing insight into the evolutionary strate-
variation which selection can act on. The pres- gies developed by cancers to evade immune
ence of devils with distinct genotypes at the dis- recognition. The strategy of immune evasion
ease front [60], some of which may offer partial employed by CTVT has many features in com-
or full resistance to the disease, may provide a mon with many human tumors including the regu-
strong selective force to the disease. Another pos- lation of MHC expression and expression of
sibility is that the disease may adapt to become immune-modulating cytokines. Thus, CTVT
more benign and slow growing. Devils which can provides an excellent model for studying these
survive longer with the disease have the capabil- features. Additionally, CTVT is one of the only
ity of infecting more devils, possibly resulting in in vivo models for studying tumor regression,
the evolution of a more benign form of DFTD. A allowing investigation into the mechanism
similar adaptation may have occurred early in the through which host immune system overcomes
history of CTVT. However, it is also possible that tumors. Understanding how the tumor controls
the modern characteristics of CTVT were present host immune response and how the host
at its origin and have gone through little forces regression of the tumor could be useful
adaptation since this time. in the development of cancer immunotherapy
Fassati and Mitchison [61] rst suggested that approaches in human patients. Following the dis-
epigenetics must be involved in the regulation of covery that host expression of IL-6 antagonises
CTVT. Although epigenetics has yet to be inves- the effects of tumor-expressed TGF1 leading to
tigated in CTVT, several studies are beginning to regression, Chou et al. [65] found that a combina-
suggest that epigenetics may have a role in the tion of IL-6 and IL-15 could induce regression in
regulation and evolution of DFTD. Increased progressing CTVT tumors. Such a therapy may be
expression of the DNA methyltransferase 1 gene useful in human cancers which also produce TGF-
in DFTD cells has been observed resulting in 1 to suppress host immune response [65]. CTVT
hypermethylation [62]. This results in different and DFTD are similar to a number of rare cancers
patterns of gene silencing in different DFTD that can be transmitted between humans and may
tumors, providing variation on which selection provide a model for these diseases. In humans, the
can act. One such area that selection is likely to most comparable diseases are cases of malignan-
act is in expression of MHC. Siddle et al. [56] cies vertically transmitted during pregnancy.
have shown that the regulation of Ag processing Mother to foetus transmissions of melanoma,
proteins, which enable DFTD cells to evade the lymphoma, leukemia and carcinomas have all
immune system, is controlled by epigenetic been reported. Parallels exist between the forma-
mechanisms. This may mean that regulation of tion of a fetus and transmissible cancers. The
MHC can vary depending on circumstances. This semi-allogenic fetus downregulates cell surface
provides a mechanism for ne tuning of the MHC class I but upregulates the nonclassical
immunology of the cancer cells, allowing DFTD HLA-G to avoid destruction by NK cells [66],
to adapt to immunologically disparate hosts. thus using a similar strategy to avoid immune
rejection as is used by both CTVT and DFTD. Fetal References

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Envisioning the Application
of Systems Biology in Cancer 23
Immunology
Julio Vera, Shailendra K. Gupta, Olaf Wolkenhauer,

and Gerold Schuler
Contents
23.1 Introduction
23.1 Introduction .............................................. 429
23.1.1 The Omics Paradigm and the Use
of Statistical Models .................................. 430 Biomedicine has evolved extremely fast in the
23.1.2 Mathematical Modeling and Systems last decade. Many challenging new insights into
Theory: Dissecting the Complexity the nature of biological systems and the avenue
Emerging Out of the Structure
of new experimental techniques have synergized
of Biochemical Networks .......................... 431
23.1.3 Bridging Biological Scales Through during this period to change our perception
the Integration of Biological Data about biomedicine. Biological systems are now-
in Multi-scale Models ................................ 431 adays envisioned as complex networks com-
23.2 One Step Further: Integrating posed of dozens to thousands of proteins, genes,
the Different Perspectives of Systems and miRNAs, which interact to control cellular-
Biology into a Unied Framework ......... 431 and tissue-level phenotypes. One can say that
23.3 Does Cancer Immunology Need biology is the science of the ultimate complex-
a Systems Biology Approach? ................. 434 ity because in one sense every single cell
23.4 A Quick View on Current Results .......... 434
23.4.1 Computational Biology, Bioinformatics,
and High-Throughput Data Analysis Used
in the Design of Immune Therapies
for Cancer .................................................. 434 S.K. Gupta, PhD
23.4.2 Mathematical Models Used in Basic Department of Systems Biology and Bioinformatics,
Oncology Research .................................... 440 Institute of Computer Science, University of Rostock,
23.5 Concluding Remarks ............................... 446 Rostock 18051, Germany
References ............................................................... 447 Department of Bioinformatics, CSIR-Indian Institute

of Toxicology Research, Lucknow, India
e-mail: shailendra.gupta@uni-rostock.de
O. Wolkenhauer, PhD
Department of Systems Biology and Bioinformatics,
J. Vera, PhD (*) Institute of Computer Science, University of Rostock,
Laboratory of Systems Tumor Immunology, Rostock 18051, Germany
Department of Dermatology, University Hospital e-mail: olaf.wolkenhauer@uni-rostock.de
Erlangen, Hartmannstr. 14, Erlangen, Germany
G. Schuler, PhD
Department of Dermatology, Faculty of Medicine, Department of Dermatology, Faculty of Medicine,
Friedrich Alexander Universitt, University of Friedrich Alexander Universitt, University of
Erlangen-Nurnberg, Erlangen-Nurnberg,
Ulmenweg 18, 91054, Erlangen, Germany Ulmenweg 18, 91054 Erlangen, Germany
e-mail: julio.vera-gonzalez@uk-erlangen.de e-mail: gerold.schuler@uk-erlangen.de

430 J. Vera et al.
contains as much complexity as entire solar sys- has given rise to several new experimental elds
tems or galaxies. In this context of increasing (e.g., genomics, transcriptomics, proteomics, and
complexity, systems biology has emerged a metabolomics, collectively known as omics
decade ago. techniques). When applied to samples obtained
Systems biology is a methodological approach from large cohorts of patients suffering complex
that combines quantitative experimental data, multifactorial diseases, especially cancer, these
mathematical modeling, and other tools from techniques have already generated massive
computational biology to address biological and amounts of clinical and biomedical data. These
biomedical questions from a systemic perspec- data are a precious resource to discover the
tive. It is almost a mandatory research strategy molecular mechanism behind the emergence of a
when: (a) analyzing massive amounts of high- disease. From an applied perspective, these tech-
throughput quantitative experimental data, (b) niques can be used to generate new protocols and
trying to understand the function and regulation tools for early diagnosis or more efcient and
of biochemical networks enriched in regulatory personalized therapeutic treatments. However,
motifs like feedback loops, and (c) integrating the data alone are not sufcient: human intuition
biological data from diverse sources across tem- and direct interpretation are not well-suited tools
poral and spatial scales. Within the methodology, for the analysis of massive volumes of high-
the use of mathematical modeling is an essential throughput data. Complex mathematical models,
step, necessary to integrate and analyze data, for- which rely on the intensive use of advanced sta-
mulate and explore biological hypothesis, or per- tistical and computational methods, are necessary
form quantitative predictions with a therapeutic to interpret and analyze the amount and type of
aim [1]. It has a clear interdisciplinary nature data generated through the omics paradigm.
because it involves expertise in biomedicine, These statistical models have been success-
quantitative experimental techniques, data engi- fully exploited in the search of biomarkers for
neering, mathematical modeling, and bioinfor- cancer progression, metastasis, or resistance [2].
matics, only to mention some of the scientic In this case, patients in a clinical study are clas-
proles of researchers that can get involved in a sied in groups according to the progression sta-
systems biology project. tus of the tumor. Expression proles of proteins,
Due to this multiplicity of disciplines, over the RNAs, or other biomolecules, obtained from
years the concept of systems biology has become patient samples, are analyzed using statistical
fuzzy and difcult to dene precisely. At the models to nd one or more disease-associated
moment, systems biology describes at least three genetic signatures. These genetic signatures
different approaches, all of them relying on the account for groups of genes having an expres-
use of quantitative experimental data and mathe- sion pattern that, considered globally, can be
matical modeling. They are briey described in used to discriminate between patient groups. The
the following subsections. ultimate aim is to use these genetic signatures
for improving diagnosis and/or prognosis. For
some tumor entities, genetic signatures have
23.1.1 The Omics Paradigm been already found that could be successfully
and the Use of Statistical associated with progression and are currently
Models used in prognosis tests [3, 4]. However, one has
to say that the statistic elucidation of this kind of
In the last few years, it has become technically signatures should never be the end point of a
and economically affordable to perform quantita- research process. It has to be followed by addi-
tive, high-throughput experiments to measure the tional in vitro/in vitro experiments and clinical
concentrations or activation state of proteins and studies to nd a mechanistic interpretation for
other biomolecules like RNAs or metabolites. This them [5].
23 Envisioning the Application of Systems Biology in Cancer Immunology 431
23.1.2 Mathematical Modeling 23.1.3 Bridging Biological Scales

and Systems Theory: Through the Integration
Dissecting the Complexity of Biological Data in Multi-
Emerging Out of the Structure scale Models
of Biochemical Networks
Evidences are growing in recent years pointing to
Accumulating experimental evidences indicate the fact that, in many cases, the inuence of the
that, at the molecular level, cells are organized surrounding media in the tumor cannot be sepa-
in large and complex regulatory networks that rated from the tumor biology [14]. The microen-
involve genes, interacting proteins, different vironment interacts with the tumor and affects its
kinds of coding and non-coding RNAs and progression via a number of selective forces
metabolites. When trying to nd a mechanistic including hypoxia, lack of nutrients, or immune-
interpretation for the behavior behind these driven apoptosis, while the tumor can modify the
large networks, simple human intuition and features of its microenvironment to subvert the
direct data analysis fail because they involve bodys protective mechanisms [15]. This notion
too many interacting variables [1, 6]. is the motivation behind the many efforts to
Furthermore, these networks contain a plethora develop data-driven mathematical models of can-
of cross-talking regulatory motifs, like feed- cer progression, able to account for the spatial
back and feedforward loops that show often organization of tumors and the interaction with
counterintuitive behavior. In engineering and the surrounding microenvironment [16]. The so-
physics, mathematical modeling has been used called cancer multi-scale models are mathematical
for a century to investigate the dynamics, regu- constructs that are able to simulate global spatio-
lation, and controllability of other physical or temporal features of tumors like growth, angio-
articial systems containing similar regulatory genesis, as well as therapy- or hypoxia-mediated
motifs. It is therefore not a surprise that bio- apoptosis and necrosis [17].
logical data-based mathematical modeling has
emerged as a powerful tool, able to dissect the
nature of biochemical networks, interpret the 23.2 One Step Further:
complex nonintuitive relations between their Integrating the Different
compounds, and provide support in the design Perspectives of Systems
of hypothesis and experiments. This strategy Biology into a Unied
has been used with remarkable success in the Framework
last years in molecular oncology and cancer
signaling. It has proved to be useful in: (a) the Although each one of these mathematical model-
detection and analysis of the nonlinear behavior based approaches has proved to be quite success-
emerging from the combination of feedback, ful in accelerating the discovery in tumor basic
feedforward, and other regulatory motifs in biology and clinics, they have limitations that
biochemical networks [7, 8]; (b) the integration cannot be ignored. Statistic models are extremely
of diverse sources of high-throughput data useful tools to analyze enormous amount of
accounting for the regulation and dynamics of patient data and nd expression patterns associ-
large cross-talked biochemical networks, with ated to given clinical phenotypes; however, those
hundreds of compounds [9]; (c) the derivation, statistical expression patterns alone suffer with
analysis, and validation of hypotheses concern- the lack of support provided by a truly mechanis-
ing the structure and regulation of cancer- tic interpretation of the data, the sort of analysis
related pathways [10, 11]; or (d) the design and that provides biological causation. Mathematical
assessment of conventional, targeted, or com- models of biochemical networks can pro-
bined anticancer therapies [12, 13]. vide insights into the biological mechanisms
432 J. Vera et al.
underlying cancer progression, but are not able to gene expression vs. the progression status
account for the effects of the tumor-microenvi- proles. In this way, one can obtain cancer-
ronment interaction. Current multi-scale models associated genetic signatures relevant to the
are accurate describing biomechanical forces, phenotype under investigation (e.g., chemore-
cell phenotypes, and spatial interactions between sistance, aggressiveness, metastatic potential).
tumor cells and their surroundings. However, These signatures account for a group of genes,
they lack a precise description of the intracellular proteins, miRNAs, or other molecules, for
mechanisms driving those phenotypic features, which a robust statistical correlation is found
as well as a connection to the clinical understand- between their combined expression pattern and
ing of the tumor biology. the investigated cancer phenotype [5].
These limitations are the motivation why STEP 2. Relevant biomedical and clinical
researches have tried to integrate the different knowledge is gathered from databases, com-
scopes into a unied conception of systems putational algorithms, and publications
biology in recent years [1821, 8]. The idea is to inspected via manual curation or text mining.
develop a unique framework that integrates tools This information is used to nd feasible bio-
and methods from statistics, bioinformatics, chemical interactions (i.e., protein-protein
computational biology, and mathematical model- interactions, transcriptional regulation, etc.)
ing with the aim of integrating biomedical data between compounds of the genetic signature,
across biological and spatiotemporal scales. This but also with other kinases, transcription fac-
approach must be able to: (a) link massive clinical tors, or microRNAs, all of them relevant to
patient data with the function and (dis)regulation the investigated cancer phenotype. In this
of biochemical networks; (b) provide a strategy way, we can construct a network of cross-
to combine different kinds of quantitative high- talked intracellular pathways relevant to the
throughput biological data into integrative investigation of the aimed cancer pheno-
pictures of cancer; (c) connect cancer genotypes types. Furthermore, similar networks can be
and phenotypes from a mechanistic, causal, data- constructed for the cell types in the tumor
driven perspective; (d) provide tools to detect and microenvironment related to the phenotype
investigate regulatory, feedback loop-like struc- investigated. Since tumor cells and cells in
tures that extend across multiple biological orga- the microenvironment secrete cytokines and
nization levels like paracrine and autocrine loops; other molecules signaling each other, the
and (e) determine the consequences of this multi- obtained network is one of cell-to-cell com-
level cross talk in the context of cancer and the munication, accounting for the tumor-
immune response. In our vision, this ultimate microenvironment interaction in the cancer
version of the systems biology method involves phenotype under investigation. The network
iterative integration of data from clinical trials obtained is often called regulatory map, noth-
and in vitro/in vivo biomedical research using ing but a visualization of the state of the art
techniques of data analysis, bioinformatics, and of the biochemical and biomedical knowl-
mathematical modeling and simulation. The pro- edge about the cancer phenotype under
posed workow is sketched in the following investigation. Tools from network biology
paragraphs (Fig. 23.1). can be used to dissect the topology of the
STEP 1. In clinical cohorts of, for example, cancer regulatory map and isolate regulatory motifs
patients vs. healthy individuals, high- relevant for the derivation of hypothesis and
throughput data of tissue and/or plasma con- experiments [22, 8].
centrations for proteins, RNAs, or other STEP 3. The parts of the network relevant to the
molecules are collected together with biometric biomedical scenarios which are related to the
data from the patients. The data are processed, investigated cancer phenotype are translated
integrated, and analyzed using statistical mod- into a mathematical model. The model consists
els aiming to group them according to their of mathematical equations, in an adequate
Fig. 23.1 Sketch of an advanced systems biology workow
modeling formalism, accounting for the time STEP 4. Additional quantitative in vitro/in vivo
evolution of the expression and/or activation experimental data are used to improve the bio-
status of the network compounds, as well as logical characterization of the model, that is,
their connection to the phenotypes. Many to make it more accurate in terms of predic-
modeling formalisms are available, all of tion of the relevant biomedical scenarios. This
which are with advantages and disadvantages is often called model calibration and allows
[6]. To circumvent some of these disadvan- assigning appropriate values to model param-
tages, one can combine them in hybrid models. eters and other model features. Alternatively,
For example, we have combined intercon- this process also allows for the validation of
nected sub-modules in ordinary differential hypothesis concerning the structure and regu-
equations and Boolean logic [23]. Ordinary lation of the network in the biomedical con-
differential equations are excellent tools to text analyzed; in this case, iterative cycles of
analyze the nonlinear behavior of signaling modeling and experimentation can be used to
pathways with multiple, nested feedback and formulate, rene, prove, or disprove hypothe-
feedforward loops, while logic models are an sis concerning the existence and relevance of
ideal representation of massive transcriptional given biochemical interactions [24]. With the
networks. The combination of both model use of the mathematical model, one can ana-
types allows the analysis of large-scale, non- lyze spatiotemporal regulatory features of the
linear transcriptional and posttranscriptional network that elude the elucidation via conven-
networks and their connection to cancer cell tional experimentation, like self-sustained
phenotypes [23]. oscillations, or bistability.
434 J. Vera et al.
STEP 5. In recent years, various studies have system secrete cytokines and antibodies (Abs)
proved that a well-calibrated, data-driven targeting the tumor cells. In addition, features of
mathematical model can be used with predic- the microenvironment in which the tumor is
tive purposes in the context of molecular hosted can affect the response of the immune
oncology. The underlying idea is to use model cells. Finally, all these processes are happening at
simulations and other tools to assess existing the same time and affecting each other at differ-
therapies in a personalized manner, design ent biological and temporal scales. Altogether,
new therapies, or detect sets of biomarkers for this suggests the use of a systemic strategy to
cancer prognosis. In a nal step, one has to go tackle the complexities of the tumor-immune
back to the bench and design additional system interaction. In the following section, we
in vivo/in vitro experiments to conrm the discuss some published results that illustrate how
model predictions. Alternatively, the model systems biology can be used in the context of
predictions can be combined with virtual oncology and tumor immunology.
screening and other techniques from computa-
tional biology and immunoinformatics and
used in the process of drug discovery or vaccine 23.4 A Quick View on Current
development. For example, potential drug tar- Results
gets, identied via mathematical modeling,
can be used as most promising candidates in a 23.4.1 Computational Biology,
drug screening procedure via protein docking- Bioinformatics, and High-
based techniques [21]. Throughput Data Analysis
Used in the Design of Immune
Therapies for Cancer
23.3 Does Cancer Immunology
Need a Systems Biology The availability of next-generation sequencing
Approach? along with omics data shifted the paradigm for
cancer treatment and opens the doors toward pos-
In our opinion, the immune system is one of the sible cancer immunotherapy. Like traditional
most complex realizations of a biological system. vaccines that stimulate the host immune system
The immune system is actually a multi-scale sys- to recognize and destroy pathogens, cancer vac-
tem (Fig. 23.2). It involves many types of cells, cines are aimed to generate an immune response
whose fate, proliferation, or activation status is that differentiates tumor cells from the normal
controlled by feedback loop-regulated pathways. cells for their possible elimination. For several of
These pathways very often cross talk creating the pathogenic cancers, such as cervical cancer
complex networks. Furthermore, the activation caused by human papillomavirus; hepatocellular
status of given immune cells depends on other carcinoma caused by hepatitis B and hepatitis
immune cells by direct contact or through secre- C virus; Hodgkin lymphoma by Epstein-Barr
tion of local or global signaling molecules, espe- virus; T-cell leukemia by human T-cell leukemia
cially cytokines. In this way, the immune system virus; and Kaposis sarcoma by Kaposis sar-
is enriched in cell-to-cell communication circuits coma herpes virus, there has been considerable
and autocrine loops. When we further consider success in designing cancer vaccines in the
the interaction between the immune system and a past, and many of them are currently in use or in
tumor, the picture becomes more systemic-like. the advanced stages of clinical trials. Most of
Tumor cells and the immune cells in the these vaccines are designed in a similar way as
surroundings communicate through chemical the traditional epitope-based vaccine-designing
signals and affect each others fate. Tumors approaches. However, for the non-pathogenic can-
secrete antigens (Ags) detected by immune cells cer, the major challenge for the immune system is
like dendritic cells, while cells from the immune to distinguish cancer cells from the healthy cells
Fig. 23.2 Tumor-immune cells interaction envisioned as global signaling molecules. Furthermore, the immune
a multilevel system. The immune system involves many system and the tumor are affected by cell-to-cell commu-
types of cells, whose fate is controlled by feedback loop- nication circuits involving tumor antigens and immune
regulated pathways, but also some immune cell types cell-secreted antibodies and cytokines
affect the activation of others by direct contact or local/
in order to activate B lymphocytes to produce self-Ags to which the immune system is

Abs or T lymphocytes. In order to trigger anti- already tolerized. Therefore, the potential
body-dependent cellular cytotoxicity or phagocy- approach is to identify non-tolerogenic, tumor-
tosis to kill cancer cells, these Abs need to associated antigens (TAAs) suitable to develop
recognize specic proteins normally on the outer Ag-specic anticancer vaccines [27]. In spite of
membrane of the cancer cells [25]. T lympho- success in other infectious diseases, the use of
cytes have the capacity to selectively recognize small self-peptides as Ags in cancer vaccines did
peptides (antigens) derived from self/ nonself not attain much interest in the past because of
proteins attached with major histocompatibility their poor immune response and minimal thera-
complexes on the antigen-presenting cells peutic benets. Most of these free peptides are
(APCs). The use of cytotoxic T cells (CTLs), likely to have a short half-life and poor pharma-
dendritic cells (DCs), and monoclonal antibodies cokinetics properties and are thus rapidly cleared
is now a well-established strategy to design before they can be loaded on the dendritic cell
potential cancer immunotherapeutics [26]. surfaces in the complex with MHC molecules to
The major challenge in the development of stimulate CD8+ and CD4+ T cells for the initia-
cancer vaccines is that Ags normally recognized tion of adaptive immune responses [28]. However,
by the immune system are expressed as the the coadministration of suitable dendritic
436 J. Vera et al.
cell-activating adjuvant along with short TAA miRNAs, etc.) [3840]. For many of these data
peptides was shown to boost immune responses mining approaches, well-established computa-
in advanced melanoma [29] and vulvar intraepi- tional pipelines already exist in the public
thelial neoplasia patients [30]. These studies gen- domain. For therapeutic cancer vaccines, the
erated the hope to design effective therapeutic idea is to either amplify or induce new immu-
cancer vaccines. nogenic responses in cancer patients based on
In order to avoid the self-recognition that CD8+ or CD4+ T-cell responses by recognizing
normally results in the weakened immune differentially expressing TAAs from microar-
responses for cancer vaccines, researchers have ray data repositories [41]. One of such database
validated the use of DNA vaccines in preclinical is Oncomine, which has a huge repository of
studies where the tumor-derived sequences were gene expression proles from microarray stud-
initially fused with the genes encoding microbial ies to identify differentially expressing genes in
proteins [31]. This strategy helped T helper cells various stages of major types of cancer [42].
in the induction of Abs against tumor Ags along These data analysis pipelines facilitate the dis-
with epitope-specic antimicrobial CD8+ T cells. covery of novel cancer biomarkers and drug/
Another example PROSTVAC, a DNA vaccine vaccine candidates. In the following section,
for prostate cancer, which includes recombinant we will describe the use of bioinformatics tools
vaccinia virus encoding prostate TAAs along and computational pipelines to discover poten-
with adhesion molecules and DCs stimulators, is tial cancer vaccine candidates with a case study.
already in the clinical trial phase III [32]. Besides,
several monoclonal antibodies (mAbs) and other 23.4.1.1 Case Study: Computational
small molecules such as kinase inhibitors, angio- Approaches to Design DNA
genesis inhibitors, proteasome inhibitors, and Vaccine for Cervical Cancer
molecular receptor blockers are also combined Caused by Human
with immunotherapy for developing targeted Papillomavirus
anticancer therapies [33]. Many Abs boost the Cervical cancer is the most common and slow-
immune response against cancer cells. growing malignant cancer present in the tissues
Ofatumumab and ipilimumab are two such mAbs of the cervix or cervical area in women. Persistent
recently approved by the US FDA. While ofatu- infection with human papillomavirus (HPV) is
mumab targets CD20 protein which inhibits considered to be one of the major etiological fac-
early-stage B-lymphocyte activation in chronic tors for cervical cancer [43]. More than 100 dif-
lymphocytic leukemia [34], ipilimumab speci- ferent types of human papillomaviruses (HPV)
cally targets cytotoxic T-lymphocyte-associated have been identied [44] and categorized into
antigen 4 (CTLA 4) that provides inhibitory high-risk and low-risk strains. A total of 16 dif-
signal for activated T cells [35]. Unconventionally, ferent high-risk strains have already been identi-
mAbs are also shown to target intracellular ed, among them strain 16 and 18 are together
oncoproteins; this nding opens a new possibility responsible for approximately 70 % of all cervi-
to predict potential targets for TAA discovery cal cancer cases [45]. Two HPV vaccines
[36, 37]. GARDASIL and CERVARIX are currently in use
Still, the detection of effective non-tolero- as prophylactic vaccines and offer no therapeutic
genic TAAs from extra-/ intracellular oncopro- benet for patients already infected with the virus
teins is one of the major challenges in cancer or those with precancerous lesions or cervical
immunotherapy. To recognize TAAs, one has to cancer [46]; also they are not completely effec-
carefully investigate sites for cancer-specic tive against all high-risk strains of this virus. In
point mutations, chromosomal aberrations, contrast, therapeutic vaccines generate a T-cell
splicing variants, alternative reading frames immune response to eliminate existing viral
along with overexpressed genes, and other infection. Epitope-based vaccines provide a spe-
regulatory elements (transcription factors, cic strategy for prophylactic and therapeutic
application of pathogen-specic immunity. The capsid proteins form the virus to detect potential
identication of epitopes suitable for diagnostic vaccine candidates. Some of the previous in vitro
use and for therapeutic or prophylactic interven- neutralization studies demonstrated high cross-
tion is clearly a crucial prerequisite of these strat- reactivity with L2 antisera. We retrieved HPV L2
egies. The selection of immunogenic, consensus, capsid protein sequences for various strains from
and conserved epitopes from proteins of major the NCBI (http://www.ncbi.nlm.nih.gov) and the
high-risk strains may provide an experimental UniProt (http://www.uniprot.org) database. To
basis for the design of very specic T-cell and identify conserved regions in the protein, we per-
DNA vaccines effective against all high-risk formed multiple sequence alignment using the
strains. Herein, we will highlight the computa- ClustalX software. Based on the multiple align-
tional pipeline adopted in one of our previously ment les, we identied conserved regions in the
published research works which was used to L2 capsid proteins using the Shannon entropy
design in silico DNA vaccines against (HPV) by function available on the Protein Variability
using consensus epitopic sequences of L2 capsid Server (http://imed.med.ucm.es/PVS). From the
protein from all high-risk HPV strains [47]. In alignment le, Shannon entropy is calculated as
addition, various computational parameters were M
optimized to increase the immunogenicity of the H = -Pi log 2 Pi
vaccine by considering multiepitopic sequences, i =1
codon optimization, CpG motifs optimization, where Pi is the fraction of residues of amino acid
and inclusion of promoter and other immune- type i and M is the number of amino acid types.
stimulatory molecules. A generalized computa- To identify the conserved regions in the L2
tional pipeline for the design of DNA vaccine is capsid proteins of all high-risk HPV strains, the
highlighted in Fig. 23.3. The work initiates with cutoff score for the Shannon entropy was set to
the detection of differentially expressing genes 2.0 (Fig. 23.4). The fragments with Shannon
in cancer (non-pathogenic) or the identication variability score 2.0 and continuous length of
of conserved immunogenic regions from patho- 9 amino acid residues were further selected for
gens involved as the major etiological agents. the epitope identication.
From the conserved regions, MHC class I and
class II epitopes are predicted followed by the Prediction of MHC Class-I and Class-II
inclusion of proteosomal/lysosomal cleavage Epitopes
sites. Various computational approaches may Epitope mapping is always the key step in vaccine
follow to lter the immunogenic peptide such as designing. Epitopes are usually thought to be
3D structure modeling to calculate the solvent derived from nonself protein Ag that interacts
accessibility of cleavage sites, post cleavage with Abs or T-cell receptors thereby activating an
conservancy of epitopes, and then long half-life immune response. Besides nonself proteins, epit-
for proper immunogenicity using molecular opic sequences from the host can also be recog-
dynamics simulations. The selected peptide can nized by MHC molecules. For an effective
then be back-translated and optimized for codons vaccine, it is important for the epitopes to invoke
and CpG motifs. In silico cloning experiments strong response from T and B cells. A large num-
may also be performed for the selection of good ber of bioinformatics algorithms were designed
expression systems to be used for vaccine for this purpose, such as Position-Specic
development. Scoring Matrix (PSSM)-based SYFPEITHI [48],
Articial Neural Networks (ANN) [49],
Retrieval of Sequence Data Stabilized Matrix Method (SMM) [50], and
and Identication of Conserved Regions Average Relative Binding (ARB) [51]. In this
in the Protein work, we used the RANKPEP server (http://
In case of previously designed HPV vaccines, imed.med.ucm.es/Tools/rankpep.html) for the
researchers thoroughly investigated L1 and L2 prediction of consensus binding epitopes (9 mers)
438 J. Vera et al.
Fig. 23.3 Generalized workow for computer-aided epitope-based DNA vaccine design
4
Shannon variability
3.5
3
2.5
2
1.5
1
0.5
0
0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675
Position of amino acid residues in the multiple alignment file
Fig. 23.4 Figure showing the Shannon variability score given position in the multiple alignment le. Blue line rep-
of individual positions in the multiple alignment les of resents the cutoff Shannon variability score. All the red
L2 capsid protein from high-risk HPV strains. Red bars bars below the blue line are potential conserved sites for
indicate the variability score of amino acid residue i at the analysis
for both MHC class-I and class-II molecules with server for CpG optimization [54]. In this process,
default parameters. In total, we used 75 MHC the consensus motif XCGY (where X is any base
class-I and 49 for MHC class-II matrices for the but C and Y is any base but G) was incorporated
prediction of potential epitopes from all the con- in the sequence as triplet (XCG or CGY) by sub-
sensus L2 capsid proteins. stituting the less frequent codons that codes the
same amino acid residues.
Reverse Translation of Immunogenic
Peptide Fragments Insertion of Cleavage Motifs
To back-translate a peptide sequence into the and Finalization of DNA Sequence
DNA sequence, a large number of bioinformatics For the purpose of generating specic epitopes,
tools are available in the public domain. Because proteasomal and lysosomal cleavage motifs were
of the degeneracy of the genetic code, the back- also included before and after each MHC class-I
translation is ambiguous as most amino acid resi- and class-II epitope, respectively. These cleavage
dues are encoded by multiple codons. To design motifs are targeted by the proteasomal and lyso-
an optimal DNA sequence, most of these tools somal cleavage machineries to generate immune
use a codon frequency table specic for the responses in the host. The corresponding nucleo-
organism of interest. We used Backtranseq pro- tide sequence of the 12-residue long peptide
gram of mEMBOSS 6.0.1 for this purpose. HEYGAEALERAG was added as proteosomal
cleavage motif before and after the optimized
Optimization of Codons and CpG Motifs DNA sequence of each MHC class-I epitope. The
Codon optimization is the process to enhance the HEYGAEALERAG motif contains all ve cleav-
efciency of DNA expression vector to express age sites Y3-G4, A5-E6, A7-L8, L8-E9, and
the foreign gene in the hosts cell environment. R10-A11 dened for eukaryotic proteasomes in
DyNAVacS server (http://miracle.igib.res.in/ which A5-E6 is the major cleavage site [55].
dynavac) was used to compute the optimal codon Similarly, the nucleotide sequence of the
for each of the amino acid residue encoded by the 5-residue long peptide KFERQ was added as
stretch of DNA. The server optimizes codons lysosomal cleave motif before and after the DNA
according to the codon usage table derived from sequence of each MHC class-II epitope. KFERQ
the Kazusa Codon Usage Database (http:// specically acts as a recognition motif toward
kazusa.or.jp/codon). We used a codon frequency heat shock proteins and facilitates further steps
table for Homo sapiens that ranks codons by ana- for the degradation of proteins by lysosomes [56]
lyzing their frequency of occurrence in 93,487 to generate MHC class-II epitopes. At the end,
coding sequences [52]. Immunogenicity of start and stop codons were added to nalize the
Ag-specic DNA vaccine was previously shown DNA vaccine. Arrangement of the epitopes is
to signicantly increase by the optimization of very crucial and one of the deterministic factors
CpG motifs [53]. We again used the DyNAVacs for the efcacy of the DNA vaccine. The folding
440 J. Vera et al.
Fig. 23.5 Arrangement of various segments of DNA vaccine constructs. The arrangement of epitopes in the sequence
is very crucial to increase the efcacy of DNA vaccine
of the protein product in the host will largely and effective vaccine candidates before being
depend on the arrangement of these epitopes and subjected to in vitro conrmatory studies.
also determine the solvent accessibility of the
cleavage motifs. Various computational tools can
be used for this purpose including molecular 23.4.2 Mathematical Models Used
dynamics simulation approaches. The overall in Basic Oncology Research
arrangement of the DNA vaccine construct is
shown in Fig. 23.5. 23.4.2.1 Pathways and Networks
The successful use of systems biology to eluci-
In Silico Cloning Experiments of DNA date the regulation and function of cancer-related
Vaccine Construct pathways is well proved by a large body of
Several expression systems have been success- literature published in the last decade. In this
fully designed in the past, for the cloning of the context, mathematical modeling has been used
number of genes encoding surface antigens to investigate the time-dependent behavior of
from pathogens to facilitate vaccine develop- biochemical systems, to integrate multiple data
ment. A good DNA vaccine vector should be sources, or to validate the existence of new regu-
designed with minimal functions so that the latory or transcriptional interactions in given
only gene expressed in mammalian cells is the regulatory pathways. A question in biochemical
antigen-encoding gene. We performed the clon- networks for which data-driven mathematical
ing experiments using clc-DNA Workbench modeling is necessary is the elucidation of the
5.0.1. For our purpose, the pVAX1 vector was nonlinear properties emerging from the combi-
selected as an expression system. pVAX1 is a nation of regulatory motifs containing positive/
nonfusion vector specically designed to stimu- negative feedback and coherent/incoherent feed-
late cellular as well as humoral immune forward loops. When biochemical pathways or
responses [57] and requires that the inserted networks hold these regulatory structures, they
gene of interest contains the Kozak translation often display behavior that evades direct reason-
initiation sequence, an initiation codon (ATG), ing. Many papers, which use a data-driven mod-
and a termination codon (TAA, TGA, or TAG). eling approach, succeeded proving how signal
When this designed DNA vaccine is injected amplication [11], sustained oscillations [58], or
into the host, the antigenic protein gets trans- bistability [59] emerged as hallmarks of signal-
lated and alerts the bodys immune system to ing and transcriptional networks.
generate immunization memory cells. To mention an example on immune-related
The methodology described above highlights pathways, Das and colleagues [60] integrated dif-
how various bioinformatics algorithms and com- ferent modeling approaches with in vitro experi-
putational tools can be combined to design novel ments to elucidate the interplay between Ras
activation and SOS proteins in the activation of T the differentiation of nave CD4+ T lymphocytes
and B lymphocytes. What makes their work into Th1, Th2, Th17, or iTreg. The regulatory
interesting is that both proteins, Ras and SOS, are map was translated into a mathematical model in
integrated in a positive feedback loop that partici- ordinary differential equations and characterized
pates in the Ag receptor stimulation of lympho- using perturbation experiments, in which differ-
cytes. In this feedback loop, Ras gets strongly ent concentrations of relevant cytokines were
activated upon membrane receptor stimulation, a used to stimulate the shift between different
process which is mediated by members of the signaling and transcriptional pathways and there-
SOS family. In turn, SOS activity at the plasma fore the distinctive differentiation of the nave T
membrane is allosterically upregulated by active cells. Once the model was calibrated and vali-
RasGTP. To validate the existence of this positive dated, model simulations and sensitivity analysis
feedback loop and its functional consequences, were combined to determine the model parame-
the authors combined model simulations and ters controlling the activation of different path-
time-dependent in vitro experiments with human ways. They found that the pathway regulating the
and chicken lymphocytic cell lines. They found nuclear receptor PPARc function plays a major
that under some stimulatory conditions, the bio- role controlling the shift between the Th17 and
chemical system displays bistability. That is, for iTreg transcriptional and phenotypic programs.
high doses of stimulus, the pathway works like an Based on these ndings, they foresee a therapeu-
all-or-nothing system: transient but intense stim- tic potential to the regulation of PPARc signaling
ulus can trigger a sustained activation of the sys- in the context of chronic inammatory and infec-
tem and the downstream pathway. When we tious diseases. In this way, the authors show how
consider a population of lymphocytes, this prop- a full systems biology strategy can be extremely
erty may induce the emergence of a bimodal useful to dissect the signaling and transcriptional
response, with a subpopulation of lymphocytes networks controlling differentiation and plastic-
getting full and sustained activation, while others ity of immune cells.
remain inactive. From an immunological per-
spective, the authors hypothesize that this system 23.4.2.2 Genotype-Phenotype
induces the emergence of a short-term mecha- Mapping
nism of molecular memory. This mechanism can Mathematical models can be used to bridge the
improve the activation of T lymphocytes which gap between intracellular pathways and the cel-
were stimulated in previous serial encounters lular phenotypes they regulate. In this case, the
with rare antigen-bearing cells. idea is to develop mathematical models that con-
In the study by Das et al. [60] the focus was to sider how genetic or epigenetic changes in criti-
elucidate the dynamics of a small signaling sys- cal cancer-related pathways can affect the fate of
tem containing regulatory loops. In other cases tumor cells and trigger (or disrupt) phenotypic
one tries to address how several pathways cross responses at the cellular level. Some authors call
talk to each other and integrate their signals to this the genotype-phenotype mapping [64].
achieve the regulation of given phenotypic We have recently applied this idea to investi-
responses. This has also been explored using gate the deregulation of critical cancer signaling-
mathematical models of large regulatory net- transcriptional networks during the emergence of
works in the context of cancer [61] and immunol- a phenotype of chemoresistance ([8], see
ogy [62]. For example, Carbo and collaborators Fig. 23.6). To this end, we constructed a data-
[63] used a systems biology approach to investi- driven mathematical model in ordinary differen-
gate the regulation of the pathways underlying tial equations (ODEs) accounting for an
CD4+ T-cell differentiation. By collecting and intracellular network around E2F1, a transcrip-
organizing the state of the art of biomedical tion factor involved in abnormal cell prolifera-
knowledge, they constructed a comprehensive tion, apoptosis, and chemoresistance. The
regulatory map of the critical pathways regulating network included the interaction of E2F1 with
442 J. Vera et al.
Fig. 23.6 Model-based genotype-phenotype mapping: additional equation describing the size of a population of
modeling genetic signatures promoting chemoresistance. tumor cells whose response upon anticancer drugs admin-
In Vera et al. (2013) [8], we derived a data-driven model istration was controlled by the E2F1 network. Model
in ODEs accounting for an intracellular network around simulations of heterogeneous tumors predicted that geno-
E2F1, involved in cancer resistance to genotoxic and cytotoxic drugs can favor the selection of subpopulations of
toxic drugs. We connected the network model with an chemoresistant tumor cells
two isoforms of p73 and the microRNA miR- tumor cells, each one of them represented with a
205, as well as a number of transcriptional targets set of model equations. Using model simulations,
whose regulation after anticancer drug adminis- we detected genetic signatures for the network
tration is controlled by these E2F1/p73/miR-205 that conferred resistance to either genotoxic or
networks. To make the genotype-phenotype map- cytostatic drugs and even double drug resistance.
ping possible, we connected our model with an Furthermore, our model predicted that genotoxic
additional equation that describes the size of a drugs, when applied to heterogeneous tumors, can
population of tumor cells whose response upon favor the selection of subpopulations of chemore-
stimulation with anticancer drugs was controlled sistant tumor cells.
by the E2F1-centered network. In this equation,
basic phenotypic traits of the modeled cells like 23.4.2.3 Multi-scale Modeling
proliferation or death rate were connected and In a more rened version of the previous strategy,
therefore controlled by the E2F1-regulated tran- systems biology and data-driven modeling can be
scriptional targets. These transcriptional targets used to account for spatial features of tumor
represent the triggering of proliferative, apop- organization and the interaction of the tumor with
totic, or antiapoptotic programs in the model. the surrounding microenvironment. This is the
This equation computationally connects the rationale for the so-called cancer multi-scale
genome of cancer cells with their phenotypic models, which has been successfully used in the
response by linking the expression of intracellular last years to investigate the detailed dynamics of
network components to the dynamics of the tumor tumor growth or angiogenesis [17]. In the recent
cell population. We could simulate tumor hetero- literature, there are several excellent reviews
geneity by considering several subpopulations of about the topic [65], as well as a number of
examples of cancer multi-scale models [16, 66], cancer-related cell and tissue phenotypes. In
many of which referred to angiogenesis. addition, systems biology can play a major role
To mention an example with a cancer immu- in translational medicine, providing tools for
nology focus, Pak and coauthors [67] derived a clinical data integration, as well as for design,
mathematical model to investigate features of the assessment, and personalization of anticancer
delivery of recombinant immunotoxins, a family therapies [68, 69]. In the following, we illustrate
of new molecules with anticancer activity. They these possibilities with several recent examples.
are composed of an Ab fragment targeting spe-
cic tumor cell Ags and a protein toxin fragment, Assessment of Conventional Therapies
which is released and triggers cytotoxic effects A very promising use for systems biology is the
upon recognition, internalization, and processing personalized assessment of anticancer therapies.
of the molecule. The authors derived a mathemat- The literature contains many recent works illus-
ical model that links recombinant immunotoxin trating how data-driven modeling can be used to
dosing and changes in tumor volume. In the maximize the efciency of current therapies but
model, a tumor is divided into a series of spheri- also to detect patient subpopulations for which
cal subunits that contain a blood vessel and a they are not suitable. For example, mathematical
number of tumor cells surrounding it, which can models can be used to determine under which
be present as normal, intoxicated, or dead conditions a conventional therapy: (a) is toxico-
tumor cells. For each one of these structures, the logically safe [70, 71], (b) does not induce further
model contains a set of differential equations resistance [8, 72], and (c) can be combined with
accounting for the dynamics of immunotoxin, other therapies [8]. Furthermore data-driven
from its release from the blood vessel until its models can be used to establish the drug dosage
internalization in a tumor cell, which becomes and timing that optimizes the anticancer effect
intoxicated. In this way, the model accounts for and/or reduce toxicity [73].
the amount of immunotoxin released, present, For example, Engel and collaborators [70]
and degraded in each tumor subunit. The other made use of data-driven mathematical modeling to
part of the model describes the dynamics of look for the optimal administration dose and
tumor cell populations existing in the subunit. timing of several conventional anticancer drugs
This part of the model considers processes like minimizing the risk of acute neutropenia, a side
cell growth, immunotoxin-related death, and cell effect of anticancer therapy in malignant lymphoma
migration to occupy the space cleared after the and other cancers. What makes therapy-associated
death of highly intoxicated cells. Using model acute neutropenia important for cancer patients is
simulations, Pak and colleagues found that Ag that they get a drastic reduction of neutrophil
shedding, a key mechanism in the dynamics of blood levels, which makes them more vulnerable
tumor-specic surface Ags, is critical for the to bacterial infections and increases the risk of life-
success of the therapy. Using model simulations, threatening sepsis. Engel and coworkers derived,
they found that Ag shedding homogenizes characterized, and tested a quantitative data-based
the distribution of the immunotoxin in solid ODE model that describes the generation,
tumors, therefore increasing the efciency of the proliferation, and differentiation of neutrophils
therapy. and other human granulocytes. The model was
extended to account for the changes in the
23.4.2.4 Mathematical Models Used granulocyte dynamics suffered by patients with
to Assess and Design lymphoma and treated with cytostatic drugs and
Therapies recombinant GSCF, an adjuvant therapy that
Previous results illustrate the potential of systems stimulates granulocyte production and accelerates
biology and data-driven modeling to explore the the recover from neutropenia. The model was
structure, function, and regulation of biochemical characterized with patient data obtained from
networks, as well as their interplay with several large randomized clinical trials, in which
444 J. Vera et al.
efcacy and safety of multidrug chemotherapies Design of New Chemo- and Immune
were assessed. The obtained model describes pre- Therapies
cisely the time response of white blood cell levels Systems biology has become a valuable approach
for ten different therapeutic regimes. Furthermore, to boost the procedure of drug discovery and the
the authors suggest that the model can be used as a design of combined therapies that integrate
predictive tool, able to assess the safety of other conventional and targeted chemotherapy. The
non-explored conventional anticancer drugs underlying idea is to combine predictive model
regimes. Although the model was characterized simulations, sensitivity analysis, and other
with data from patients suffering malignant advanced model-based computational methods to
lymphoma, they claim the model can be adapted to help detect single or combined potential drug tar-
assess the risk of therapy-associated neutropenia gets. These model-obtained potential drug targets
in other tumor entities. can later direct the search from new drugs [21,
This idea can be extended to other conven- 75, 76]. In a quite remarkable example of this
tional anticancer therapies. For example, Ribba strategy, Schoeberl and colleagues combined
and colleagues [71] developed a multi-scale high-throughput and time series data with math-
model to investigate the effect of some tumor ematical modeling of the receptor tyrosine kinase
features in the efciency of radiotherapy. The signaling family to detect new anticancer drug
authors constructed a model for colorectal cancer targets [12, 77]. They derived, calibrated, and
progression that links cell cycle progression, validated an ODE-based mathematical model
DNA damage level, and other signaling pathways describing the known features of the ErbB/PI3K
to the sensitivity of individual cells to the irradia- signaling network in the context of cancer pro-
tion doses. Their model integrated four modules, gression. Predictive model simulations were
implemented using different modeling frame- combined with computational sensitivity analysis
works. Some of the key features of the model are: to identify which members of the ErbB family
(a) it includes regulatory pathways controlling have a major effect in the activation of AKT sig-
cell cycle, cell division, and apoptosis; (b) these naling in cancer cell lines. They later synthesized
pathways are connected with the fate of individ- a human monoclonal antibody that inhibits the
ual tumor cells and actually control tumor cell phosphorylation and subsequent activation of
death and proliferation; (c) the model also con- their top one model-detected drug target, the
siders the spatial structure of the tumor, that is, ErbB3 receptor. The model predictions were vali-
how cells get distributed and interact with the dated by showing that this antibody stops the
tumor microenvironment through gradients of growth of human tumor xenografts in mice mod-
growth and antigrowth factors and hypoxia; (d) els. Interestingly, the team is entirely composed
additional model equations describe how differ- of researchers from a biotech company devoted
ent irradiation dosing (time and dose) triggers to the use of systems biology in drug discovery
DNA damage in proliferative tumor cells. When (Merrimack Pharmaceuticals, Cambridge, USA).
they simulated radiotherapy administration with This strategy has also delivered some interesting
their model, they found that the efcacy of con- results in the context of immune anticancer thera-
ventional irradiation protocols can be improved if pies. Kim and Lee [78] used data-driven modeling
the cell cycle-regulated dynamics of tumor of the lymph node-tumor interaction to analyze
growth is considered when planning the schedule whether preventive vaccination with cytotoxic T
of irradiation sessions. This result is in line with lymphocytes (CTLs) can be employed to promote
others suggesting similar optimal schedules of the clearance of microtumors before clinical detec-
chemotherapy sessions, something known as tion (Fig. 23.7). Toward this end, they derived a
cancer chronotherapy [74]. hybrid mathematical model composed of two
Fig. 23.7 Data-driven modeling of the lymph node-tumor cytotoxic T lymphocytes by the matured antigen-presenting
interaction and the clearance of microtumors with cytotoxic cells. In addition, the model describes tumor cell detection by
T-lymphocyte (CTL) vaccination. The model describes the CTLs and CTL-mediated tumor cell death. The model can
dynamics of CTL activation, including tumor Ag production, simulate variations over time for the populations of the dif-
its detection by antigen-presenting cells, and the activation of ferent immune cells and the tumor cells
interconnected modules. The rst module describes quent proliferation, maturation and migration, as
the dynamics of CTL activation, including the well as the emergence of memory T cells. The sec-
tumor antigen production at the tumor site, its ond module describes the interplay between active
detection by antigen-presenting cells, and the sub- cytotoxic T lymphocytes and tumor cells, including
sequent maturation and their migration to the lymph tumor cell detection, recruitment of additional
node. Furthermore, the module includes the activa- CTLS, and CTL-mediated tumor cell death. The
tion of CTL by the matured APCs and its subse- model was characterized using data from breast
446 J. Vera et al.
cancer. The authors used the mathematical model to and genotoxic drug administration using either
determine a threshold in the size of the anticancer normal MCF-7 cell lines or mutant cell lines
memory CTL pool able to promote an effective overexpressing proteins involved in the efux of
clearance of microtumors. Furthermore, the model anticancer drugs. Using data from these experi-
predictions attribute an important role in the success ments, they characterized the rates of growth and
of the immune response to the rapidity in which drug sensitivity of both tumor cell subpopulations
CTLs detect the tumor site. Paradoxically, the in the model. Later, model simulations were per-
model simulations suggested that tumors with fast formed to analyze the tumor growth rate when
growth rate are more prone for CTL destruction due different versions of their adaptive therapy were
to the faster production of tumor antigens and, used; they compared the results with the tumor
hence, faster detection by CTLs. growth rate under conventional genotoxic chemo-
therapy. They found that the combination of their
Unconventional Therapies adaptive therapy (which tunes the timing and dose
A fascinating option with data-driven mathemati- of conventional chemotherapy) with the adminis-
cal modeling is to explore therapies inspired in tration of non-chemotherapeutic membrane pump
not yet experimentally proven concepts and ideas. substrates (a kind of competitive inhibitors of
In this sense, modeling is used to formulate new drug efux) and 2-deoxyglucose (an inhibitor of
hypothesis on the origin and progress of cancer, as glucose transporters and glycolysis) provokes a
well as to foresee how one could derive new ther- fourfold increase in the progression-free survival
apies based on this. In the recent literature, there in their computational models.
are some examples of this procedure [79, 80]. In
a series of recent papers, Gatenby and coworkers
hypothesized that adaption to chemotherapeutic 23.5 Concluding Remarks
agents has an energetic cost for cancer cells, and
this can be exploited to design anticancer thera- Systems biology emerged a decade ago as a
pies [80, 81]. In fact, the starting point of their methodological approach that combines quanti-
hypothesis is that chemoresistant cells need addi- tative experimental data, mathematical modeling,
tional energetic resources to keep working the and other tools from computational biology, aim-
resistance mechanisms against drugs. Their adap- ing to understand the regulation of these complex
tive therapy relies on considering the existence of biochemical systems. The interaction between
several coexisting subpopulations of cancer cells tumors and the immune system is not an excep-
in the tumor, with different genetic and pheno- tion to this scenario. The immune system is by
typic backgrounds regarding chemoresistance. In denition a multi-scale system not only because
their hypothesis, one can favor the proliferation it involves biochemical networks that regulate the
of chemosensitive cells by manipulating the tim- fate of immune cells but also because immune
ing and dose of conventional chemotherapy, in a cells communicate with each other by direct con-
manner which these cells can effectively compete tact or through secretion of local or global sig-
with chemoresistant ones for space and resources nals. Furthermore, tumor and immune cells
and delay the development of a fully resistant communicate, and this interaction is affected by
tumor. To substantiate their hypothesis, they have the features of the microenvironment in which
derived a series of in vitro data-driven mathemati- the tumor is hosted. Altogether, we are envision-
cal models, which describe the growth of tumors ing a complex multi-scale biological system,
composed by chemosensitive and chemoresistant whose analysis requires a systemic view to suc-
cancer cell subpopulations. For the most updated ceed integrating massive amounts of quantitative
version of the model, they performed in vitro experimental data coming from different tempo-
experiments under conditions of normal growth ral and spatial scales.
Acknowledgements This work was supported by the Feldhaus MJ, Kudla AJ, Nielsen UB. Therapeutically
German Federal Ministry of Education and Research targeting ErbB3: a key node in ligand-induced activa-
(BMBF) as part of the projects eBio:miRSys [0316175A tion of the ErbB receptor-PI3K axis. Sci Signal.
to JV] and eBio:SysMet [0316171 to SKG]. 2009;2(77):ra31.
13. Chmielecki J, Foo J, Oxnard GR, Hutchinson K,
Ohashi K, Somwar R, et al. Optimization of dosing
for EGFR-mutant non-small cell lung cancer with
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Principles of Immunological
Diagnostic Tests for Cancers 24
Amber C. Donahue and Yen-lin Peng
Contents
24.1 Introduction
24.1 Introduction .............................................. 451
24.2 Overview of Antibodies ........................... 452 Through the use of deliberate mutation of
24.2.1 Monoclonal vs. Polyclonal Antibodies ...... 452 immunoglobulin genes, the immune system has
24.2.2 Antibody Fragments .................................. 453
24.2.3 Reporter Labeling ...................................... 454 evolved the ability to produce antibodies (Abs)
24.2.4 Primary and Secondary Antibodies ........... 454 able to bind targets with exquisite specicity
24.3 Immunoprecipitation ............................... 454
(i.e., recognition of ONLY the target) and impres-
sive afnity (i.e., strong binding to the target).
24.4 Immunoblotting ....................................... 455 These abilities explain why Abs remain an
24.5 Radioimmunoassays ................................ 457 invaluable tool for the detection and measure-
24.6 Enzymatic Immunoassays ....................... 457 ment of biological phenomena and already repre-
sent some of the treatment modalities of the
24.7 Immunocytochemical
and Immunohistochemical Assays ......... 460 present and near future. While most of the work
with Abs is currently ex vivo, their use in vivo has
24.8 Flow Cytometry........................................ 461
already shown signicant progress and benets.
24.9 Bead-Based Assays................................... 464 Antibodies are currently used for biosensing of
24.10 Antibody Arrays ...................................... 465 specic targets in the body, in order to deliver
radioactive isotopes or cytotoxic drugs (reviewed
24.11 Concluding Remarks ............................... 468
in Ricart and Tolcher [1]). Antibodies have also
References ............................................................... 468 been used for visualizing specic biological pro-
cesses such as tumor shrinking and tumor growth
[25] or to aid in the imaging of tumors. These
types of applications for antibodies will likely
become more common as immunoglobulin engi-
neering becomes more sophisticated, increasing
the potential of using Abs in vivo for the targeting
of specic lesions or tumors or even for the neu-
A.C. Donahue, PhD (*) Y.-l. Peng, MS tralization of specic biological processes. In the
Department of Hematology/Oncology Research and meantime, Abs are widely used in multiple for-
Development, Quest Diagnostics-Nichols Institute, mats and platforms to aid in the detection of a
33608 Ortega Highway, San Juan Capistrano, CA
wide range of cancers. This chapter will intro-
92675, USA
e-mail: amber.c.donahue@questdiagnostics.com; duce the structure of the immunoglobulin pro-
yen-lin.x.peng@questdiagnostics.com tein, including the most commonly used altered

452 A.C. Donahue and Y.-l. Peng
and engineered variants created by researchers, by the injection of a puried full-length recombi-
and provide detail on how these various Abs can nant protein into an animal, which can lead to the
be labeled to allow their detection. A number of generation of Abs that recognize many portions
different applications then become possible. The of the protein. In other instances, a short peptide
principles of these applications and the ways in comprising a more specic region of interest is
which they can be combined to create diagnostic used, creating a number of different Abs that
tests will be outlined, including how diagnostic recognize a very specic region or epitope. In
assays are increasingly being designed to include most cases the rabbit is used to generate poly-
the detection of large numbers of targets simulta- clonal Ab mixtures. Many other species can also
neously, a technique known as multiplexing. be used to create these Abs, contributing to the
multiplexing exibility of Abs. The injected pep-
tide or protein, known as an immunogen, is
24.2 Overview of Antibodies selected to include a very specic, and preferably
unique, region of interest in a target molecule.
Antibodies, or soluble forms of immunoglobulin When the injected animals immune system rec-
(Ig), possess a vast array of possible specicities ognizes the peptide or recombinant protein as
and a structure that is one of the more stable among foreign, the resulting immune response will gen-
mammalian proteins. Researchers have capitalized erate multiple immunogen-specic Abs, which
on the large pool of specicities provided by nave can then be isolated from the animal to yield a
B lymphocytes as well as on the renement of polyclonal antiserum. In some cases, this antise-
specicities for the recognized motif, or epitope, rum is puried further using afnity chromatog-
provided by the process of somatic hypermutation raphy [6].
during clonal expansion of activated B cells. Because of a higher degree of confidence in
However, the ex vivo generation of Abs is becom- their affinity and specificity, mAbs are often
ing the standard for the purposes of research, diag- chosen over polyclonal preparations when
nostics, and therapy. This allows for an increased possible. Kohler and Milstein developed the
amount of versatility through a large number of first mAbs in the mid-1970s by expanding on
sources and formats. Clinicians and researchers the techniques used to generate polyclonal Ab
have the choice of intact Ab molecules or frag- preparations. As with polyclonal Ab stimula-
ments, as well as polyclonal or monoclonal anti- tion, an immune response is elicited to an
bodies (mAbs) from a number of different species. injected immunogen. In this case, however,
Each of these various Ab molecules can also be multiple antibody-producing daughter B cells
chemically linked to a multitude of reporter mole- are isolated from the spleen of the injected
cules, allowing the use of Abs in a wide range of animal after several days. Myeloma cells are
assay platforms. The most common of these plat- then fused with the harvested antibody-
form variants are described below. producing B lymphocytes to generate hybrid-
omas. These hybridomas can produce large
amounts of the Abs expressed by the original
24.2.1 Monoclonal vs. Polyclonal activated daughter B cells and are capable of
Antibodies proliferating in culture indefinitely. Single
hybridomas are separated and expanded in
A polyclonal Ab preparation consists of a mix- culture to create monoclonal populations. The
ture of immunoglobulin molecules with multiple Abs produced by the monoclonal populations
specicities, all of which are directed against the are then screened for affinity and specificity
target. Most polyclonal Ab mixtures are created [7, 8].
24 Principles of Immunological Diagnostic Tests for Cancers 453
a Ag b NH2 H2N c NH2 H2N

Binding H2N VH VH NH2 VH VH
NH2 H2N H2N NH2
H2N VH VH NH2 VL CH1 VL VL VL

CH1 CH1
VL VL
ss
1 1 CL ss CL CL ss CL
s s COOH HOOC
ss s s
CL ss CL HOOC COOH HOOC COOH
s s
s s
HOOC COOH F(ab)2 fragment Fab fragment
CH1-3 2 2
Fc
3 3
HOOC COOH
Intact Ig
Fig. 24.1 Intact immunoglobulin and common antibody are circled in red. The Fc portion of the molecule, con-
fragments. (a) Schematic representation of an intact sisting of CH23, is indicated. Domain labels are con-
immunoglobulin molecule. Each heavy chain (blue) constant throughout the gure. (b) The F(ab)2 antibody
sists of three constant domains (CH1-3) and the variable fragment. Enzymatic digestion of intact immunoglobu-
domain (VH). CH1 and CH2 are linked by the exible lin with pepsin results in the cleavage of the molecule at
hinge region, which forms two disulde bonds with the the hinge region, maintaining the disulde bonds and
hinge region of the complementary heavy chain. Each yielding the F(ab)2 fragment. (c) Papain cleaves the
light chain (purple) consists of one constant domain (CL) hinge region of intact immunoglobulin just above the
and one variable domain (VL) and is associated with the disulde bonds, generating two Fab fragments. Fab frag-
heavy chain through a disulde bond proximal to the ments can also be created through genetic manipulation.
carboxy-termini of the two chains (COOH). The anti- The heavy and light chains can associate non-covalently
gen-binding regions of the molecule (Ag Binding) are (right) or may maintain a disulde bond near the car-
found at the amino-termini of the VH/VL pairs (NH2) and boxy-termini (left)
Several technologies for more cost-effective, 24.2.2 Antibody Fragments

rapid, and simpler generation of mAbs have since
been developed. Chimeric or humanized Abs Depending on the requirements of the assay plat-
have been made possible by recombinant tech- form, Abs can be used in a number of different
niques, combining human Ab DNA with the formats, including the intact immunoglobulin
sequence encoding the binding site of a mouse molecule as well as multiple types of smaller
mAb [9]. Recent years have also seen the emer- fragments (Fig. 24.1). The Fab fragment includes
gence of bacterial expression of antibodies, the entire light chain, as well as the variable and
which allows for the selection of advantageous rst constant region of the heavy chain, and can
Ab specicities via phage display. The displayed form stable H/L heterodimers without being
Ab fragments are generated from the plasma covalently linked. In some cases Fab fragments
cells of human donors or from the spleen of an can remain joined through a C-terminal disulde
immunized animal. Increasingly, however, these bond (Fig. 24.1c) [9]. Fab fragments can be created
phage libraries and other screening tools are gen- by papain digestion of intact immunoglobulin
erated by genetic engineering (discussed in molecules, or more recently, through genetic
greater detail in Donzeau and Knappik [9]). The manipulation. The F(ab)2 fragment is similar, in
highly specic high-afnity mAbs required for that it also retains the disulde bond which cova-
therapies, diagnosis, and basic research are cre- lently links the two chains of the Fab fragment
ated using these methods. (Fig. 24.1b). In the case of the F(ab)2 fragment,
however, a portion of the exible hinge region 24.2.4 Primary and Secondary
remains intact following its creation by digestion Antibodies
of intact Abs with pepsin. Additional small frag-
ments and multivalent engineered Abs can also Some diagnostic assay formats require the use of
be created through genetic engineering and may Ab pairs for detection (see Fig. 24.6b for a sche-
enjoy increasing use in diagnostic assays and matic representation). The rst, or primary, Ab is
possibly cancer therapy in the coming years. specic for the target. A secondary reporter-
conjugated Ab can be used in cases where the
primary Ab does not include a reporter. Anti-
24.2.3 Reporter Labeling species Abs, which are directed against immuno-
globulin molecules produced by a different
There are a number of reporter molecules avail- species, are commonly used as secondary Abs.
able for use in visualizing and even quantifying For example, mouse immunoglobulin is injected
the binding of an Ab to its target [10]. One such into a goat to produce an immune response,
class of reporters is the group of laser-activated resulting in a polyclonal goat anti-mouse Ab
uorescent molecules called uorophores or u- preparation that can be labeled with a reporter
orochromes, commonly used in ow cytometry molecule. The goat anti-mouse Ab preparation is
(see Sect. 24.8). Other reporters can be enzymatic used to detect the presence of the primary mouse
and therefore depend on chemical reactions to be mAb wherever it may be bound to the target.
detected. For these reporters, the Abs are chemi- However, in order to avoid possible cross-
cally linked, or conjugated, to an enzyme such as reactivity and to minimize the complexity of the
alkaline phosphatase (ALP) or horseradish per- assay, simpler assays in which the primary Ab is
oxidase (HRP). An intense color is generated by directly conjugated to a reporter are preferred
the product created when these enzymes are when the assay system permits.
incubated with chromogenic substrates, allowing
measurement with a spectrophotometer. It is also
possible to incubate these enzyme-linked Abs 24.3 Immunoprecipitation
with a chemiluminescent substrate, the product
of which gives off light, which can then be quan- For many years, specic Abs have been used as a
tied by a number of different instruments and means to bind and concentrate targets in solution
even captured on lm. [13]. This process, known as immunoprecipita-
A common third approach, often used to allow tion (IP), involves the mixing and incubation of
greater exibility for the multiplexing of targets, the specic Ab with a solution containing the
includes biotin-conjugated Abs [11]. Biotin rec- molecule of interest (Fig. 24.2). After sufcient
ognizes streptavidin with a high level of specic- time to allow the Ab to bind the target, the Ab
ity and afnity, forming one of the strongest itself can be captured through binding to beads
known non-covalent bonds. Streptavidin can be coated with bacterial protein A, protein G, or a
linked either to uorophores or to enzymes like mixture of both. The solution can then be centri-
HRP and ALP, providing the exibility to use a fuged to pellet the beads at the bottom of the
particular biotinylated Ab across multiple assay tube, allowing the supernatant to be transferred
platforms. Similarly, within a single platform, the or discarded. Through this process, the target has
same biotinylated Ab can be used in multiple been isolated and greatly concentrated and is now
wells or tubes and, if necessary, be identied by more readily detected.
different colors by using varied streptavidin- When searching for comparatively rare pro-
conjugated reporters, as with the multiple uoro- teins, which are present at much lower concentra-
phores used in ow cytometry [12]. tions, a larger number of cells or volume of bodily
Removal/transfer
a b c of supernatant
Addition of Centrifugation
protein A/G
microbeads
Target
Other protein
Other protein
Antibody
Target bound to antibody
Protein A/G microbead
Fig. 24.2 Immunoprecipitation. (a) Cell lysate or other coating the bead. (c) Following centrifugation, the beads
biological sample is incubated with specic antibody (Ab), and their cargo of Ab and target protein will form a pellet at
which binds to the target in solution. (b) Microbeads coated the bottom of the tube. The supernatant, now depleted of
with bacterial protein A, protein G, or a combination of the target protein, can be transferred to another tube or dis-
both are added to the solution. The Abs, whether bound to carded. These schematic representations of Abs and their
target protein or free, will be bound by the bacterial proteins targets will be used for all subsequent gures
uids like plasma are required. This larger amount bodily uid such as plasma, to a solution of
of material often presents problems for the detec- cellular proteins released from cells by treatment
tion system, which can be solved through the with a lysis buffer. These proteins are separated
capture and concentration of the target by IP. In according to mass via polyacrylamide gel elec-
other cases, IP is used to diminish the amount of trophoresis (SDS-PAGE) and transferred to a
background detected by the assay system. The membrane for detection (Fig. 24.3). The specic
background can be minimized either by pulling primary Ab is washed over the surface of the
the target out of the sample mixture for detection membrane for a prolonged incubation period,
or by specically depleting the mixture of an allowing it to bind the target protein, followed by
unwanted protein(s) that has been found to conict incubation with a secondary enzyme-conjugated
with the detection of the target. IP is often used as anti-species Ab. After the addition of a chemilu-
a rst step before detection by immunoblotting. minescent substrate, a band of light will be gen-
erated at the position where the primary and
secondary Abs are bound to the membrane. The
24.4 Immunoblotting amount of protein present dictates the amount of
primary and secondary Ab bound to the mem-
Also known as Western blotting, immunoblotting brane, which in turn dictates the intensity of the
(IB) makes use of specic Abs for the detection light generated. This light signal is traditionally
of proteins of interest [14]. Sodium dodecyl detected by exposure to autoradiography lm,
sulfate (SDS) and heat are used to denature the but advances in low-light camera-based systems
proteins in a sample, which can range from a have led to increasing use of these documentation
a b c
Size Sample(s) Size Sample(s)

Size Charge
ladder ladder
Larger
Denatured proteins Enzyme

migrate toward cathode & 2 antibody
1 antibody
separate based on size
Target
Smaller +
Separate proteins by SDS- Incubate membrane with Incubate membrane with

PAGE primary and secondary chemiluminescent substrate
antibodies and capture image
Fig. 24.3 Immunoblotting. (a) Samples are denatured in secondary antibodies and reporters is given in Fig. 24.5).
lysis buffer, loaded onto a polyacrylamide gel, and separated A molecular weight standard containing multiple proteins
by electrophoresis (PAGE). The presence of sodium of known molecular weights is usually included in each
dodecyl sulfate (SDS) in the buffer masks the native experiment (size ladder), to provide an estimation of the
charges of the proteins and lends an overall negative charge, distribution of the sample proteins. The proteins in these
allowing the proteins to migrate toward the cathode ladders are often dyed, sometimes with multiple colors, to
according to size, with smaller proteins traveling farther allow visualization on the membrane. (c) The target is
through the matrix than large proteins (SDS-PAGE). visualized by incubating the membrane with the chemilu-
Proteins can also be analyzed by their native conforma- minescent substrate of the reporter enzyme, which emits
tions under non-denaturing conditions in the absence of light. The signal is captured by exposure to autoradiogra-
SDS (not shown). (b) Separated proteins are transferred to phy lm or by a camera-based gel-documentation system.
a nitrocellulose or polyvinylidene uoride (PVDF) The quantity of target can then be extrapolated from
membrane via the application of electrical current. The signal intensity and/or band size, with larger bands corre-
membrane is then probed with primary Ab specic for sponding to more bound target, although this measure is
the target protein or residue, followed by an enzyme- not truly quantitative, but relative to the other samples in
conjugated secondary anti-species Ab (more detail on that experiment only
methods. On a traditional immunoblot exposed to with a number of cancers. The proteins of interest
lm, lower-intensity signals correspond to fainter, in these Western-based tests are actually Abs them-
thinner bands, while larger amounts of signal selves. The Ri immunoblot detects the anti-Ri Ab
create fatter, darker bands (Fig. 24.3). present in patients with paraneoplastic myoclonus/
Due to the fact that it provides an opportunity to opsoclonus syndrome, which is most often associ-
physically view the interactions of an Ab with the ated with gynecological cancers, breast cancer, and
proteins present in a sample matrix, immunoblotting small cell lung cancer. The Yo, or Purkinje cell, Ab
is still widely used in a research setting despite is also found in patients with breast, ovarian, and
being an older technique. This characteristic can other gynecological cancers, in this case suffering
help researchers determine the specicity of an Ab from paraneoplastic cerebellar degeneration. Hu
during the development of a cancer test, even if antineuronal nuclear Abs are detected by Western
another technique will ultimately be used for detec- blot in a small percentage of patients with small
tion. However, despite the fact that the method is cell lung cancer and are associated with paraneo-
comparatively time-consuming and labor inten- plastic sensory neuropathy and encephalomyelitis.
sive, there are still some cancer-related diagnostic The highly specic Abs used in these Western blots
tests which make use of Western blotting. Examples provide conrmation of the identity of the Hu, Yo,
include conrmatory tests for Ri, Hu, or Yo, which and Ri Abs initially detected by rst-line screening
are found in paraneoplastic syndromes associated tests.
24.5 Radioimmunoassays of TSH-secreting pituitary adenomas. In the

interest of laboratory safety, however, technology
One of the rst highly sensitive methods for has moved away from techniques requiring the
measuring the levels of proteins such as hormones handling of radioactivity, and the RIA method
in the blood was the radioimmunoassay (RIA) has largely been replaced by enzymatic
[15]. In a classic RIA, a known quantity of puri- immunoassays.
ed target protein is radiolabeled, most often
with a gamma radioisotope of iodine. This hot
protein is mixed with a specic Ab that has been 24.6 Enzymatic Immunoassays
immobilized on a surface, and then the biological
sample containing unlabeled or cold protein is Enzymatic immunoassays, or EIAs, are the
added to the mixture (Fig. 24.4). In a standard archetypal antibody-based detection format and a
competition assay, the cold protein will then foundation of basic cellular biology research.
compete with the radiolabeled protein for bind- The best known EIA format is the enzyme-linked
ing to the Ab, leading to the displacement of a immunosorbent assay (ELISA) [17], which has
fraction of the radiolabeled protein. The amount been used for the detection of targets in both cell
of target protein present in the sample can then be lysates and in nearly every bodily uid, ranging
extrapolated by measuring the amount of dis- from whole blood to sputum to cerebrospinal
placed radioactivity. uid. Most commonly, ELISA assays are per-
RIA technology allowed some of the rst spe- formed in microtiter plates containing 96 or more
cic and sensitive tracking of important hor- wells, providing the opportunity to test a large
mones like insulin in human blood [16] and is number of samples in a single run. Further, as the
still used in some cancer-related diagnostics treatment of each well is often identical, the for-
today, including thyroid hormone testing. Some mat of the ELISA assay lends itself to a high
thyroid hormone tests, including reverse T3, free degree of automation using liquid handling
T4, and especially thyroid-stimulating hormone robots and plate washers. Since the ELISA often
(TSH), are still offered via RIA. These thyroid contains multiple lengthy incubation steps, the
hormone tests are included as diagnostic tests in ease with which it can be automated provides
the preliminary characterization of thyroid nod- valuable time and labor savings in a high-
ules as malignant or benign and in the diagnosis throughput cancer diagnostics laboratory.
a b Measure displaced labeled protein
Radiolabeled purified target protein
Add unlabeled

sample
Fig. 24.4 Radioimmunoassay. (a) Puried target protein the Abs, displacing some of the radiolabeled protein when
is radiolabeled, often with the gamma isotope of iodine present at high enough concentrations. The unbound pro-
() and incubated with immobilized specic antibody tein is removed from the well, and the radioactivity of the
(Ab). Sample containing unlabeled target protein is then displaced radiolabeled protein is measured to give an indi-
added to the well. (b) The unlabeled target protein com- rect measure of the amount of unlabeled target protein
petes with the puried radiolabeled protein for binding to present in the sample
a Substrate Signal b Anti-species

Ab
Detection
Ab
Well Specific Detection
Enzyme Ab Ab Capture
Target Ab
Capture Anti-species
Ab Ab
Target
protein
Fig. 24.5 ELISA. (a) The simplest ELISA consists of ELISA and its possible variations. The specic capture
proteins adsorbed to the surface of a well and incubated Ab can be directly coated onto the surface of the well or
with specic enzyme-conjugated Abs. After binding of be bound itself by an anti-species Ab. After capture of the
the Abs to the target protein, the well is washed, and the target protein, the target is bound by the detection Ab,
colorimetric or chemiluminescent substrate is added. The which can be conjugated to a reporter itself or bound by a
reporter enzyme acts on the substrate, generating signal in reporter-conjugated secondary anti-species Ab. Each of
the form of color or light, respectively. (b) The sandwich these permutations is represented
ELISA formats can range from simple to different species, to prevent the binding of the
complex, incorporating from one to four Abs secondary detection Ab to both.
(Fig. 24.5) [17]. At the most basic end of the The exibility made possible by the sandwich
spectrum is the direct ELISA, which uses a ELISA allows the detection of specialized pro-
single reporter-labeled primary Ab to detect the tein motifs. Examples include the differentiation
target that has been adsorbed to the surface of the between isoforms created by alternative splicing
well or plate (Fig. 24.5a). More commonly used, [18] or detection of posttranslational modica-
however, is the sandwich ELISA, which can tions such as phosphorylation, acetylation, glyco-
use from two to four Abs as shown in Fig. 24.5b. sylation, methylation, ubiquitination, and even
In many cases the sandwich format is preferred protein cleavage [1823]. The turnover rate of
due to the greater level of specicity conferred by important proteins, the activation status of spe-
requiring two different specic antibodies to bind cic pathways, and other important cellular
the target before detection is achieved. The rst activities can be inferred from the posttransla-
Ab which binds the target is referred to as the tional modications of important cell signaling
capture Ab and is bound to the plate/well either proteins. For detection of these modications, the
through direct adsorption or through interaction target protein can be bound by the capture Ab,
with a corresponding anti-species Ab that is the unbound background protein is washed away,
bound to the plate instead. The capture Ab will and then a detection antibody specic for the
bind the target during incubation with the lysate modication of interest can be used to determine
or bodily uid, after which the irrelevant proteins whether the protein contains that posttransla-
are washed away, leaving the enriched and puri- tional change. The opposite approach can also be
ed target. The second, or detection, Ab is now taken, in which a detection Ab specic for the
incubated in the well and allowed to bind to the target protein can be used to probe the proteins
target wherever it has been captured in the well. pulled out of solution by a capture Ab specic for
The detection Ab can be directly labeled with a phosphotyrosine, for example. In some cases, the
reporter or can be detected itself by a secondary posttranslational modication at a specic amino
reporter-conjugated anti-species Ab. The impor- acid residue is even included in the immunogen,
tant consideration to remember when designing a in order to generate an Ab specic ONLY for the
sandwich ELISA is that if a secondary anti- version of the protein containing a phosphory-
species Ab will be used for detection, the capture lated residue at a given position rather than the
and detection Abs must have been generated in non-phosphorylated version.
a b
A B C D
1 2
E F G H
3 4
I J K L
Spot 1 Spot 2
M N O P
Fig. 24.6 Multi-spot ELISAs. (a) A schematic represen- multi-spot assays without robotic spotting of the capture
tation of a 16-spot multi-spot ELISA well. Each spot, or antibodies, allowing mixing and matching of desired ana-
letter, corresponds to a different capture Ab that is carefully lytes. Each capture Ab is conjugated to one of several
applied to the plate in one discrete area, usually by robot. A chemical linkers and incubated simultaneously in the well.
single sample can then be incubated in the well and 16 Each linker binds only to its corresponding spot, isolating
different sandwich ELISAs performed simultaneously on each capture Ab in one specic region of the plate. Multiple
one small volume of analyte. (b) Chemical linkers can create sandwich ELISAs can then be performed as in (a)
It is also theoretically possible, though match the capture Abs in a given well and do it
generally technically difcult, to use a sand- in-house (Fig. 24.6b). These linker-conjugated
wich ELISA to detect the protein product of a capture Abs are used with specialized plates, in
gene fusion, such often happens in cancer. One which the binding partner of each chemical linker
such example is the BCR-ABL fusion protein has already been spotted in a specic position on
which is the result of the so-called Philadelphia the bottom of the well. Each capture Ab will
chromosome, or the reciprocal translocation therefore only bind to one particular spot within
t(9;22);(q34;q11), that occurs most often in the well, and the sample can then be added to the
chronic myeloid leukemia (CML). In this exam- well and interrogated for the presence of many
ple, a capture Ab specic for the BCR protein target proteins at once.
would immobilize both wild-type (WT) and These sorts of multiplexed ELISA platforms
fused BCR, while only the fusion protein would generally require camera-based detection sys-
be bound by the anti-Abl detection Ab. tems that include sophisticated software capable
The ability to detect multiple targets side by of discriminating and parsing the signal gener-
side in a single aliquot of sample can provide a ated by multiple spots in a single small well.
great deal of important information, as well as Adding an ever greater level of control over the
maximize the information derived from the often process, some more advanced ELISA platforms
inadequate and precious samples received in can- now include computer-controlled initiation of the
cer diagnostic laboratories. Newer ELISA tech- chemiluminescent reaction. In this system the
nologies have emerged in the last decade that reporter is a true electrochemiluminescent (ECL)
make multiplexing possible through the use of reagent, requiring an electrical current to undergo
multi-spot wells. In this assay layout, a number the chemical reaction, and the assay is performed
of different capture Abs are bound to the bottom in a specialized plate containing a small electrode
of each well in discrete spots, ranging from 2 to 4 in each well. The computer controls the applica-
up to 100 (Fig. 24.6a). Flexibility has been fur- tion of current, usually breaking the plate down
ther increased by breakthroughs in chemical link- into sections read in sequence. These sorts of
ers, which allow assay designers to mix and adaptations to the ELISA platform represent
some of the advances made in the last decade and can be compared to that baseline and used to
will likely see increasing uptake in the design of monitor the efcacy of therapy.
cancer tests.
This versatility in the sandwich ELISA plat-
form, as well as the exibility provided by the 24.7 Immunocytochemical
large number of available reporter/detection for- and Immunohistochemical
mats, suggests that similarly ingenious ELISAs Assays
will continue to be developed. Most commonly in
cancer diagnostics, however, more straightforward Immunohistochemistry (IHC) and immunocyto-
sandwich ELISAs are used for the purposes of chemistry (ICC) are similar techniques used by
quantitative detection and monitoring of relevant researchers and pathologists to recognize particu-
proteins. An example is the HER2 ELISA, which lar cell types or to determine the location of
measures the level of HER2/neu present in the important proteins within the cell. These proteins
serum of breast cancer patients. With the inclu- can include indicators of apoptosis or prolifera-
sion of a standard curve on the ELISA plate, the tion, as well as tumor markers. IHC and ICC
amount of HER2/neu protein present in the well assays can provide a wealth of information to the
can be quantied, and the concentration of the trained observer (Fig. 24.7) [24, 25]. The cells
protein circulating in the body can be extrapo- being studied can be found in an intact tissue
lated. These data can be used by the clinician to section as is the case in IHC or taken from sus-
assess the patients prognosis and to determine the pension or from a smear as in ICC. As with an
likely response of the patient to a given therapy. ELISA, these cells are incubated with the pri-
Further, if a baseline concentration of the circu- mary Ab specic for the protein of interest and
lating protein is established prior to administering can be detected either through direct conjuga-
therapy, subsequent longitudinal measurements tion of that primary Ab or by the binding of a
Tissue section
Fig. 24.7 Immunocytochemistry and immunohisto- (b) Simplied schematic of IHC, depicting a slide-mounted
chemistry. (a) Simplied schematic of ICC, depicting a sin- tissue section. Only a few cells in the tissue section express
gle cell probed for two specic proteins. One protein is found the protein for which the sample has been stained (dark
to be localized to the cytoplasm (green), while the other pro- blue). IHC and ICC can make use of both colored stains and
tein is localized to the nucleus (red). This nuclear localization uorescent markers and often require microscopes with
is conrmed by a co-stain which identies the nucleus (blue). multiple excitation and/or emission lters (not shown)
secondary reporter-conjugated anti-species Ab. One of the best known and most commonly
ICC and IHC can use both enzymatic and uores- used IHC tests in cancer diagnostics is the stain-
cent reporters; the use of uorescent reporters is ing of breast cancer sections for the presence of
also sometimes referred to as immunouores- the estrogen receptor protein (ER). As a predic-
cence, differentiating the technique slightly due tive marker, ER is currently the most useful test
to the requirement for a uorescent or confocal for establishing patient prognosis. In addition, it
microscope, as opposed to the light microscope continues at this time to be the best predictor of
that can be used to visualize enzymatic reporters. patient response to hormone therapies. ER is
Additional common antibodies or dyes are often ordered in tandem with IHC staining for the
often used to identify structures within the cell, progesterone receptor (PR) as well, which pro-
such as the nucleus. The prepared samples are vides similar, if less statistically signicant pre-
viewed using advanced microscopy techniques dictive information.
and often computer-based image analysis systems
as well.
In recent years, advances in automation have 24.8 Flow Cytometry
generated higher-throughput solutions for IHC
and ICC. One such advance, tissue arrays, allows One of the most powerful techniques to make use
the placement of multiple patients samples on a of the versatility of Abs is ow cytometry [26].
single slide, which leads to a signicant increase An ever-increasing number of uorophores are
in the uniformity and speed of slide preparation. available as reporters, allowing high orders of
Further, increasingly sophisticated software and multiplexing with newer instruments; in some
new automation systems reduce the amount of cases, up to 11 different parameters can be
time that is required to screen slides, thereby recorded simultaneously. These reporter uoro-
greatly increasing throughput. An example is the phores absorb the energy provided by laser light
InScape system, which includes the scanning of at a specic excitation wavelength and then
the slide to create a high-resolution digital image, emit energy at a different emission wavelength.
and automated determination of results using This emitted light is captured by the cytometer
marker-based algorithms after the region of inter- using an elegant and elaborate series of optical
est is chosen by a pathologist. The result is then lters and photomultipliers (Fig. 24.8). In newer
veried by the pathologist, saving a great deal of cytometers, multiple lasers are used to increase
time in the analysis of IHC stains. the available excitation spectrum and thus take
ICC and IHC continue to be valuable tools for advantage of the range of available uorophores;
pathologists due to the ability of the technique to these cytometers therefore require computer-
map the location of the target protein to a specic controlled timing of the lasers and optical lters.
position within the cell. Some types of proteins, The combination of these numerous reporters
such as transcription factors, are regulated wholly with the adaptability provided by streptavidin
or in part by localization. For example, many conjugation of the uorophores and pairing with
transcription factors are found in the cytoplasm biotin-conjugated Abs provides an impressive
when inactive and shuttled to the nucleus follow- number of possible analyte combinations that can
ing activation. Mutations in some proteins that be studied for a particular cell type or biological
lead to improper localization within the cell have uid.
been demonstrated to contribute to malignancy. Initially, and perhaps still predominantly, ow
ICC/IHC assays for the visualization of the local- cytometry was used as a platform for the study of
ization of these proteins, as well as assays that intact cells, intended to measure the levels of pro-
detect the presence or absence of posttransla- teins present on the surface of the cell. The multi-
tional modications, different isoforms, and even plexing ability provided by the range of uorophores
mutant proteins, are all valuable diagnostic and and number of possible parameters allows the anal-
prognostic tools for pathologists. ysis of several surface markers simultaneously and
a b
Excitation Emission
wavelengths wavelengths
Lasers Ab-labeled Detectors

Flow cell cell
c
Parameter 6
Counts
Sample Waste
input output Parameter 2 Parameter 3
Fig. 24.8 Basic principles of ow cytometry. (a) Cells, detectors after passing through a complex system of optics
which have been incubated with uorophore-conjugated (not shown). (c) Software manipulation of the recorded
Abs, are drawn from the sample tube into the machine, light signals results in data that can be analyzed in many
where they pass the beam(s) of laser light in single le and ways and combinations. Each target assayed, or parame-
continue on to a waste receptacle. (b) As the cells pass the ter, can be analyzed in tandem with any other in a dot plot
interrogation point, any bound uorophores are excited by (left; see Fig. 24.9 for more details) or analyzed singly in
the laser light. The excited uorophores then emit light at the form of histograms and then compared to the histo-
slightly different wavelengths, which are captured by grams of other samples (right)
has made possible the characterization of the actually offer the average response of the entire
numerous subsets of cell types present in the human population tested. Even the most carefully puri-
body. However, advances in the technology in the ed cell preparations generally contain a mix-
last few decades have also allowed the detection ture of different cell types, and this
and quantitation of both intracellular and soluble heterogeneous population may very well
proteins using ow cytometry, as well as cellular express the protein of interest at different levels
DNA content, greatly expanding the possibilities or even exhibit a differential reaction to the
afforded by this platform. conditions being studied. This heterogeneity
The events occurring inside a given cell can can make it difcult to interpret results and rep-
provide valuable insights, including whether resents a major roadblock for the study of rare
the cell is activated, in the process of proliferat- cell types, which are in short supply and often
ing or in the process of dying under particular difcult to adequately purify. For these reasons,
conditions. In more traditional cell biology the ability of ow cytometry to discriminate
research, these questions would generally be between lineages by surface marker expression,
answered using Western blotting or perhaps and combine this with intracellular cytokine
even ELISAs. Despite being powerful methods staining in preparations of xed and permeabi-
which characterize the response of a population lized cells, is an important advance in studying
of cells to a given condition, both techniques intracellular events in mixed populations of
a A b
Surface marker A
Surface marker B
B c
B+ population
Counts A+ population
Intracellular marker
Fig. 24.9 Surface and intracellular cytokine staining of cell density. Surface marker A (green reporter; y-axis)
permeabilized cells. (a) Mixed cell populations are labeled is present at high levels on the upper cell, while surface
with Abs specic for surface markers that identify subsets marker B (red reporter; x-axis) is absent, indicating that
such as different lineages, different activation states, and these cells will fall in the top left corner of the dot plot.
others. Two different cell subsets are indicated here by Conversely, the lower cell shows high levels of marker B
binding to two different surface marker Abs, represented and low levels of marker A, placing them in the lower
here by green (A, upper) and red (B, lower) reporters right corner of the dot plot. These expression patterns cre-
which will be seen by the cytometer as different param- ate two distinct populations in the dot plot. Gates can
eters. The cells are then permeabilized to allow passage then be drawn around the populations (rectangles), telling
of Abs across the membrane, represented by the dashed the software to consider only those cells falling within the
line surrounding the cell. Permeabilized cells are incu- gate in downstream analyses. (c) The cells within each gate
bated with Abs specic for the intracellular target (purple are analyzed for levels of the intracellular protein (purple
reporter), which will be seen by the cytometer as a third reporter). Levels are suggested by the intensity of the stain-
parameter that is the same for all cells. (b) After sample ing for the third parameter (Intracellular Marker, x-axis).
acquisition by the ow cytometer, the different cell sub- The diagram in (a) depicts the upper cell as having a lower
sets are differentiated by their expression of the surface level of the target intracellular protein, and this is reected
markers for which they were stained. Comparison of two by the green histogram falling farther to the left on the
parameters is generally done with a dot plot, in which each scale than the red histogram, indicating a higher intensity
dot represents a single cell; the dot plot shown here is col- of staining in the surface marker B-positive cells than in
ored like a heat map to indicate areas of greater and lesser the marker A-positive cells
cells (Fig. 24.9) [2732]. These sorts of intra- tively containing the protein of interest. Following
cellular cytokine staining protocols have capture by the beads, the protein can then be
allowed the study of cell signaling cascades in bound by a specic detection Ab. As with sand-
intact normal cells [33], as well as characteriza- wich ELISAs, the bead-based assay can use up to
tion of aberrant signaling in mutation-bearing four Abs, but again, fewer Abs are generally pre-
cancer cells and in cancer cells exposed to ferred (Fig. 24.10). One successful application of
emerging therapies. this technology is the detection of soluble proteins
Further advances in ow cytometry have even released into the bloodstream by dying leukemia
made it possible to mix samples from two differ- cells [3537]. Despite the similarities of the tech-
ent sources, including from two discrete patients nique to the sandwich ELISA, the bead-based
or from a single patient pre- and posttreatment, assay benets from greater multiplexing possi-
using a barcoding method [34]. Each sample is bilities, including the Luminex and cytometric
mixed with a different uorescent dye that emits bead array technologies.
at a distinct signature wavelength, which, when As stated above, the most advanced cytome-
the samples are mixed, allows discrimination of ters can measure upwards of 11 or more parame-
each through sorting based on the detection of the ters. This often presents calibration issues due to
signature. Although a boon for researchers, this the slight spectral overlap of the uorophores
technique has yet to become standard practice in available. One approach to avoiding this problem
clinical oncology diagnostics laboratories. Flow is to use a single uorophore to measure different
cytometry itself, however, is rmly entrenched, analytes, rather than a large number of different
primarily as a valuable tool for hematopatholo- colors. The cytometric bead array (CBA)
gists, who use ow cytometry to examine the makes use of beads of different sizes, one size for
populations of circulating cells in the blood in each of the different capture antibodies to be
order to discover subsets of abnormal cells, such used. All detection antibodies can then be conju-
as those present in hematological malignancies gated to the same reporter uorophore, because
like leukemias and lymphomas. Flow cytometry the discrimination between the different proteins
panels for differential diagnosis of leukemia/ detected will be provided by the size of the bead,
lymphoma can contain upwards of 20 cell surface which is one of the parameters measured as the
markers, and algorithms characterizing the particle ows past the cytometers detector. These
patterns of these markers on the surface of cell different bead sizes will result in easily distin-
populations in the blood help pathologists iden- guishable populations and thus analytes, as
tify the particular type of leukemia or lymphoma shown in Fig. 24.11a, while the level of protein
present. captured and detected by a given antibody pair
will be quantied by the intensity of the report-
ers uorescence (not shown). In this way, the
24.9 Bead-Based Assays CBA assay allows the measurement of multiple
analytes side by side in the same sample.
As with the detection of intracellular proteins, the Beyond just determining the relative amounts
study of soluble proteins present in bodily uids of protein captured by the CBA assay, however,
and in cell culture supernatants was traditionally researchers have applied a standard curve to the
performed by immunoblots or ELISA. But again, assay, allowing the quantitation of detection Ab
as with intracellular proteins, ow cytometry now molecules bound to a bead. Each experiment
represents an additional platform for the detection includes a tube containing four groups of beads,
of soluble proteins through the use of bead-based each with a different known level of bound
assays. In a design that combines the best features reporter uorophore. The data derived from this
of IP and sandwich ELISAs, Abs are coated onto sample is used to generate a standard curve, plot-
microbeads rather than plates, and these beads ting the known number of reporter molecules
can then be incubated with the sample uid puta- against the mean uorescence intensity (MFI)
a b c
Wash and analyze
Add sample Add detection Ab
Target
Other protein
Other protein
Antibody
Target bound to antibody
Microbead
Detection antibody
Fig. 24.10 Bead-based ow cytometry assays. (a) protein. (c) The target protein is bound by uorophore-
Capture Abs are coated on microspheres. (b) The beads conjugated detection antibody, the sample is washed to
are incubated with proteins in solution (e.g., lysate, cell remove unbound detection antibody, and the beads are
culture supernatant, or plasma) and bind only the target analyzed by ow cytometry
measured by the cytometer. Using this curve and Abs are added, all conjugated to the same
the MFI value recorded for a given sample, the reporter uorophore as in the case of the CBA
number of bound reporter-conjugated detection assay. The data are then collected using the basic
Abs can be calculated. This technique provides principles of ow cytometry, in that the dyes
an even more accurate quantitation of the level of inside the beads are excited with a red laser to
the target protein present in the matrix and can reveal the signature identifying which target
even be applied to the more traditional non-bead- should be captured by that particular bead, and a
based ow cytometry methods of intracellular green laser is used to excite the reporter uoro-
and surface protein detection. phore to allow the measurement of the levels of
The Luminex technology makes use of a protein actually captured [38]. The multiplexing
combination of the advantages of both micro- capabilities of this platform provide the potential
bead assays and ow cytometry, creating a for Luminex to provide as much information
method ostensibly able to analyze up to 100 tar- about a sample as some types of antibody micro-
gets in one well (see Luminex Corporation for arrays or multi-spot ELISAs (see below) and is
examples). Luminex makes use of polystyrene therefore currently more often used in a cancer
microspheres impregnated with carefully con- research or clinical trial setting.
trolled levels of both red and infrared dyes.
These different titrations create different color
signatures for each population of beads, much 24.10 Antibody Arrays
like the barcoding technique described above
(Fig. 24.11b). These different beads can then be The antibody microarray makes possible the
coated with discrete capture Abs, mixed together, detection of a very large number of analytes in
and incubated with the biological matrix. a complex sample, similar to its predecessor,
Following capture of the target proteins, detection the DNA microarray [39, 40]. Most antibody
Side scatter
Forward scatter
Counts
Parameter X
(bead color)
Fig. 24.11 Cytometric bead array and Luminex technol- of the same size which have been impregnated with dyes of
ogies. (a) The CBA platform consists of the Abs specic slightly different wavelengths. Each set of beads is coated
for each target being conjugated to beads of a different with a different capture Ab, incubated with sample to capture
size. The beads are incubated with the sample at the same target protein, and detected with a uorophore-conjugated
time, allowing capture of the target proteins. The beads are detection Ab (left). The cytometer-based analysis instru-
then incubated with detection Abs for each target, all con- ment detects the slight variations in the color of the bead
jugated to the same uorophore (left). When analyzed, the (Parameter X), creating discrete populations based on bead
different bead sizes are recognized by the cytometer via color which can be gated (right). The reporter uorophore
the forward and side scatter parameters and are identiable intensities within each population can then be analyzed,
as discrete populations that can be analyzed separately via yielding information about the concentration of each target
gating (right). (b) Luminex technology makes use of beads analyte
microarray formats are essentially ELISAs on a mats, including the variable of whether it is
necessarily grand scale, as shown in Fig. 24.12. protein or antibody bound to the array itself.
These arrays are valuable both for basic research In its infancy, antibody array technology most
and in the search for diagnostic and prognostic closely paralleled that of DNA microarrays by
markers of cancer. A small volume of biological spotting the surface of the array with probes con-
material can yield a substantial amount of infor- sisting of mAbs. Universally labeled proteins are
mation using this technique, and often of greater then incubated with the array, and the captured
importance, relationships and patterns within the protein is identied by its binding position on the
data can be recognized and characterized in a array (Fig. 24.12a) [39]. The protein-labeling pro-
single snapshot experiment. Antibody microar- cess includes either direct labeling with reporters
rays can be designed in a number of different for- or indirect detection using biotin or digoxigenin.
Direct array Competitive direct array
Sandwich array
Pre-therapy Post-therapy
Reverse phase array
Fig. 24.12 Antibody array formats. (a) Direct antibody depicted in Fig. 24.5b, simply with a large number of cap-
arrays involve the spotting of specic Abs onto a surface. ture Ab specicities combined into a single assay and
The array is then incubated with reporter-labeled proteins requiring one small volume of analyte. (c) The reverse-
(left). The identity of a target protein that binds to the array phase array consists of the proteins in a sample being
is determined by matching the location of the signal to the adsorbed to the array surface, followed by detection with
known layout of the Abs. In a competitive direct array, the reporter-conjugated Abs as in Fig. 24.6a. Although the
proteins in two separate samples are labeled with distinct number of targets that can be analyzed simultaneously is
reporters (red and green) and incubated with the array limited here, the value of the reverse-phase array is that it
simultaneously (right). The target proteins will compete for allows multiple samples to be analyzed side by side. The
binding to the Abs on the array, and the relative signal example represented here is pre- and post-therapy, and the
intensities will indicate which sample contained greater changes in protein expression resulting from the treatment
quantities of each protein assayed. (b) The sandwich anti- are clear
body array is highly similar to the sandwich ELISA
Through the use of multiple reporters, it is also is always the concern that the direct labeling of
possible to compare two samples by incubating the proteins may interfere with recognition of the
them together in a classic competition assay protein by the Ab due to the physical masking or
(Fig. 24.12a). This antibody array format is gen- alteration of the epitope.
erally referred to as a direct array and is the best With these limitations in mind, additional
option for assaying truly large numbers of ana- antibody microarray formats were developed to
lytes in a single array, as the only major limita- include both capture and detection antibodies
tions are space and the availability of specic (Fig. 24.12b) [41]. Specicity is greatly enhanced
antibodies for the desired targets. To date, most when relying on the recognition of the target pro-
arrays offered commercially contain analytes tein by two different Abs for detection, as one
numbered in the hundreds. The primary technical source of background is minimized. In addition,
hurdles encountered when using direct Ab arrays the problem of possible epitope masking is also
include limited specicity and sensitivity and solved by removing the necessity of labeling
ltering out background signal. In addition, there the proteins. One limitation of this sandwich
approach, in both basic ELISAs and the antibody molecular assays dont tell the whole story. For
array, is the occasional lack of good matched example, it has been amply demonstrated that
antibody pairs. Another concern is the problem the level of mRNA, though often useful as a
of cross-reactivity among the detection antibod- marker in and of itself, does not always directly
ies, which generally serves to limit the number of correlate to the level of the protein that will be
possible targets when using a sandwich microar- translated. Similarly, molecular assays reveal
ray in contrast to a direct array. However, as the nothing about the posttranslational modica-
targets of greatest interest or benet for a given tions that can dictate subcellular localization or
model or cancer type are determined, highly cus- activation of a protein, which can be a more tell-
tomized arrays are being developed for diagnosing measure of aberrant function than the
tic, prognostic, and research uses. For example, sequence of the gene. The ability to study the
some arrays are designed to study groups of puta- actual protein of interest itself is an important
tive or known breast cancer markers, while others aspect of learning as much as possible about the
are used to screen the effects of drug candidates malignancy, to better ght and defeat it. To this
on their target cells. end, researchers have harnessed the power of the
There is also, as might be expected, an anti- immune system to create clever tools for the
body microarray design in which it is the protein study of proteins via the exquisite sensitivity of
mixture that is immobilized on the surface of the Abs, and these tools continue to be absolutely
array (Fig. 24.12c) [41]. These protein spots can invaluable in the diagnostic workup of cancer
then be probed with reporter-conjugated specic patients.
Abs. This reverse-phase array allows the immo-
bilization of multiple samples proteins on a
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Flow Cytometry in Cancer
Immunotherapy: Applications, 25
Quality Assurance, and Future
Ccile Gouttefangeas, Steffen Walter,

Marij J.P. Welters, Christian Ottensmeier,
Sjoerd H. van der Burg, Cedrik M. Britten,
and Cliburn Chan

25.1 Introduction................................................. 471
Cancer immunotherapy seeks to elicit or augment
25.2 Main Flow Cytometry Assays
in Cancer Immunotherapy ........................ 472
the antitumor immune response in a patient with
detectable tumor or remaining tumor cells in the
25.3 Panel Development and Quality
adjuvant setting in order to enlist the help of the
Assurance .................................................... 474
patients own immune system for tumor control.
25.4 Prociency Programs Addressing In this context, active cancer immunotherapy
Flow Cytometry Assays .............................. 477
refers to the use of cytokines (e.g., IL-2 in melanoma
25.5 Structured Reporting of Immune
Assay Experiments ..................................... 478
25.6 Organization of Immune Monitoring
in Multicenter Trials ................................... 479 C. Ottensmeier, MD, PhD, FRCP
25.7 Towards Automated Analysis .................... 480 University of Southampton, Mailpoint 824,
Cancer Science building, Tremona Road,
25.8 New Methods and Technologies................. 482 Southampton SO16 6YD, UK
e-mail: c.h.ottensmeier@soton.ac.uk
25.9 Concluding Remarks .................................. 485
S.H. van der Burg, PhD
References ............................................................... 486
Experimental Cancer Immunology and Therapy,
Department of Clinical Oncology (K1-P),
Leiden University Medical Center,
C. Gouttefangeas, PhD Albinusdreef 2, 2333 ZA Leiden, The Netherlands
Department of Immunology, e-mail: S.H.van_der_Burg@lumc.nl
Institute for Cell Biology, University of Tbingen,
C.M. Britten, MD
Auf der Morgenstelle 15, 72076 Tbingen, Germany
TRON, Translational Oncology at the University
e-mail: cecile.gouttefangeas@uni-tuebingen.de
Medical Center of the Johannes-Gutenberg
S. Walter, PhD University gGmbH and Association for Cancer
Immatics Biotechnologies GmbH, Immunotherapy (CIMT),
Paul-Ehrlich-Strae 15, 72076 Tuebingen, Germany Building 708, Langenbeckstr. 1, 55131 Mainz,
e-mail: walter@immatics.com Germany
e-mail: Cedrik.Britten@TrOn-Mainz.DE
M.J.P. Welters, PhD
Experimental Cancer Immunology and Therapy, C. Chan, MBBS, PhD (*)
Department of Clinical Oncology (K1-P), Department of Biostatistics and Bioinformatics,
Leiden University Medical Center, Duke University Medical Center,
Albinusdreef 2, 2333 ZA Leiden, 11078 Hock Suite 1102, Hock Plaza,
The Netherlands 2424 Erwin Road, Durham, NC 27705, USA
e-mail: mjpschoenmaekers-welters@lumc.nl e-mail: cliburn.chan@duke.edu

472 C. Gouttefangeas et al.
and renal cell carcinoma), immunomodulatory to harmonize the assays across multiple laborato-
monoclonal antibodies (e.g., antibodies (Abs) ries. This chapter describes the main ow cytom-
against CTLA-4, PD-L1, and PD-1), cell-based etry methods being applied in cancer
products (e.g., sipuleucel-T for metastatic hormone- immunotherapy, with an emphasis on recent prog-
refractory prostate cancer), or experimental vac- ress in the eld, challenges associated with quality
cines based on various antigen (Ag) formats. control, its promise to reveal biomarkers of clinical
When evaluating immunotherapies, particularly efcacy, and further developments that are likely
in experimental settings, it is essential to monitor to be rapidly implemented in routine cancer
the immune response elicited by the treatment. immunology.
Immunomonitoring delivers evidence of immu-
nogenicity, guides the choice and dosage of
antigens, assesses the effects of immune modulators 25.2 Main Flow Cytometry Assays
and therapy combinations, and has the potential in Cancer Immunotherapy
to reveal early biomarkers of clinical efcacy. In
this respect, immunomonitoring is helpful for Together with immunohistochemistry, immuno-
rational clinical development and supplements phenotyping by ow cytometry is probably the
clinical efcacy parameters such as disease-free most commonly used assay to investigate immune
period or survival, which are often only available and other cell subsets of interest in cancer immu-
at later clinical trial stages. nology. Flow cytometry distinguishes human
In view of their role in the anticancer immune immune cells via a combination of physical prop-
response, the quantity and quality of tumor antigen- erties and uorescent markers such as labeled
specic effector CD4+ and CD8+ T cells are of par- monoclonal antibodies (mAbs) targeted against
ticular interest. In addition, the role of immune cell-specic molecules. Physical properties mea-
regulatory cells, e.g., regulatory T cells (Tregs) or sured by the cytometer are forward scatter light
myeloid-derived suppressor cells (MDSCs) that (FSC) which is roughly proportional to the cell
can suppress the effector immune response to a size and side-scattered light (SSC) which reects
tumor, is increasingly recognized. Informative the granularity of cells. Markers targeted by uo-
analysis requires multiple markers for identica- rescent mAb are mostly categorized in Clusters
tion of phenotypic and functional properties and of Differentiation (CD) nomenclature [1]. To
the accurate quantication of cell subsets that are date, the Human Cell Differentiation Molecules
typically found at relatively low frequencies in Association (http://www.hcdm.org) has indexed
the peripheral blood. These characteristics call for more than 360 CD markers. Commonly used
an assay that is multiparametric, robust, and sensi- basic CD markers are CD3, CD4, and CD8 for
tive enough to characterize rare individual cells. T-cell subsets, CD19 for B cells, CD14 for mono-
The canonical multiparameter assay for the cytes, CD11c for subsets of dendritic cells, CD56
characterization of single cells in solution is poly- for natural killer (NK) cells, and CD15 for granu-
chromatic ow cytometry, and hence, it is ubiqui- locytes. In addition to whole blood and PBMC
tously used for immune monitoring in preclinical samples, enumeration of the number and fre-
tumor immunology and in cancer immunotherapy quencies of immune cell types can also be per-
trials. While the rst uorescence-based ow formed on single-cell suspensions obtained from
cytometer dates back to 1968, the past several tissues (for instance malignant tumors) [2, 3].
years have brought major advances in cytometer When analyzing tumors, further markers can be
technology, reagents, range of applications, auto- added to identify endothelial cells (CD31), bro-
mated analysis techniques, and minimal informa- blasts (ER-TR7), epithelial cells (EpCAM, i.e.,
tion standards. Much has also been learnt from CD326), and particular tumor cells (e.g., CAIX
large-scale prociency testing programs about the for renal cell carcinoma).
challenges facing th use of increasingly complex Many cell populations can currently only
ow cytometry assays, and what needs to be done be identified by the use of multiple mAb
25 Flow Cytometry in Cancer Immunotherapy: Applications, Quality Assurance, and Future 473
simultaneously; this is the case for natural by the TCR) and its HLA-restriction (i.e., the
regulatory T cells (nTregs) [CD4+/CD25high/ HLA-molecule which binds and presents the
Foxp3+/CD127low or various subsets of MDSCs peptide to the TCR) must be known in advance.
[4]. Polychromatic ow cytometry is also neces- To date, there also remains a lack of general
sary to characterize the activation status, matu- availability of class II multimers for CD4+ T-cell
rity, clonality, and differentiation status of T detection [26].
lymphocytes. Commonly used markers for this Intracellular cytokine staining (ICS) is another
purpose include CD25, CD27, CD28, CD45RA/ common assay used for antigen-specic T-cell
RO, CD69, CD137, and CD154, as well as anti- immune monitoring. It is the ow cytometric
bodies to different TCR V family members [5 method of choice when HLA-multimers are not
9]. A combination of mAbs against activation available, if the exact T-cell epitope is unknown,
markers and chemokine receptor (i.e., CCR7) and for routine monitoring of CD4+ T-cell
can be used to identify nave, effector memory, responses. ICS enables monitoring of multiple
central memory, terminally differentiated effector effector functions of both CD4+ and CD8+ T-cell
memory (TEMRA), and memory T cells with subsets [2729], including polyfunctional T cells
stem cell-like features [1012]. These differ- that have been associated with pathogen protec-
entiation stages are associated with changes in tion [30, 31]. A few groups have described poly-
functional and proliferative properties [13], are functional T cells after cancer vaccination in
altered in the elderly [14], and hence are relevant patients, but whether these cells are associated
for adoptive transfer therapy and for possibly with benecial and long-lasting antitumor T-cell
predicting response to vaccination in aging responses remains an open question [32, 33].
cancer patients. However, up to now, there is no Optimized Ab combinations, protocols, and
gold standard for markers that are necessary and standardization approaches have been published
sufcient to identify most immune cell subsets; [3436], and ICS assays are widely used in clinical
this is not surprising as our appreciation of the studies.
complexity and plasticity of human immune cell Cytotoxicity or proliferation assays, which
subsets is constantly evolving. have traditionally relied on the detection of
A major interest in immunotherapy clinical radioactivity (i.e., 51Cr release or 3H thymidine
trials is to characterize the specicity of tumor incorporation) can also be conducted by ow
antigen-specic T cells, most notably in settings cytometry. For assessment of killing activity,
of active immunotherapy with dened Ags. The target cells (control and antigen-loaded cells
most direct characterization of antigen specicity or tumor cells expressing the antigen endoge-
is via the use of HLA-peptide multimers, which nously) are differentially labeled using fluo-
bind directly to the peptide-specic T-cell receptors rescent dyes (e.g., Paul Karl Horan (PKH) or
(TCR). First described more than 15 years ago 6-carboxyuorescein diacetate succinimidyl
[15], the HLA-class I multimer assay currently ester (CFSE)) and incubated with the effector T
serves as a versatile tool for enumerating, charac- cells to be tested. Apart from the obvious safety
terizing, and following CD8+ T cell immune aspects over radioactivity-based assays, advantages
responses, and staining protocols are broadly of ow cytometry methods are that (1) several
available [1618]. Hence, HLA-multimers are targets can be tested in the same tube; (2) as com-
widely used to monitor T-cell responses, espe- pared to a classical 51Cr release assay, effector-
cially in the context of peptide-based vaccination target incubation time can be signicantly
approaches [1922]. They can easily be combined prolonged (up to 24 h); and (3) the assay has been
with mAb panels to determine the phenotype and reported as being sensitive and effective even
differentiation status of antigen-specic CD8+ T when low numbers of effectors are available [37,
cells [2325]. Limitations of HLA-multimers are 38, 39]. Another approach to indirectly deter-
that both the precise T-cell epitope (i.e., the exact mine the cytotoxic capacities of T cells is the use
amino-acid sequence of the peptide recognized of a mAb directed against CD107a (LAMP-1)
which becomes extracellularly detectable after the need for both robustness and sensitivity to
cytotoxic granules have fused with the cellular detect tumor antigen-specic T cells and/or rare
membrane (degranulation) [40]. For measuring cell subsets poses specic challenges for the use
proliferation by ow cytometry, effector cells are of this complex tool in clinical research applica-
rst labeled with uorescent dyes (CFSE or other tions. This is addressed in the following sections.
tracking dyes such as CellTrace reagents) and
cultured for several days in the presence of rele-
vant stimuli. Since the dyes are diluted from the 25.3 Panel Development
mother to the daughter cells, the number of cell and Quality Assurance
divisions is visible in the number of uorescent
peaks detected [41]. The frequency of proliferat- Current state-of-the-art polychromatic ow
ing cells can also be assessed directly ex vivo by cytometry in cancer immunotherapy involves
staining of the proliferation-associated nucleus multistep, multi-reagent assays followed by sam-
Ag Ki67, expressed at all phases of the cell cycle ple acquisition on sophisticated instruments that
except the resting G0 stage [4 , 42 ]. These are able to capture up to 20 parameters per cell at
measurements of target killing or cell division by a rate of tens of thousands of cells per second.
uorescent dyes have rarely been used in large- Analysis of these data can be a challenge, as stan-
scale vaccine studies so far [38, 43], probably dard tools require multistep gating strategies and
because they are time-consuming and require preselection of the parameter combinations to be
careful optimization and technical expertise to investigated. Obtaining reproducible results from
achieve reproducible results. such a complex assay requires well-trained staff,
Finally, cell-free cytokine analysis can also be stringent quality management, and detailed
performed by ow cytometry with the use of standard operating procedures (SOPs) for panel
multiplex beads, a method that has been recently development, cytometer calibration, reagent
adapted to meet GCLP standards [4446]. The qualication, sample preparation, use of appro-
method uses mixes of beads of different size and priate technical and biological controls, and
uorescence that are each coated with Abs spe- careful data analysis.
cic for the different cytokines of interest. The We start by considering the factors important
soluble cytokines present in the sample (i.e., cul- to understand when developing a mAb staining
ture supernatant, serum, or plasma) bind to these panel. Target molecules in ow cytometry for
Ab-coated beads, and a second Ab coupled to cancer immunotherapy can have vastly different
another uorescent label is used to visualize the expression levels. While lineage markers such as
amount of bound cytokine. Simultaneous quanti- CD45, CD3, or CD8 can be expressed at very
cation of several soluble factors in one sample high copy numbers per cell, some important mark-
can be done by comparison to standard curves ers such as transcription factors (e.g., FOXP3 for
provided by the manufacturer, for example, to CD4 Tregs) or chemokine receptors (e.g., CCR5
evaluate Th1/Th2 proles [28]. The assay is as on CD4 Th1 cells) are expressed at much lower
sensitive as ELISA, with detection limits in the levels. In addition, the available probes (such as
range of 20 pg/mL for most cytokines, and can be mAb or HLA-peptide multimers) can have vari-
even more sensitive when an enhanced sensitivity able afnities for their respective targets. Probes
system is used. are labeled with different chemical classes of
The examples above clearly show that ow uorescent dyes that must be matched to the
cytometry is a versatile tool for investigations of instrument, considering factors such as the avail-
the phenotype, frequency, and functional proper- ability of a high-power laser line with a wave-
ties of immune cell subsets. Furthermore, assays length close to the maximum absorption of the
can often be combined for multiparametric uorescent dye and with a detector (photomul-
probing of cell properties which is benecial as tiplier plus lters/mirrors) that has a high
precious patient samples are spared. However, sensitivity in the spectral emission range of the
given dye. Complicating matters further, cellular to the absorption maximum can reduce errors in
autouorescence (i.e., uorescence due to cellu- photon counting, and narrow bandpass lters
lar molecules such as NADPH even in the can reduce spillover; both these measures will
absence of all dyes) limits the sensitivity that can reduce spreading error. Finally, probe combina-
be achieved with a given uorescent probe, laser, tions should be designed so that overlapping u-
and detector. In practical terms, autouorescence orochromes are chosen for labeling markers
of lymphocytes is usually limited to a distinct which are expected to be expressed on different
range of emission and absorption wavelengths cells.
[47, 48]. In general, the degree of autouores- In practice, panel development usually starts
cence determines the limit of detection, which in with the denition of a wish list of cellular targets,
earlier reports was of 3,000 molecules for a stan- followed by the prioritization of these cellular
dard ow cytometer [49]. Consideration of all targets, characterization of their expression
these factors leads to the following recommen- levels, and checking for the availability of probes
dations for detecting cellular markers expressed and conjugated dyes appropriate for the cytometer
at very low levels: use a high afnity Ab conju- to be used. Guidance documents [54] and helpful
gated to a uorescent dye with high quantum software (CytoGenie: www.woodsidelogic.com,
yield with emission spectral range far away from Fluorish: www.uorish.com, Chromocyte: www.
cellular autouorescence, for which the cytome- chromocyte.com) are available. A practical limi-
ter has an appropriately matched high-power tation can be the lack of commercially available
laser line and detector. uorochrome conjugates for individual antibody
For polychromatic ow cytometry, additional clones. Indirect staining with secondary reagents
constraints are set by the phenomena of optical (such as the biotin-streptavidin system) is
spillover and spreading. In ow cytometry, cells possible but often not practical for multicolor
are analyzed in a near-physiological aqueous applications. A better alternative is the use of new
solution to preserve the structural properties of methods now available for the self-conjugation
biomolecules. Due to the spectral absorption of of small amounts of Ab to uorescent dyes
water and air, the useful spectral space is limited [55, 56]. Based on the discussion above, the cor-
to the range from Near-UV (ca. 200 nm) to nerstones of panel development guidance are the
Near-IR (ca. 1,000 nm). Also, in aqueous solu- assignment of bright probes for dim targets
tions, both the absorption and emission of uoro- and strategies to avoid spreading error and
chromes show relatively broad spectral lines. autouorescence in channels relevant for dim
Together, this means that the number of uoro- targets. It is also possible to change the optical
chromes that can be analyzed at the same time is pathway of the ow cytometer to optimize the
ultimately limited: the combination of 1520 instrument (e.g., choice of lters) according to
different uorochromes appears to be the upper the requirements of the panel. As the amount of
feasibility limit [50]. potential interference between dyes rapidly
As a further consequence, spectra of uores- increases with the number of colors in the panel,
cent dyes routinely overlap (spillover) [51], and as a large number of critical parameters
requiring software deconvolution of true and should be optimized, development of large
observed signals (compensation). However, (8 colors) panels and especially those
compensation cannot correct other errors caused involving separate staining steps for intracellular
by measurement, binning, and photon noise, and and extracellular targets can be an expensive iter-
these errors accumulate to give an irreversible ative process requiring several man-months
effect termed as spreading error [52] or spillover of dedicated work. Hence, the ow community
spreading [53]. Spreading error will cause the pres- is encouraged to share rigorously calibrated
ence of one bright uorochrome to reduce sensi- and optimized polychromatic panels via the
tivity for spectrally close uorochromes present Optimized Multicolor Immunouorescence
on the same cell. Use of a high-power laser close Panels (OMIPs) project [57].
Quality assurance of a ow cytometry assay can help with setting gate boundaries at the
starts with the ow cytometer itself, consisting of analysis stage by dening the negative region.
optimization, calibration, and standardization Pretested, aliquoted, cryopreserved samples with
of the machine, and we refer the reader to the prescreened, predictable properties (such as
technical report by the Roederer group for details being positive or negative for individual markers
[58]. These optimization steps must not be in the mAb panel) can serve as valuable biological
neglected, as they may identify faulty parts that controls which can be used in each assay run to
need replacement, such as a photomultiplier tube track the variations in assay performance between
(PMT) with reduced sensitivity or suboptimal operators and over time.
lters, and are important to optimize general As ow cytometry-based methods become
instrument parameters. Conveniently, some (but incorporated into clinical trials, the need for a
not all) of these steps have been incorporated in stable and unlimited source of cell specimens
vendor software packages, such as the Cytometer that contains dened numbers of functional
Setup and Tracking (CS&T) application within antigen-specic T cells as batch controls becomes
BD FACSDiva 6 that uses a proprietary mixture paramount. Moreover, cell samples containing a
of calibration beads. For long-term immuno- known number of T cells specic for a dened
monitoring, it is essential to maintain accurate Ag would allow easy assessment of the quality
records of daily monitoring checks to track and accuracy of assays and provide standard con-
reproducibility and stability. trols for comparison of results across laboratories
For cell staining, reagent quality can be an or time. Currently available sources for reference
issue, especially if the assay is performed repeat- samples are either (i) based on leukapheresis or
edly over time. Often, reagents used are classied buffy-coat material from healthy donors which
as research use only (RUO) and can show con- are restricted to reactivity against immunogenic
siderable batch-to-batch variation in important viral Ags, expensive and available in limited
properties, such as concentration of antibody-dye amount, or (ii) dependent on the ability to gener-
conjugate, concentration of free dye, and even ate and propagate T-cell lines/clones on a
in the spectral properties of the dye (as in the case repetitive basis which is a burdensome task. The
of tandem dyes). In addition, the shelf life desig- Cancer Immunotherapy (CIMT) Immunoguiding
nated by vendors is not always based on quantita- Program (CIP) group has recently established a
tive specications. As a result, individual reagent process for the generation of reference samples
batches have to be pretested and pre-titrated, and (RS) that can be used in T-cell assays. In a rst
tests repeated even during the designated shelf proof-of-principle study, we showed that retrovi-
life of a reagent. As batch sizes available from rally TCR-transduced T cells spiked at dened
vendors are often limited, this can result in the numbers in autologous PBMC can be used as
requirement of reagent bridging (demonstration standard samples. The T cells could be accurately
of the comparability of reagent batches) during detected at all dilutions in a linear fashion,
the course of a study, leading to complex logistic down to frequencies of at least 0.02 %, and the
and tracking processes. Reagent quality assurance feasibility of RS was conrmed in a small-scale
may be facilitated by the preparation of mixtures prociency panel [60]. Subsequently, we estab-
of lyophilized reagents (lyoplates) [59] that can lished, optimized, and standardized the produc-
reduce pipetting error and lead to increased tion of RS obtained by transfection of modied
reagent stability. and stabilized RNA. Such a platform offers a
Appropriate use of technical and biological simple, virus-free, and scalable process for the
controls is also vital for assay interpretation. In manufacturing of reference samples. In proof-of-
addition to instrument calibration beads, unstained concept studies for HLA-multimer experiments,
and single-stained beads are used to determine the feasibility of using such RNA-engineered RS
the spillover matrix for compensation. Isotype was shown. RS offered favorable properties
and uorescence minus one (FMO) controls across a variety of CD8+ and CD4 T-cell-derived
TCRs against multiple Ags, including clear clus- pros and cons of assay harmonization vs. stan-
tered populations, reproducible results, high sta- dardization have been discussed in detail else-
bility over time, and the potential for linear where [62, 68].
dilution. Moreover, the analysis of the RS is simi- Assay harmonization is based on the participa-
lar to that of the tested cell samples in that the tion of single laboratories in iterative testing exer-
same gating strategy (and even the same gates) cises called prociency panels. Pretested PBMC
can be used. This suggests that RS are a useful samples, synthetic peptides, and/or HLA-peptide
tool to control T-cell assay performance. The multimers are shipped from a central lab to all
suitability of these RS samples was subsequently panel participants who then use their own
tested in a prociency panel organized recently reagents, protocols, and analysis strategies for
(manuscript submitted). detecting antigen-specic T cells. Participants
A nal, critical aspect of quality management then report their data, which are centrally ana-
is the careful documentation of each procedure lyzed, allowing comparison of individual assay
performed, as well as provision of detailed variables and performance to detect T cells. Thus,
standard operating procedures (SOPs) for each parameters involved in assay performance may be
stage including data analysis. Technical staff successively identied, corrected, and conrmed
needs to be well trained and perform the analyses to exert an impact in subsequent panels (i.e., mul-
on a regular basis to keep up performance. tistep approach). Finally, benchmarks and guide-
Participating in prociency panels will also help lines are formulated and disseminated to the
improve laboratory standards. community. Participating laboratories benet by
being able to measure their own performance in
reference to peer laboratories, and regularly tak-
25.4 Prociency Programs ing part in prociency panels over time can also
Addressing Flow be seen as a quality control of assay performance
Cytometry Assays for individual labs. Additionally, the working
group can guide laboratories to improve perfor-
While HLA-multimers and ICS are commonly mance if needed, while providing an exchange
used for monitoring experimental vaccines or platform for assays and their application.
other anticancer immunotherapies such as adop- Prociency panels can in principle be applied
tive transfer of in vitro expanded T cells, there are for any T-cell assay, including those based on ow
still notable obstacles to the advancement of these cytometry [6971]. In 2005, two consortia, the
T-cell monitoring assays as robust biomarkers for European Cancer Immunotherapy (CIMT)
clinical trials [61, 62]. First, there is no gold stan- Immunoguiding Program (CIP) and the Cancer
dard protocol for any of these assays. Second, Immunotherapy Consortium of the Cancer
correlations between in vitro immunomonitoring Research Institute in the USA (CIC/CRI) launched
results and patient clinical benets have rarely a large program of prociency panels and syner-
been reported [4, 28, 6367]. The reality is that gistically pioneered the concept of assay harmo-
assays performed at different institutions are not nization [62, 68]. From 2005 to 2012, the CIP
equal; this results in difculties in comparing the (www.CIMT.eu/workgroups/CIP) has organized
efcacy of the various immunotherapy approaches 15 small- to large-scale prociency panels, dedi-
tested for recruiting a meaningful anticancer cated to the measurement of antigen-specic
T-cell response, in turn hampering progress in CD8+ T cells by HLA-multimers, ELISPOT, and
the eld. intracellular cytokine staining.
One approach for addressing these problems Prociency panels have taught us that there
is by assay validation and standardization and/or are large variations in the performance of T-cell
centralization of the immunomonitoring at a assays among the ow community. While the
dedicated core facility. An attractive alternative majority of labs do detect antigen-specic T cells
to these strategies is assay harmonization. The present at quite high frequencies in PBMC samples
(approx. >0.2 % of CD8+ cells), the detection rate (unpublished data). This is not a surprise, since
drastically decreases for low-frequency effectors manual gating is subjective and highly dependent
(<0.05 % of CD8+ cells). This is very relevant for on the experience of the experimenter and tradi-
cancer immunotherapy, as tumor-specic T cells tion in the lab. Further work is therefore needed
are expected to be present at low frequencies in with a focus on both data acquisition and analy-
the blood, even after patient vaccination. Another sis, including the potential for automated analy-
lesson is that comparable performance is achiev- sis strategies to reduce the subjectivity inherent
able with different laboratory-specic protocols in gating as described in Sect. 25.7.
and reagents, and full interlaboratory standard-
ization is not necessary for good results.
Surprisingly, we also found that operator experi- 25.5 Structured Reporting
ence in a method does not necessarily predict of Immune Assay
performance, underlining the utility of regular Experiments
quality control of established methods. Finally,
adoption of simple measures can lead to signicant An increasing number of minimal information
improvements in assay performance. For exam- projects have emerged in the last years to provide
ple, staining and acquiring larger numbers of guidance for structured reporting of biological
CD8+ cells increase the ability to detect low-fre- assays. The rst minimal information project that
quency HLA-multimer-positive cells, and inclu- set the scene was the Minimal Information About
sion of a cell-resting phase improved sensitivity Microarray Experiments (MIAME) published in
in the IFN-ELISPOT. In contrast, a high back- 2001 [77]. It is now an established and manda-
ground production of the cytokine (IFN-) both tory standard for publishing microarray data for a
in ICS and ELISPOT is clearly associated with growing list of highly recognized journals (http://
decreased performance [72, 73]. www.mged.org/Workgroups/MIAME/journals.
Over several prociency panel iterations, it html). More than 30 such guidelines have emerged,
also became clear that all steps of the assays, asking for minimal information on reported
starting from cell handling (freezing/thawing/ results, including minimal information for cellu-
resting), assay conditions (reagents and protocols lar assays (MIACA) (http://miaca.Sourceforge.
for mAbs and HLA-multimer staining, conditions net/), specication for in situ hybridization and
of antigenic stimulation in ICS), result acquisi- immunohistochemistry experiments (MISFISHIE)
tion including instrument settings, down to the [78], and ow cytometry experiments (MIFloCyt)
data analysis, can benet from harmonization for [79]. Information on the majority of available MI
achieving comparable results between laborato- projects can be found in a central portal for minimal
ries. In ow cytometry specically, instrumenta- information on biological and biomedical investi-
tion performance may be an issue, as we recently gations (MIBBI) (http://mibbi.Sourceforge.net/).
observed in a panel dedicated to the simultaneous These guidelines aim at achieving two major
detection of four Ag T-cell specicities by HLA- goals: rst, to annotate data to such extent that
multimers (manuscript in preparation). Both CIC they give transparent evidence on the quality,
and CIP have also observed in independent pan- reliability, and possible error sources of reported
els conducted for ICS [73, 74], as well as for results and, second, to use the reporting standard
HLA-multimer staining [75, 76], that suboptimal to systematically feed public databases [80].
gating strongly inuenced the ultimate results More recently, structured reporting guidelines
i.e., the detection and deduced frequencies of have also been provided for the specic context
antigen-specic T cells. We also showed that of immune assay experiments. As outlined before,
analysis (gating) performed by a unique user sub- the continuous conduct of prociency panels
stantially decreased the variation in the frequen- over several years led to the identication of
cies of specic cells as compared to those steps in the assay that critically impact the results,
reported by single labs analyzing their own data namely, (i) the sample, (ii) the assay, (iii) the data
acquisition, (iv) the data analysis, and (v) certain interest and citations over time. The authors
characteristics of the lab environment. In concor- therefore recommend considering structured
dance with these ndings, a ow chart of deci- reporting of results from T-cell assays whenever
sions that can affect the quality of data produced possible.
in clinical trials in which immunological param-
eters are monitored by ow cytometry was listed
in a landmark publication [81]. Although the 25.6 Organization of Immune
variables critically affecting the quality of results Monitoring in Multicenter
are for most of them well known, only very Trials
few scientic publications provide sufcient
information on these aspects in their material and Clinical trials will often require the recruitment
method descriptions. This lack of transparency is of patients at multiple sites in order to reduce the
one of the major reasons preventing meaningful overall duration and costs of the trial. The labora-
comparison of published results generated across tory data generated from all patients and at different
institutions. In contrast, study results reported sites should be comparable, but as the regulatory
with transparent information on the essential framework for the conduct of clinical trials
variables of assay conduct, explicitly indicate (ICH-GCP) is not very detailed with respect to
awareness of the investigator to control critical standards of laboratory analyses, further details
variables, thus can be much better interpreted and are specied by the more recent concept of good
reproduced. clinical laboratory practice (GCLP) [8688].
To reduce the discrepancy between available Two general strategies emerge on how analyti-
knowledge on immune assay conduct and lack of cal assays can be performed among different sites
critical information in scientic publications, a [89]: in the distributed analysis paradigm, each
group of T-cell immunologists from the cancer site analyzes its locally derived samples. In con-
immunology, infectious diseases, autoimmunity, trast, in the central lab paradigm, all samples are
and transplantation elds initiated the Minimal transported to a central lab for analysis. In either
Information About T-cell Assays (MIATA) case, ow cytometry poses additional chal-
project [82]. The group conducted an intensive lenges due to the fragility of the sample and the
vetting process with two public consultation peri- complexity of the assay.
ods, two open consensus workshops, and several For distributed analysis, the assay and instru-
webinars [83]. The process towards reaching a mentation at different sites must be comparable.
broadly acceptable guideline on the minimum This can be achieved via full interlaboratory stan-
information that should be provided for T-cell dardization, as is already routinely performed in
assays [84] can be found at the projects webpage clinical ow cytometry with in vitro diagnostic
www.miataproject.org. With the MIATA consensus (IVD)-certied reagents and instruments [90].
guidelines becoming available, the implementation Due to the high development costs, the number of
of more structured reporting for T-cell immune clinical ow cytometry products for IVD on the
monitoring can begin and should be considered market is limited and focuses on the clinically
by all investigators, especially for conducting most relevant tasks as, e.g., the quantication
T-cell assays in clinical trials [85]. So far, three of CD4+ T cells in blood. In many cases, these
peer-reviewed journals endorse the MIATA applications lack the technical capabilities of
guidelines and assign the MIATA label. The modern polychromatic ow cytometry. Full-scale
label indicates that authors of accepted manu- interlaboratory standardization (with demon-
scripts take great care about reporting on and strated low interlaboratory variation) of research
control of variables that matter for T-cell assays. assays with RUO-grade reagents and customized
All MIATA compliant manuscripts will be listed ow cytometric instrumentation has been dem-
on the MIATA homepage leading to greater expo- onstrated by some groups but requires great
sure of the published work, which may increase efforts [91]. An alternative to full interlaboratory
standardization discussed in Sect. 25.4 is harmo- As an example demonstrating feasibility of

nization which can be achieved via regular this approach, an international, multicentric
participation in prociency panels. immunotherapy trial was conducted recently
For highly complex ow cytometric assays including T-cell immunomonitoring in which
within clinical trials, having all samples analyzed more than 40 clinical sites were trained in blood
by the same central laboratory eliminates the sampling, labeling, and shipping, with labels and
need for full-scale interlaboratory standardiza- collection tubes provided by a central laboratory.
tion of participating institutes and may be less Local PBMC isolation laboratories were centrally
demanding. However, maintaining sample quality supplied with pretested kits containing all critical
becomes a critical issue with this strategy. The reagents required for isolation and cryopreserva-
initial sample material for ow cytometry contains tion of PBMCs. All laboratory technicians were
living cells (in most cases derived from blood trained and qualied on central SOPs describing
with the addition of anticoagulants). From this in detail the PBMC isolation and cryoconservation
sample material, cells have to be isolated before processes. Where required, the fresh blood was
the start of the ow cytometric assay. Cells are transported from the clinical sites to the PBMC
usually more fragile compared to biomolecules isolating labs using temperature controlled ship-
or small molecules. Several studies have been ments. The isolated frozen PBMCs were shipped
performed to determine how long blood can be to the central lab in validated dry ice containers.
stored or transported before peripheral blood Patient visits involving a PBMC sampling were
mononuclear cell (PBMC) isolation (mostly carefully coordinated in advance among the
using density gradient centrifugation) and how clinical sites, the PBMC isolating laboratories,
stable isolated cells are before the assay is started and the logistic service providers to ensure that
[34, 92, 93]. For simple phenotyping (e.g., CD4 the blood could be processed within 8 h after
counting), a 48 h delay before centralized analysis venipuncture of a patient. This process led to a
is acceptable, while the most demanding applica- successful logistic chain for 361/362 (99.7 %)
tions (such as some functional T-cell assays) PBMC samples and an overall evaluability rate
require isolation of the cells within 8 h of veni- of 64/68 (94 %) patients for T-cell immunomoni-
puncture, followed by immediate analysis or toring [4].
cryopreservation of the cells [94]. Shipment to a
central lab followed by processing of blood
samples within 8 h is however not feasible in 25.7 Towards Automated Analysis
international multicenter trials. Therefore, a
mixed model may be chosen [4], whereby cells As discussed in Sects. 25.4 and 25.5, the standard
are isolated and cryopreserved from peripheral approach for analyzing ow cytometry data is by
blood at individual labs close to the patient and the visual identication of cell subsets of interest
then shipped in the frozen state to the central lab on histograms or two-dimensional scatter plots.
where they are stored frozen before analysis. All With multiparameter data, gating consists of rst
stages of isolation, cryopreservation, and trans- choosing a gating strategy a sequence of dot
port conditions should be fully standardized in plots that is designed to allow identication of
this model. Standardized labeling of samples that the cells of interest. For example, a possible
allow the unambiguous assignment of a sample gating strategy for identifying HLA-multimer-
to a trial, site, patient, and visit is also critical. positive CD8+ T cells might be FSC-A/FSC-H
GCP regulation also requires special care to (singlets), FSC-A/SSC-A (lymphocytes), CD3/
protect the privacy of patients, and this may be viability dye (viable T lymphocytes), CD4/CD8
achieved by pseudonymization. These proce- (basic T lymphocyte subsets), and CD8/multi-
dures have to be clearly dened in the clinical mer. In each dot plot, cells of interest are included
trial protocol and are usually further detailed in and other events excluded by the use of elliptical
the clinical trial laboratory manual. or polygonal gates or sometimes by splitting the
dot plot into quadrants. The exact location and channels may be explicitly excluded from analysis
shape of these gates may be based on experience at this stage if they are not likely to be informa-
or by comparison with negative (e.g., isotype, tive for the cell subset targets of interest. Often,
FMO, or unstimulated control in ICS) and pos- a quality control lter is also applied at this
itive (reference sample or T-cell clone or super- stage, and data sets with inconsistent annotation,
antigen stimulation) controls. After a gating too few events, and anomalous event distribu-
strategy has been set, it is typically applied in tions or signatures may be agged for manual
common to all ow cytometry samples in the evaluation [99].
batch being analyzed. Some researchers will The core of most automated analysis is the
also adjust gates for individual samples to take unsupervised partitioning of events into cell
individual variability into account. In general, subsets. There are a variety of approaches that
there is no consensus or accepted standard gating can be taken to partition or cluster events, as sum-
strategy, and individual laboratories may apply marized in a recent publication [98]. One popular
different gating strategies to identify the same approach is the use of statistical mixture
target cell subset. Notably, prociency panels have models, either identifying cell subsets with indi-
made it very clear that the subjectivity of gating vidual mixture components (which are typically
forms a signicant source of assay variability multivariate Gaussian, student T, or skewed
between laboratories in the absence of a harmo- versions of these distributions) or using features
nization program [72, 95]. of the estimated density to assign events to cell
To increase the objectivity of ow cytometry subsets [100102]. Such probabilistic approaches
analysis, automated methods in which cell sub- provide a declarative framework to model domain
sets are directly quantied by machine algorithms knowledge and support formal statistical infer-
have been proposed [9698]. In broad terms, ences for structure learning, classication, and
these algorithms have to rst partition all the prediction. The underlying statistical model for
events in a data sample into disjoint subsets, based the domain knowledge can also be naturally
on properties of each individual event and its extended in different contexts for example, to
relationship to other events, and then to assign incorporate specic assay details for combinato-
these subsets to biologically meaningful categories rial multimer encoding [103] or to incorporate
(e.g., HLA-multimer-binding CD8+ lymphocytes). multilevel effects via hierarchical modeling
In the context of cancer immunology, a specic [104]. The power of probabilistic models comes
challenge for automated approaches is the high at a price, in that these models tend to be much
sensitivity required, since antigen-specic responses more computationally demanding than non-
(e.g., HLA-multimer positivity or polyfunctional probabilistic approaches [105108], and the run-
cells) may be relevant at relative frequencies of time for analysis of high-volume, high-dimensional
0.010.1 %. Data from multiple laboratories sig- data sets may be prohibitive. However, recent
nicantly increases the challenges for automated developments in the use of highly parallel graphical
analysis, since the algorithms have to also account processing units (GPU) [109] have accelerated
for the variability across laboratories and issues run-times by orders of magnitude, making the
with harmonization of sample annotation. probabilistic approaches a viable approach for
A typical automated analysis preprocessing many applications in cancer immunology.
pipeline starts with the extraction of the essential The essential step in postprocessing is the
matrix of information stored in a ow cytometer alignment of cell subset clusters across multiple
FCS le, where each row represents an event and data samples, since comparative analysis of
each column represents a detector channel, either equivalent cell subsets is a necessary requirement
scatter or uorescent intensity. Preprocessing of ow cytometry analysis in clinical research.
algorithms may apply compensation or specic Perhaps the most straightforward approach is to
transformations to regularize the data distribution align each data sample with respect to either a
(e.g., bi-exponential transformation). Specic reference or consensus clustering via an optimization
routine that minimizes some distance between ow operator with the following developments
pairs of clusters (e.g., Euclidean distance between developers of these tools will continue to improve
cluster centroids). Other possible approaches their ease of use; the most successful algorithms
skirt the problem entirely by enforcing a common will be incorporated into commercial software
clustering across all data samples or partition the analysis packages; and more workshops will be
clusters from tting all data samples into super- organized to train people in the use and potential
clusters all clusters in the super-cluster are pitfalls of these exciting new technologies.
then assigned to the same cell subset. The nal
step of assigning meaningful cell subset labels to
the aligned clusters is typically done manually, 25.8 New Methods
although there have been recent efforts to develop and Technologies
heuristics that can automatically label clusters
by establishing a concordance between cluster Flow cytometry has played an instrumental role
features and cell phenotype characteristics in the in our comprehension of the immune system and
Cell Ontology. Innovations in the visualization of its interplay with human tumors. The technique
high-dimensional cytometry data have also greatly has recently experienced dramatic advances and
increased our ability to interpret the results of the methods and technologies are evolving con-
automated analysis [110112]. tinuously. Due to space limitations, we focus
The detection of antigen-specic cells poses a here on the recent innovations that in our opinion
specic challenge for automated algorithms have the potential to transform the eld of general
because of the extremely low frequency of these cytometry and are directly relevant for cancer
cell subsets in many patient samples for exam- immunotherapy.
ple, as few as 0.010.1 % of the CD8+ T lympho- Since the rst description of a tumor Ag targeted
cyte population may be specic for a particular by human T cells [114], many tumor-associated
tumor Ag multimer. Two nonexclusive approaches proteins and HLA-class I- and class II-restricted
for improving the ability of automated algorithms epitopes have been identied. However, the
to improve the limit of detection are biased sub- antitumor T-cell immune response as a whole,
sampling to enrich the sample for rare events i.e., the repertoire of Ag specicities recognized
[111, 113] or to increase the complexity of the by T cells of individual patients, has only rarely
statistical model [104]. The development of algo- been dissected [115, 116]. This is indeed a dif-
rithms that can accurately and robustly identify cult task, due to the inherent complexity of such
rare cell populations is a driving motivator for projects (many Ags and HLA-allele restrictions
much current research in automated ow analysis, have to be taken into account), along with the
and we expect rapid advances in this area. limited amount of patient material generally
Illustrative examples comparing manual and auto- available, and high requirements in terms of cost
mated analysis of antigen-specic cells for HLA- and time. Two groups simultaneously described a
multimer and ICS assays are shown in Fig. 25.1. combinatorial encoding method which is a very
Finally, we note that most of these automated elegant way to circumvent most of these hurdles
analysis tools are developed under open source [117, 118]. The technique is based on the combi-
licenses and so free to use without restriction. nation of many HLA-peptide multimers, whereby
Some packages require a modicum of program- a single multimer is coupled to several (two or
ming ability to use effectively (e.g., R or Python three) uorochromes, generating a color code for
scripting skills) and others are available online, each tested TCR specicity. Currently, up to 27
but in general, these algorithms are probably not HLA-multimers labeled with eight uorochromes
easily used by the average ow operator in a can be combined in routine analysis [117].
clinical research laboratory. In the coming years, Coupled to the production of HLA-monomers by
we expect that these automated analysis tools the UV exchange technology, this high-throughput
will become increasingly accessible to the average method represents an important technical
a
8,000 250k 250k
105
Aqua livedead
200k 200k
6,000 104
FSC-H
Count
SSC-A
150k 150k
4,000
103
100k 100k
0
2,000 50k 50k
0 0 0
0 50 100 150 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
Time FSC-A FSC-A FSC-A
105 105 0.0233 % 105 0.0047 % 105 0.1927 %
104 104 104 104
QDot605
CD19
APC
PE
103 103 103 103
0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
CD8 CD8 CD8 CD8
105
Aqua livedead-A
104
FSC-H
SSC-A
103
FSC-A FSC-A FSC-A
105 105 0.0229 % 105 0.0046 % 105 0.1933 %
104 104 104 104

QDot605-A
APC-A
CD19
PE-A
103 103 103 103
0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
CD8 CD8 CD8 CD8
Fig. 25.1 (a) Manual and automated identication of [103]. Essentially identical frequencies of peptide-MHC
antigen-specic MHC class I multimer-positive CD8+ T multimer positive cells are found with manual and auto-
lymphocytes among PBMC of a HLA-A2+ healthy donor. mated analyses. (b) Manual and automated analysis of
Top panel shows a manual gating strategy to identify CD8+ antigen-specic T cells among PBMC of a second HLA-
T cells specic for three HLA-A*0201-restricted epitopes A2+ healthy donor tested in an intracellular cytokine stain-
derived from a EBV, inuenza, and CMV viruses. From ing (ICS) assay after incubation with a synthetic peptide
left to right, the plots show gates to exclude artifacts due corresponding to an HLA-A*0201-restricted epitope of
to ow stream bubbles or clumps (count/time), nd sin- pp65 CMV. Manual analysis nds cells positive for IFN
glets (FSC-A/FSC-H), exclude nonviable cells (FSC-A/ and TNF, and a few events positive for IL-2. Without
Aqua LiveDead), identify lymphocytes (FSC-A/SSC-A), further gating, it is not possible to tell if the IFN- and
exclude B lymphocytes (CD8/CD19), and quantify CD8+ TNF-positive events come from two separate or a single
T cells binding to EBV BRFL1 peptide-MHC multim- bifunctional population. Automated analysis reveals that
ers (CD8/PE), inuenza matrix peptide-MHC multim- there is indeed a single-cell population positive for IFN
ers (CD8/APC), and CMV pp65 peptide-MHC multimers and TNF, with no evidence for an IL-2-positive popula-
(QDot605). Bottom panel shows the corresponding pep- tion. Again, the frequencies of antigen-specic events iden-
tide-MHC binding CD8+ T cells identied using an auto- tied by expert gating and automated analysis are almost
mated analysis approach that tted a Dirichlet Process equivalent
Gaussian Mixture Model with 256 components to the data
b
5,000 250k 250k
105
Aqua livedead
4,000 200k 200k
104
FSC-H
Count
SSC-A
3,000 150k 150k
103
2,000 100k 100k
0
1,000 50k 50k
0 0 0
0 50 100 150 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
Time FSC-A FSC-A FSC-A
105 105 0.0777 % 105 0.0005 % 105 0.0773 %
104 104 104 104

CD4
TNF
IFN
IL2
103 103 103 103
0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
CD8 CD8 CD8 CD8
105
Aqua livedead-A
104
SSC-A
FSC-H
103
FSC-A FSC-A FSC-A
105 105 0.0804 % 105 0.0000 % 105 0.0804 %
104 104 104 104

IFNy
CD4
TNF
IL2
103 103 103 103
0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
CD8 CD8 CD8 CD8
Fig. 25.1 (continued)
achievement for the T-cell immunology eld and binding of cytokines to their specic cell surface
has started to deliver precious information by receptors generally results in the activation (i.e.,
dissecting the anti-melanoma TIL repertoire in phosphorylation) of the downstream signal trans-
melanoma patients [119, 120]. Combinatorial ducers and activators of transcription (STATs),
staining could easily be implemented for moni- which in turn regulate the expression of many
toring vaccination trials, for example, when genes involved in cell growth, survival, differentia-
applying cocktails of antigenic peptides for tion, and polarization. Next to cytokines, the effect
which many specicities need to be tested in a of unspecic mitogenic stimuli such as phorbol
single PBMC sample. myristate acetate (PMA), phytohemagglutinin
The combination of extracellular phenotyping (PHA), or MHC-peptide complexes binding to the
with determination of intracellular changes in T-cell receptor (TCR) can be studied by measuring
phosphorylation patterns upon stimulation is start- the level of other key signaling molecules such as
ing to provide new insights into signaling pathways phosphorylated (p)-Erk, p-S6, and p-NF-B in
in healthy and disease conditions [121, 122]. The T and B cells, whereas Toll-like receptor (TLR)
ligand-induced activation can be followed with and of CD8+ T lymphocytes subsets and will
p-Akt, p-Erk, and p-NF-B in B cells and mono- certainly mature to become an indispensable
cytes. The proof of principal for a single-cell net- technique in cancer immunology and immuno-
work proling (SCNP) method was obtained on therapy [129, 130].
healthy donors PBMCs [123]. In this initial study,
age as well as race differences were observed,
whereas intra-donor variability needs to be estab- 25.9 Concluding Remarks
lished by testing blood samples taken at different
time points over time. As T-cell signaling defects Flow cytometry is the prototypical multiparame-
have been described in cancer patients [124, 125], ter single-cell assay, with applications in cancer
insights in the intracellular phosphorylation pat- immunotherapy ranging from epitope screening
terns of T cells, including during immunotherapy, to immune monitoring of clinical studies. Due to
may soon deliver precious information. its ability to characterize complex immune phe-
A fundamental advance in ow cytometry in notypes and exibility in measuring multiple
recent years is an increase in the number of immune functions such as Ag binding, expression
parameters that can be simultaneously evaluated of activation and inhibitory markers, cytokine
on single cells. Access to an increasing number of production, cytotoxicity, and proliferation, ow
reagents and uorochromes including tandem cytometry is indispensable in cancer immunology
conjugates, semiconductor nanocrystals (quantum research. However, because of the complexity of
dots or eFluors), and organic polymers (brilliant the assay and the fragility of the sample, it is chal-
violet family) [126128], together with the wide lenging to apply and maintain robustness, sensi-
availability of sophisticated ow cytometers, is tivity, and reproducibility, especially across
making polychromatic analysis mainstream. multiple laboratories. Factors to consider when
However, spectral overlap ultimately limits using ow cytometry in clinical research include
the number of uorochromes in a single panel to understanding the range of ow-based assays
an upper bound of approximately 20, as described available, as well as best practices for instrument,
in Sect. 25.3. An exciting new technology that reagent, sample, and data analysis.
has the potential to greatly increase the number In order to harmonize laboratory protocols,
of measurable parameters is mass cytometry practices, and analysis strategies, ow cytometry
(CyTOF), which uses stable heavy metal ions prociency testing programs have been orga-
tagged to Abs (or, e.g., MHC multimers) in place nized to learn and raise awareness of best prac-
of uorochromes. These isotope labels are tices. We believe that participation in prociency
detected by time-of-ight mass spectrometry testing programs, along with other initiatives
after vaporization of the cell. Although isotope delivering protocols, assay guidelines and reporting
labels generally produce a signal of low intensity, frames, is critical for raising the standard of ow
they have a lower background and virtually no cytometry analysis and strongly recommend that
spillover, making the measurement of a much all clinical research laboratories that perform
larger number of markers feasible. immune monitoring for clinical trials join such
Mass spectrometry has been reported to be programs.
qualitatively and quantitatively equivalent to ow
cytometry, with the simultaneous analysis of Acknowledgments CG, SW, MJP, SvB, CO, and CB
are members of the steering committee of the CIMT
more than 30 parameters being already possible Immunoguiding Program (CIP). The CIP and CC are sup-
[129]. However, this promising new technology ported by a grant of the Wallace Coulter Foundation (Miami,
has the current following limitations as compared Florida). CG is supported by a grant of the Deutsche
to traditional flow cytometry: lower label Forschungsgemeinschaft SFB685. CC is supported by
grants to the Duke University Center for AIDS Research and
sensitivity, substantial cell loss, low acquisition EQAPOL program funded by NIH grant 5P30 AI064518
rate, and the impossibility to sort living cells. and NIH contract HHSN272201000045C, respectively. We
Nevertheless, this method has started to reveal thank S Heidu for excellent technical assistance.
the complexity of healthy hematopoietic cells
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Immunohistochemistry of Cancers
26
Alireza Ghanadan, Issa Jahanzad, and Ata Abbasi
Contents 26.3.2 Tumors of the Larynx, Nasopharynx,

and Oropharynx ......................................... 500
26.1 Introduction .............................................. 492 26.3.3 Tumors of the Salivary Glands .................. 501
26.2 Immunohistochemistry 26.3.4 Immunohistochemistry of Salivary
of Skin Tumors ......................................... 492 Gland Tumors ............................................ 502
26.2.1 Markers of Normal Skin ............................ 492 26.3.5 Tumors of Thyroid and Parathyroid
26.2.2 Epithelial Tumors ....................................... 494 Glands ........................................................ 505
26.2.3 Sweat Gland Tumors .................................. 495 26.4 Immunohistochemistry
26.2.4 Trichogenic Tumors ................................... 495 of Lung Tumors ........................................ 505
26.2.5 Sebaceous Tumors ..................................... 496 26.4.1 Adenocarcinoma ........................................ 505
26.2.6 Melanocytic Tumors .................................. 496 26.4.2 Mesothelioma............................................. 506
26.2.7 Prognostic Markers of Melanoma.............. 497
26.2.8 Specic Mesenchymal Tumors 26.5 Immunohistochemistry
of the Skin .................................................. 497 of Gastrointestinal Tumors ..................... 507
26.5.1 Liver ........................................................... 508
26.3 Immunohistochemistry of Head 26.5.2 Esophagus .................................................. 509
and Neck Tumors ..................................... 499 26.5.3 Stomach ..................................................... 509
26.3.1 Tumors of the Nasal Cavity 26.5.4 Small Intestine ........................................... 511
and Paranasal Sinuses ................................ 499 26.5.5 Colon.......................................................... 511
26.5.6 Anal............................................................ 511
26.5.7 Appendix.................................................... 511
26.5.8 Pancreas ..................................................... 511
26.5.9 Gastrointestinal Stromal Tumor ................. 513
26.5.10 Neuroendocrine Carcinomas...................... 513
A. Ghanadan (*) 26.6 Immunohistochemistry

Department of Dermatopathology, Razi Dermatology of the Urinary Tract ................................. 513
Hospital, Vahdate Eslami Ave., Tehran, Iran 26.6.1 Kidney ........................................................ 513
26.6.2 Bladder ....................................................... 514
Department of Pathology, Cancer Institute, Imam
Khomeini Complex Hospital, School of Medicine, 26.7 Immunohistochemistry of Female
Tehran University of Medical Sciences, and Male Genital Tumors ....................... 516
Keshavarz Blvd., Tehran, Iran 26.7.1 Uterine Cervix............................................ 516
e-mail: dermpath101@gmail.com 26.7.2 Vulva and Vagina ....................................... 516
26.7.3 Uterine Corpus ........................................... 516
I. Jahanzad, MD A. Abbasi, MD, MPH 26.7.4 Ovary.......................................................... 517
Department of Pathology, Cancer Institute, Imam 26.7.5 Breast ......................................................... 517
Khomeini Complex Hospital, School of Medicine, 26.7.6 Prostate....................................................... 520
Tehran University of Medical Sciences, 26.7.7 Testis .......................................................... 521
Keshavarz Blvd., Tehran, Iran
e-mail: jahanzad@yahoo.com; ata.abasi@gmail.com, 26.8 Immunohistochemistry
aabbasi@razi.tums.ac.ir of Lymphoma............................................ 521

492 A. Ghanadan et al.
26.9 Immunohistochemistry of Soft Peroxidase Polymer

Tissue and Bone Tumors ......................... 522
Link Ab
26.9.1 Epithelial Markers...................................... 523
26.9.2 Myogenic Markers ..................................... 526
Primary Ab
26.9.3 Nerve and Schwann Cell Markers ............. 530 Ag
26.9.4 Endothelial Markers ................................... 530
26.9.5 Fibrohistiocytic Markers ............................ 531
26.9.6 Lipocytic Markers ...................................... 533
26.9.7 Chondrocyte Markers................................. 533 Biotin
26.9.8 Osteogenic Markers ................................... 533 Biotinylated Ab(link) Labeled streptavidin
26.9.9 Unknown-Origin Soft Tissue Tumors ........ 534
Peroxidase
26.10 Immunohistochemistry
of the Nervous System ............................. 534 Primary Ab
26.10.1 Neuroepithelial Tumors ............................. 535
26.10.2 Non-neuroepithelial Tumors ...................... 536
26.10.3 Undifferentiated Tumors ............................ 538 Ag
26.10.4 Proliferative Markers ................................. 538
26.11 Immunohistochemistry Fig. 26.1 Schematic mechanism of two immunohisto-
of Pediatric Tumors ................................. 538 chemistry methods. Top: secondary antibodies and
enzymes link to polymer molecule. Bottom: biotinylated
26.12 Immunosurveillance, secondary antibody and labeled streptavidine
Immune Editing, Immune Constant
of Rejection, Immune Contexture,
and Immune Scoring of Cancers ............ 541 Immunohistochemistry has wide application
26.13 Concluding Remarks ............................... 545 including research uses, diagnostic purposes, and
prognostic and therapeutic aims. IHC is a nice
References ............................................................... 545
technique for tracking of proteins and haptens, so
it is used to dene expression of specic genes at
the level of proteins. It is also very useful in diag-
nostic pathology including denition of cellular
26.1 Introduction lineage (epithelial, vascular, lymphoid, etc.) or
subtyping of some specic lesions and malignan-
Immunohistochemistry (IHC) is the art of using cies such as malignant lymphomas. Prognostic
antibodies (Abs) to detect specic antigens and therapeutic applications have gradually
(Ags) in tissues. Histopathologic evaluation of become widely popular such as the denition of
diseases has been altered and enhanced by the hormone receptor status of breast cancer (ER,
advent of IHC, and some sophisticated tech- PR, and AR) and oncogene products (e.g., Her2,
niques have been replaced by IHC due to its easy EGFR, c-kit, etc.) which could be a part of guide-
and versatile immunohistochemical techniques. lines for targeted therapy of the tumors.
Of course, disorganized application of IHC
could be misleading.
Immunohistochemistry is based on specic 26.2 Immunohistochemistry
Ab-Ag interactions. The Abs which are used to of Skin Tumors
detect Ag(s) are called primary Abs. Primary Abs
are linked to enzymes (main part of chromogenic 26.2.1 Markers of Normal Skin
system) via another Ab called link Ab. This link-
age to enzymes is mediated by polymers or some Skin tissue is composed of epidermal and
molecules such as streptavidin-biotin complexes. adnexal components as well as mesenchymal
Peroxidase is the enzyme mostly used in immu- dermal components. All epithelial cells in the
nohistochemistry. Alkaline phosphatase is also epidermis, folliculosebaceous unit, and sweat
used (but less frequently). Some mechanisms are glands reveal pan-keratin markers such as AE1/
shown in Fig. 26.1. AE3 (Fig. 26.2a). Keratinized squamous cells
26 Immunohistochemistry of Cancers 493
a b
c d
e f
Fig. 26.2 Normal skin. (a) Pan-keratin of AE1/AE3 (c) and CK20 (d). EMA (e) reacts with sebaceous glands
stains the epidermis, folliculosebaceous unit epithelium, rimming cytoplasmic vacuoles, and CD1a highlights den-
and sweat glands. Basal keratinocytes are highlighted by dritic Langerhans cells in the epidermis (f)
CK5 (b). Sweat glands are immunostained by CK7
and proliferative keratinocytes express cytokera- comprise sweat structures of the skin. Normal
tin (CK) 6/16, nonkeratinized squamous cells eccrine glands show reactivity with CD7, CD20
reacts with CK4/13, and basal keratinocytes (Fig. 26.2c, d), CEA, and S100, while apocrine
exhibit reactivity for CK5/14/15 (Fig. 26.2b). glands exhibit immunostaining for CEA and
Squamous cells in palm and sole are reactive for GCDFP15 [3, 4]. Sebaceous glands exhibit
CK1/9/10 [1, 2]. Eccrine and apocrine glands reactivity for CK10 as well as EMA rimming
Markers
Stratum
corneum
Filaggrin, involucrine
Grannular
layer
Nonkeratinized squamous cells
Spinous CK4/13
layer Keratinized squamous cells
CK6/16
Basal
layer CK5/14/15
Keratinocyte Langerhans cell CD1a, S100, langerin

S100, HMB45, MART1,
Melanocyte
Melan-A
Fig. 26.3 Immunohistochemistry antibodies in schematic normal epidermal components
Table 26.1 Immunoprole of normal epidermis, folliculosebaceous, and sweat gland structures in comparison with
respective tumors
Cell Antibodies Tumor Markers
Keratinocyte CK6/16 Squamous cell carcinoma EMA, p63
Basal keratinocyte CK5/14/15 Basal cell carcinoma BerEp4
Eccrine cell CK7, CK20, CK5/14, Eccrine carcinoma EMA, CEA, CD15, p63,
CK1/10, CEA, S100 S100
Apocrine cell CEA, GCDFP15 Apocrine carcinoma EMA, CEA, CD15, p63,
CA72.4, GCDFP15
Trichogenic cell CK14/15/19 Trichilemmal carcinoma CEA, S100
Proliferating trichilemmal carcinoma EMA, CD34
Sebaceocyte CK5/14/15, CK8/18 Sebaceous carcinoma EMA
cytoplasmic lipid vesicles (Fig. 26.2e) [5]. 26.2.2 Epithelial Tumors

Normal melanocytes express S100, HMB45, and
MART-1/melan-A but do not react with tyrosi- Squamous cell carcinoma (SCC) and basal cell
nase [6]. Langerhans cells are stained with CD1a carcinoma (BCC) are derived from the spinous
(Fig. 26.2f), S100, langerin, and CD31 [7]. layer and basal layer of the epidermis, respec-
Displaying neurotactile differentiation, Merkel tively. Well-differentiated SCC expresses high
cells of normal skin are reactive for CK20, molecular cytokeratin, while those with poor dif-
MOC-31, neurolament, and CD56 [810]. ferentiation express low molecular cytokeratin.
Markers of the normal epidermal components Cytokeratin, p63, and vimentin are present in the
are depicted in Fig. 26.3. The immunoprole of sarcomatoid variant of SCC [11]. EMA, one of the
normal skin components and respective cancers human milk fat globule proteins not expressed in
is summarized in Table 26.1. normal keratinocytes, is expressed on malignant
squamous cells. Basal cell carcinoma expresses eccrine tumors, but not in apocrine tumors. A
BerEp4 (Fig. 26.4) but does not demonstrate reac- remaining challenge is distinguishing primary
tivity with EMA and p63, distinguishing it from eccrine carcinoma from metastatic carcinoma
SCC [12]. by immunoprole of CK5/6 and p63 which are
positive in eccrine carcinoma, but not in meta-
static carcinoma [13]. Paget disease is an
26.2.3 Sweat Gland Tumors intraepidermal extension of neoplastic cells into
the epidermis which shares similar histopatho-
Malignant eccrine tumors are distinct from logic features with malignant melanoma and
benign eccrine tumors by displaying reactivity Bowen disease. Immunohistochemistry study
with EMA. Eccrine tumors display CEA, CD15, can be a helpful method in differentiating these
and p63 which are also common with apocrine tumors as denoted in Table 26.2 [14]. CK20 and
tumors. Differentiating markers of apocrine GCDFP-15 are useful markers in distinguishing
tumors are TAG-72 (CA72.4) and GCDFP15 primary and secondary perianal Paget diseases,
(Fig. 26.5) which are not expressed on eccrine respectively [15].
tumors [4]. S100 is demonstrated in 50 % of
26.2.4 Trichogenic Tumors
Tumors with trichilemmal differentiation display

reaction with CK14/15/19, BerEP4, and p63 but
do not react with EMA (except proliferating
trichilemmal tumor), CEA, S100, CD15, CA72.4,
HMB45, and GCDFP15 [3]. Trichilemmal carci-
noma displays reactivity with CEA and S100,
and proliferating trichilemmal carcinoma (malig-
nant proliferating tumor) shows reactivity with
EMA and CD34 [17]. Desmoplastic trichoepithe-
lioma shares histopathologic similarities with
inltrating BCC and microcystic adnexal carci-
Fig. 26.4 Immunoreaction of basal cell carcinoma with noma. The immunoprole of these tumors are
BerEP4 demonstrated in Table 26.3.
a b
Fig. 26.5 Primary skin apocrine carcinoma (a) immunostained by GCDFP15 (b)
Table 26.2 Immunophenotype of mammary and extramammary Paget disease (PD), Bowen disease, and malignant
melanoma
Extramammary PD (apocrine Bowen disease
Makers Mammary PD carcinoma in situ) (SCC in situ) Melanoma (in situ)
CK7 + +
CEA + +
CAM5.2 + +
GCDFP15 + +
MUC1 + +
MUC5AC +
CA15-3 +
CA72.4 +
KA-93 +
CK5/6 +
S100/HMB45/MART +
Refs. [1416]
Table 26.3 Immunoprole of desmoplastic trichoepi-

thelioma (DTE), inltrating basal cell carcinoma (IBCC), 26.2.6 Melanocytic Tumors
and microcystic adnexal carcinoma (MAC)
Tumor DTE IBCC MAC Being a sensitive but a nonspecic marker of
Panel EMA, CK5/6, CK5/6, CD10 EMA, melanoma, S100 is a calcium-binding protein
antibodies CD10 (epithelial), p63, CK7, given its name because of solubility in 100 %
(stroma), Bcl-2, BerEP4, Ck5/6, saturated ammonium sulfate solution. Other
CK15, CK20, stromelysin-3, CK15,
S100-positive tumors include undifferentiated
p63, Bcl-2, p53 p63,
BerEP4 SMA carcinoma, nerve sheath and glial tumors, adi-
Refs. [1820] pose tumors, and histiocytic and Langerhans cell
proliferations [26, 27]. Considering as highly
specic marker of melanocytes, the gp100 group
26.2.5 Sebaceous Tumors includes HMB-45 and MART-1/melan-A with 60
and 80 % sensitivity, respectively. Melanoma
Sebaceous tumors exhibit reactivity with antigen recognized by T-cells-1 (MART-1) is a
CK5/14/15, CK8/18, EMA, CD15, anti- protein which serves as a potential target for
adipophilin (ADP) and androgen receptor. CK15 cytotoxic T lymphocytes recognized by two
is positive in sebaceoma but does not exhibit reac- monoclonal antibodies (mAbs), A103 and melan-
tivity with sebaceous carcinoma [21]. Sebaceous A [28]. Desmoplastic/spindle cell variant of mel-
tumors do not express CEA, S100, CA72.4, and anomas does not show reactivity with HMB45
GCDFP-15 in comparison with sweat gland and MART/melan-A. Instead, these melanomas
tumors, which are positive for these markers [4, are more reactive with S100, p75-NGF-R, and
22]. Sebaceous carcinoma is differentiated from tyrosinase [29]. Small cell melanoma is another
BCC by showing reactivity for EMA (Fig. 26.6) variant of the melanoma which could be distin-
and negative reaction to BerEP4, vice versa of guished from other small cell undifferentiated
BCC [23]. Proliferating markers are good mark- tumors of the skin and subcutaneous tissue by
ers to differentiate sebaceous adenoma from Abs panel (Fig. 26.7). The immunoproles of
sebaceous carcinoma (Table 26.4). these tumors are summarized in Table 26.5.
a b
Fig. 26.6 Sebaceous carcinoma (a). Sebocytes are stained with EMA (b). Nuclear reactivity of tumor cells for andro-
gen receptor (c)
Table 26.4 Immunoprole of sebaceous adenoma (SA)

metastatic melanomas, followed by primary
and sebaceous carcinoma (SC)
melanomas and nevi [32, 33]. Other prognostic
Tumor Ki-67 (%) p53 (%) Bcl2 (%) p21 (%)
markers correlated with melanoma progression
Sebaceous 10 11 56 34
adenoma
and prognosis include MIB-1 (Ki-67), Bcl2, p53,
Sebaceous 30 50 7 16
p16, cyclin-D1, cyclin-D3, osteopontin, NM23,
carcinoma E-cadherin, beta-catenin, Wnt5a/frizzled, Cdc42,
Refs. [24, 25] and CXCR4 [3440].
26.2.7 Prognostic Markers 26.2.8 Specic Mesenchymal Tumors

of Melanoma of the Skin
Detection of BRAF p.V600E mutation by immu- Mesenchymal tumors are discussed in soft tissue
nohistochemistry in melanomas could be used as tumors, but some tumors which are more seen in
a rst step to identify patients with melanoma as skin are discussed here. Kaposi sarcoma which
candidates for BRAF inhibitors. Displaying by originates from endothelial cells is an intermedi-
immunohistochemistry, melanoma progression is ate malignant potential vascular tumor of the skin
correlated with MERTK expression: highest in positive for a highly sensitive and specic Ab
a b
c d
e f
Fig. 26.7 Small round cell tumor in the skin. Malignant melanoma (a) reacts with S100 (b) and melan-A (c) antibod-
ies. Merkel cell carcinoma (d) immunostained by CK20 as paranuclear dots (e) and shows weak reaction with CD99 (f)
called HHV8 latent nuclear antigen-1 [41]. broxanthoma is a brohistiocytic tumor exhibit-
Dermatobrosarcoma protuberance is an interme- ing reactivity with vimentin, CD10, and CD99
diate tumor of brohistiocytic cell origin which is (Fig. 26.9) [43]. Among tumors with smooth
diffusely positive for CD34 (Fig. 26.8) and nega- muscle differentiation, leiomyoma and leiomyo-
tive for factor XIIIa separate from dermatobroma sarcoma are reactive for SMA, desmin, and calde-
which is in reverse of DFSP (CD34, factor smon similar to extracutaneous equivalents
XIIIa+) [42]. Considering it as a supercial vari- [44, 45]. Neurothekeoma (NTKs) is a distinctive
ant of malignant brous histiocytoma, atypical neoplasm of the skin showing schwannian and
Table 26.5 Immunopanel of small cell melanoma (SCM), Merkel cell carcinoma (MCC), small cell squamous carci-
noma (SSCC), small cell eccrine carcinoma (SEC), peripheral neuroectodermal tumor/extraskeletal Ewing sarcoma
(PNET/ES), lymphoma, rhabdomyosarcoma (RMS), and metastatic pulmonary small cell carcinoma (MPSC)
Panel antibodies SCM MCC SSCC SEC PNET/ES Lymphoma RMS MPSC
S100/HMB45/MART +
CK20/CD56/SYN/CGN +
CK/EMA + + +
CD15/MOC31/TAG-72 +
CD99/CD56/SYN/CGN + +
LCA/CD3/CD20 +
DES/MSA/MYG +
CEA/TTF-1 +
Refs. [2631]
Note: CGN chromogranin A, DES desmin, MYG myogenin, MSA muscle-specic antigen. LCA leukocyte common
antigen, SYN synaptophysin
a b
Fig. 26.8 Dermatobrosarcoma protuberans. Spindle brohistiocytic cells, entrapping subcutaneous fat tissue (a)
highlighted by CD34 (b)
neuroectodermal differentiation which typically small cell neuroendocrine carcinoma, and ES/
labels with S100 (conventional variant), CD99, PNET (Table 26.6). Undifferentiated carcinomas
and NKI-C3 (cellular variant) [46]. include sinonasal undifferentiated carcinoma,
undifferentiated nasopharyngeal carcinoma
(Fig. 26.10), and undifferentiated neuroendo-
26.3 Immunohistochemistry crine carcinoma (Fig. 26.11) [47, 48]. All poorly
of Head and Neck Tumors differentiated and undifferentiated carcinomas
express cytokeratin [49]. Undifferentiated naso-
26.3.1 Tumors of the Nasal Cavity pharyngeal carcinoma reacts with EBV, and
and Paranasal Sinuses undifferentiated neuroendocrine carcinoma is
positive for neuroendocrine markers and S100
Tumors of the nose and paranasal sinuses can be [50]. NUT midline carcinoma (NMC) is an
categorized in two groups of small cell carcino- aggressive tumor with translocation of the NUT
mas and undifferentiated carcinomas. Small cell (nuclear protein in testis) gene resulting in the
carcinomas of the nasal cavity and paranasal formation of BRD4-NUT fusion gene. Recently,
sinuses include olfactory neuroblastoma (ONB), new mAbs against the NUT Ag have been
melanoma, lymphoma, rhabdomyosarcoma, designed which will improve the diagnosis of
a b
Fig. 26.9 Atypical broxanthoma. Atypical pleomorphic cells with vesicular nuclei in the dermis (a, b) are immunos-
tained by CD10 (c)
Table 26.6 Immunohistochemistry of small cell carcinomas of nasal cavity: olfactory neuroblastoma (ONB), rhabdo-
myosarcoma (RMS), Ewing sarcoma/peripheral neuroectodemal tumor (ES/PNET), and small cell neuroendocrine car-
cinoma (SNEC)
Tumor ONB Melanoma Lymphoma RMS SCC ES/PNET SNEC
Immunoreactive SYN HMB45, LCA, Desmin, AE1/AE3, CD99, SYN Cytokeratin,
markers S100, vimentin Myogenin, EMA, SYN neuroendocrine
vimentin vimentin markers
Refs. [49, 5357]
NMC [51]. Immunohistochemistry of poorly dif- 26.3.2 Tumors of the Larynx,

ferentiated and undifferentiated carcinomas are Nasopharynx,
denoted in the Table 26.7. and Oropharynx
26.3.1.1 Theranostic Application Squamous cell carcinoma (SCC) is the most com-
In olfactory neuroblastoma, immunoreactivity mon malignancy in the head and neck. Typically,
with bcl-2 may predict response to neoadjuvant head and neck SCCs are positive for cytokeratin
chemotherapy and seems to be associated with cocktails, AE1/AE3, and pan-cytokeratin. Human
worse survival [52]. papilloma virus (HPV) is detected in some SCCs
a b
c d
Fig. 26.10 Undifferentiated nasopharyngeal carcinoma highlights malignant cells (b), and intermixed lympho-
shows inltration of large undifferentiated cells with cytes react with LCA (c). Ki-67 antibody reacts with
intermixed small lymphocytes (a). Cytokeratin antibody about 20 % of malignant cells (d)
of the oropharynx and known as a risk factor of can be a novel marker for the prediction of
head and neck SCCs [60, 61]. Being as a variant of metastasis [66].
SCC, basaloid squamous cell carcinoma (BSCC)
is another tumor with predominance of basaloid
components. Basaloid squamous cell carcinomas 26.3.3 Tumors of the Salivary Glands
express p63 which is relatively specic but also
found in other squamous tumors (Fig. 26.12). Salivary glands are tubuloacinar exocrine glands
Neuroendocrine markers are negative in BSCC having two-layered epithelium which comprise of
[62]. Spindle squamous cell carcinoma (SSCC) is luminal (acinar and ductal cells) and abluminal
a cytokeratin-negative SCC in which spindle cell (myoepithelial and basal cells). Luminal cells are
component is uniformly and strongly positive for positive for low molecular cytokeratin, whereas
vimentin [63]. Undifferentiated nasopharyngeal myoepithelial and basal cells react with high
carcinoma shows reactivity to EBV immunostain- molecular cytokeratin and myoepithelial markers.
ing as well as some SCCs and BSCCs [64, 65]. The majority of salivary gland carcinomas can be
diagnosed by routine hematoxylin and eosin
26.3.2.1 Prognostic Marker (H&E)-stained slides, and immunohistochemical
As a transcription repressor of E-cadherin, (IHC) staining has only a limited role in the
Snail-1 is expressed in more than half of the diagnosis of salivary gland tumors [47, 67].
cases of SSCC but not in SCC. In addition, it Figure 26.13 summarized the various components
a b
Fig. 26.11 Neuroendocrine carcinoma (a). Tumor cells are immunostained with synaptophysin (b) and NSE (c)
Table 26.7 Immunohistochemistry of poorly differenti-

ated and undifferentiated carcinomas of nasal cavity: sino-
nasal undifferentiated carcinoma (SNUC), undifferentiated
neuroendocrine carcinoma (UNEC), and undifferentiated
nasopharyngeal carcinoma (UNPC)
Markers SNUC UNPC UNEC (Fig. 26.11)
Cytokeratin + + +
EBV +
Neuroendocrine +
CD99 +/
S100 +
Refs. [49, 58, 59]
of the normal salivary glands with an emphasis on Fig. 26.12 P63 immunoreaction in basaloid squamous
the immunohistochemistry Abs. cell carcinoma
cystic carcinoma (Fig. 26.14), basal cell adeno-

26.3.4 Immunohistochemistry carcinoma, epithelial-myoepithelial carcinoma,
of Salivary Gland Tumors mucoepidermoid carcinoma (Fig. 26.15), myo-
epithelial carcinoma, polymorphous low-grade
The most common malignant tumors of salivary adenocarcinoma, and salivary duct carcinoma.
glands consist of acinic cell carcinoma, adenoid All tumors are cytokeratin positive; however,
different immunoprole patterns exist [68]. C-kit positive for both epithelial and myoepithelial
(CD117) is positive in acinic cell carcinoma and markers but do not exhibit reaction with EMA
adenoid cystic carcinoma [69, 70]. Acinic cell and CEA [73]. Malignant monophasic salivary
tumor and mucoepidermoid carcinoma demon- gland tumors include acinic cell carcinoma,
strate reactivity with membrane-bound mucin myoepithelial carcinoma, mucoepidermoid car-
(MUC) [71, 72]. Myoepithelial carcinomas are cinoma, and polymorphous low-grade adenocar-
cinoma. Immunophenotype proles of
monophasic and biphasic tumors are denoted in
Acinar cell Ductal cell Tables 26.8 and 26.9. Application of CK7 and
MUC1, CK20 is a useful panel in distinguishing primary
EMA, CEA, CK7, 8, 18
Markers CK7, 8, 18 amylase MUC2, salivary gland carcinoma (CK7+, CK20) from
19, EMA,
MUC4,
SOX10 CEA
MUC5AC metastatic carcinoma (CK7, CK20+) [74].
CK5, 7, 14, vimentin,

Markers
p63, S100, GFAP
CK5, 14, vimentin, p63 26.3.4.1 Prognostic Marker
In mucoepidermoid carcinoma, MUC1 expres-
Myoepithelial cell Basal cell
sion is correlated with tumor progression and
Fig. 26.13 Normal salivary gland components with worsened prognosis, whereas MUC4 expression
immunohistochemistric antibodies is related to a better prognosis [72].
a b
Fig. 26.14 Adenoid cystic carcinoma with typical cribriform pattern (a) shows immunoreaction with EMA (b) and
CEA (c)
a b
Fig. 26.15 Poorly differentiated mucoepidermoid carcinoma with polygonal atypical epidermoid cells (a) exhibits
immunostaining with CK7 (b) and EMA (c)
Table 26.8 Immunophenotype of monophasic malignant salivary gland tumors: acinic cell carcinoma (ACC), myoepi-
thelial carcinoma (MC), mucoepidermoid carcinoma (MEC), and polymorphous low-grade adenocarcinoma (PLGC)
Tumor AC MC MEC PLGC
Epithelial CAM5.2, CK7/8/18, AE1/AE3, CAM5.2, CAM5.2, CK7/8/14/18/19, EMA, CAM5.2, CK7,
Markers EMA, CEA, MUC3 CK14, 34E12 CEA, MUC1/4/5 AC, 5B 14, EMA
Myoepithelial/ N p63, calponion, p63 (epidermoid component) p63
basal markers SMA, myosin
Other markers C-kit, S100 Vimentin, S100, S100
GFAP
Refs. [68, 69, 7173, 75, 76]
Table 26.9 Immunophenotype of biphasic malignant salivary gland tumors: adenoid cystic carcinoma (ACC), basal
cell adenocarcinoma (BCA), epithelial-myoepithelial carcinoma (EMC), and salivary duct carcinoma (SDC)
Tumor ACC BCA EMC SDC
Epithelial CAM5.2, CK7/14/19, AE1/AE3, CAM5.2, AE1/AE3, CAM5.2, AE1/AE3, EMA, CEA
markers EMA, CEA CK7, EMA, CEA CK14
Myoepithelial/ p63, calponin p63, calponin, SMA p63, calponin, SMA p63
basal markers
Other markers C-kit, S100 C-kit, S100 S100 AR, GATA3, HER2/neu
Refs. [68, 69, 71, 7782]
a b
c d
Fig. 26.16 Thyroid papillary carcinoma. Papillary projections with intranuclear inclusions (a) and Orphan Annie
nuclei (b) are highlighted by thyroglobulin in the cytoplasm (c) and TTF1 in the nuclei (d)
26.3.5 Tumors of Thyroid in parathyroid adenomas, whereas its expres-

and Parathyroid Glands sion is often reduced in parathyroid carcinomas.
Table 26.10 shows an immunopanel of thyroid
The functional unit of thyroid is the follicle and parathyroid tumors. Figure 26.17 depicts
which is composed of follicular cells and C cells. thyroid medullary carcinoma.
Follicular cells exhibit reactivity with thyroglob-
ulin, TTF1, PAX8, AE1/AE3, EMA, and CK7
and CK8/18/19, whereas C cells are positive for 26.4 Immunohistochemistry
calcitonin, TTF1, CK7, synaptophysin, and chro- of Lung Tumors
mogranin. Being as a nuclear transcription fac-
tor, TTF1 is expressed on follicular and C cells. 26.4.1 Adenocarcinoma
A follicular cell-specic marker is thyroglobulin
which does not react with C cells (Fig. 26.16). The most frequent IHC pattern observed in lung
As a member of the paired box (PAX) gene fam- tumors is positivity for CK7, TTF1, and Napsin
ily, PAX8 is a sensitive marker of thyroid tumors A, along with negative staining for CK20, CDX2,
similar to TTF1. Among intermediate laments, and MUC2. It is highly advocated to consider the
CK19 is more expressed in papillary carcinoma fact that there are recently increasing reports of
than other tumors. Parathyroid hormone (PTH) primary pulmonary adenocarcinomas with intes-
and parabromin are markers of parathyroid tinal differentiation which are CK7 and TTF1
tumors. Parabromin is uniformly expressed negative but CK20 positive which can be highly
Table 26.10 Immunopanel of thyroid and parathyroid misinterpreted as metastatic colorectal adenocar-
tumors
cinomas. Therefore, the importance of physical
First-choice Second-choice examination and imaging studies is highlighted.
antibody panel antibody panel Consistent with
It should be noted that neuroendocrine markers
CK+, TTF1+, PAX8+, CK19+ Papillary
including chromogranin, synaptophysin, NSE,
TGB+ carcinoma
(Fig. 26.16) and Leu7 (CD57) can be positive in lung non-
PAX8, VIM+ Follicular neuroendocrine carcinomas such as adenocarci-
carcinoma nomas and SCC. Recent studies have shown
CK+, TTF1+, TGB Calcitonin+, Medullary EGFR, Her2, and BRAF mutations in lung can-
SYN+, CGN+ carcinoma cers which can increase the chance for targeted
(Fig. 26.17)
therapies in these cancers [103106].
CK, TTF1+, TGB p53+, VIM+, Anaplastic
PAX8 carcinoma
CK+, TTF1, TGB PTH+, CGN+, Parathyroid
parabromin tumor 26.4.2 Mesothelioma
Refs. [83102]
Note: CGN chromogranin, SYN synaptophysin, TGB thy- Neoplasms of the pleura are very rare, and most
roglobulin, VIM vimentin
tumors in this area are usually metastatic lesions.
a b
Fig. 26.17 Thyroid medullary carcinoma. Solid nests with medium-sized atypical cells (a) exhibit immunoreaction
with calcitonin (b) and chromogranin (c)
Table 26.11 Immunohistochemistric differentiation of pulmonary adenocarcinoma (PAC) and malignant mesothelioma
Marker Pulmonary AC Mesothelioma Comment
Calretinin R Usually + The most specic and reproducible positive marker in
mesothelioma
CDX2 R About 13 % positive, in pulmonary mucinous carcinomas
Cytokeratin AE1/AE3, CK5/6 (S), CK7 CK7: Most common CK in primary lung cancer (About
CK5/6 (R), (used to 100 % in AC, 40 % in small cell carcinoma, about 20 % in
CK7, differentiate carcinoid tumor, and none of SCC arising from lung)
mesotheliomas CK5+ specially in lung SCC
from sarcomas)
D2-40 + Usually positive specially in sarcomatoid variants of
mesothelioma
EMA S (cytoplasmic) S (membranous)
TTF1 +
Mesothelin +
p63 Positive in pulmonary SCC
pCEA +
S100 +
SMA 5060 %
SP-A (surfactant 50 %
protein A)
Thrombomodulin +
Vimentin +
WT1 60 %
Note: pCEA polyclonal CEA, SMA specic muscle antigen
One of the most important applications of IHC is site and the patients clinical history [110].
to assist pathologists in differentiating mesothe- Previous studies show that blinded use of an IHC
liomas from lung adenocarcinomas [107109]. panel for differential diagnosis can primarily
Table 26.11 shows the most frequent markers identify about 83 % of tumor origins vs. 65.6 %
stained by IHC staining in mesothelioma of metastasis. Several publications on IHC stud-
compared with pulmonary adenocarcinoma ies are available, and each recommends its own
(Fig. 26.18). IHC panel for differential diagnosis. This makes
it clear that there is no single IHC panel, or stan-
dard of care, for tissue determination, and pathol-
26.5 Immunohistochemistry ogists have long known that tissue of origin
of Gastrointestinal Tumors identication is inherently a multiplex problem
[111113].
Immunohistochemistry is used in gastrointestinal Here, the authors have briey tried to intro-
and colon cancers to particularly determine the duce the major and common IHC markers used to
tumor subtype and origin, especially for poorly differentiate frequent gastrointestinal tumors. It
or undifferentiated cancers for which morphol- should be noted that the average positivity of a
ogy alone cannot determine the origin. Generally, marker in a specic tumor differs from one study
it should be noted that denite tissue diagnosis in to another, as well as in different textbooks. In
clinical practice needs combination of IHC this chapter the most prevalent and reliable data
results and clinical information, including biopsy are provided.
a b
c d
Fig. 26.18 Mesothelioma. Adenomatoid type (a) shows immunostaining for mesothelin (b), and tubular type (c)
shows immunoreaction for calretinin (d)
Table 26.12 Immunohistochemistry of normal liver

Markers
Normal tissue Hepatocellular Adenocarcinoma Carcinoma Canalicular Others
Hepatocytes HepPar1, TTF1 MOC31 CAM5.2 CD10, pCEA B-catenin
(cytoplasmic)
Bile duct cells CK7, CK19 (+/), CAM5.2, CKAE1/ B-catenin
MUC6 AE3, EMA, BerEp4
26.5.1 Liver the least, certain differential diagnoses can be

excluded [114118]. Immunophenotype of
The most common primary hepatic cancer is normal liver is summarized in Table 26.12
hepatocellular carcinoma which is well known to (Figs. 26.19 and 26.20).
have a wide spectrum of histologic differentia- Cholangiocarcinoma is a malignant tumor
tion and a great diversity of appearances. It with characteristics mostly similar to other
necessitates the application of IHC as an ancil- types of adenocarcinomas. The tumor is usually
lary aid for better diagnosis of the lesion. It is positive for CK7, CK19, CAM5.2, CK AE1/
important to reiterate that IHC is after all an AE3, pCEA, mCEA (noncanalicular pattern),
ancillary aid. A signicant clinicopathologic cor- and MOC31. MUC4, MUC5AC, and MUC6
relation seems mandatory for the nal diagnosis. can also be useful not in diagnosis but in
If a denitive diagnosis cannot be clinched, at classication and predicting the prognosis.
Additionally, CD56 which is positive in benign the esophagus is immunophenotypically simi-

bile ductular proliferations and negative in lar to gastric adenocarcinomas, and there is no
cholangiocarcinomas can be useful in differen- IHC panel to distinguish these two. Esophageal
tiating malignant lesions from benign prolifera- SCC is usually positive for most CK markers
tion. The exception for this rule is clear cell including CK AE1/AE3, CK 34bE12, CK5/6,
cholangiocarcinoma which is positive for CK19 (positivity increases with tumor grade
CD56. Staining for CK7 and CK19 in cholan- whereas benign squamous lesions are negative
giocarcinoma can help to differentiate this for this marker), and p63. Additionally, most
tumor from HCC, which is negative for the SCCs are negative for CK7 and CK20 which
mentioned markers [119, 120]. Table 26.13 can be useful in distinguishing poorly differen-
indicates the immunophenotypes of hepatocel- tiated SCCs from poorly differentiated adeno-
lular carcinoma and cholangiocarcinoma. carcinomas positive for these two CK markers
[121123].
26.5.2 Esophagus
26.5.3 Stomach
The most common esophageal cancers are ade-
nocarcinomas and SCC. Adenocarcinoma of Stomach glandular epithelium expresses CK20
and less commonly CK7 (CK7+, CK20+) and
MUC5AC, distinguishing it from small intes-
tine and colorectal epithelium. Immunoprofile
of normal gastrointestinal mucosa is denoted
in Table 26.14. Gastric adenocarcinoma has
many histologic variants, but they have almost
similar immunophenotyping. It should be
mentioned that synaptophysin and chromo-
granin as neuroendocrine markers can be posi-
tive in gastric adenocarcinomas; therefore,
positive staining with these markers is not suf-
ficient for the diagnosis of neuroendocrine
carcinoma [124126]. Immunoprofile of gas-
Fig. 26.19 Normal liver stains with HepPar1 showing tric adenocarcinoma is demonstrated in
typical cytoplasmic coarse granules of hepatocytes Table 26.15 (Fig. 26.21).
a b
Fig. 26.20 Hepatocellular carcinoma with huge bizzare giant nuclei making diagnosis simple as malignant (a) exhibits
reactivity with HepPar1 (b)
Table 26.13 Immunohistochemistry of hepatocellular carcinoma and cholangiocarcinoma

Markers
Tumor Hepatocellular Adenocarcinoma Carcinoma Canalicular Sinusoidal
Hepatocellular HepPar1, TTF1 CAM5.2, CD10, pCEA CD34, FVIII
carcinoma (cytoplasmic) EMA (/+)
Cholangiocarcinoma MOC31, CK7, CAM5.2, pCEA, mCEA
CK19, MUC4, CKAE1/AE3 (noncanalicular)
MUC5AC, MUC6
Table 26.14 Immunoprole of normal gastrointestinal mucosa

Simple epithelial marker MUC
Intestinal CDX2
Gastric (MUC2, (intestinal
Normal tissue CK7 CK20 AE1/AE3 CAM5.2 CEA (MUC5AC) MUC4) marker) CD15
Stomach +/ + + + + + +
Small intestine + + /+ + + +
Large intestine/ + + + + + + +
appendix
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
Table 26.15 Immunoprole of gastric, small intestine, and colorectal adenocarcinoma (AC)
Tumor associated marker MUC
Intestinal CDX2
CK Gastric (MUC2, (intestinal
Tumor type 18/19 CK7 CK20 AE1/AE3 CAM5.2 CEA (MUC5AC) MUC4) marker) CD15
Gastric AC + +/ /+ + + + /+ /+ /+
Small intestine + +/ +/ + /+ /+ /+ +/ +/
AC
Large intestine/ + + + + + /+ +/ +
appendix AC
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
a b
Fig. 26.21 Adenocarinoma of the stomach with atypical glands and nuclear pleomorphism (a) immunostained with
CEA (b)
Table 26.16 Immunoprole of colon adenocarcinoma based on chromosomal instability and MSI pathways
Chromosomal instability pathway (8085 %) MSI pathway (1520 %)
CK20 100 % CK20 Can be negative in about 30 %
MUC2 Usually positive MLH1 Complete absence of staining
MUC5AC Usually negative (about 30 % positive, especially MSH2 with a sufcient internal
in mucinous carcinomas) control is needed for a positive
CAM5.2 Usually positive MSH6 result
MOC31 Usually positive PMS2
CDX2 About 90 % CDX2 Can be negative in about 20 %
CK7 510 %
CEA Usually positive specially monoclonal type
CK8 Usually positive
CKAE1/AE3 Usually positive
MSI-related markers These markers are usually positive in this subtype
of colon carcinomas
26.5.4 Small Intestine CDX2, and CK5/6 which helps to differentiate

them from adenocarcinomas of colon origin
Immunophenotyping of adenocarcinoma is also [135, 137, 138].
valuable in neuroendocrine tumors (NET) [127
129]. Tables 26.14 and 26.15 summarize the
immunoprole of normal small intestine, its ade- 26.5.7 Appendix
nocarcinoma, as well as their comparison with
stomach and colon adenocarcinoma. Mucinous adenocarcinomas of appendix origin
can be distinguished from mucinous colorectal
carcinomas with immunostaining for CK7 and
26.5.5 Colon MUC markers [139141].
In contrast to older studies which have discussed

colon cancers generally, recent studies reveal that 26.5.8 Pancreas
colon cancers arise from two different pathways
(chromosomal instability of APC gene vs. mic- Pancreas is composed of glandular/ductal, aci-
rosatellite instability (MSI) pathway) with differ- nal epithelium, and endocrine cells. Pancreatic
ent immunophenotypic features [116, 130136]. neoplasms can be roughly divided into two
Immunoprole of normal and colon adenocarci- categories of exocrine and endocrine system
noma is denoted in Tables 26.14, 26.15, and 26.16. neoplasms. This part mostly discusses the exo-
crine system and mostly adenocarcinomas of
this area. Additionally, tumor suppressor genes
26.5.6 Anal including DPC4 and SMAD4 are inactivated
in about 5060 % of the adenocarcinomas of
The most frequent anal cancers are SCC and ade- this site [116, 142, 143]. Immunoprole of
nocarcinoma. Anal SCC is almost similar to SCC normal pancreas and some pancreatic tumors
of other origins; nonetheless, the role of HPV is are summarized in Tables 26.17 and 26.18.
highlighted. Adenocarcinomas of the anus are Figure 26.22 depicts solid pseudopapillary
usually positive for CK7 and negative for CK20, neoplasm.
Table 26.17 Immunoprole of normal pancreas

Marker Normal tissue
Exocrine Glandular/ductal Epithelial CAM5.2, AE1/AE3, CK7, CK8/1/8/19
MUC MUC1, MUC6
ONP
Acinar Trypsin, chymotrypsin, lipase, amylase, elastase
Endocrine CGN, SYN, NSE
Table 26.18 Immunoprole of some pancreatic tumors: pancreatic ductal adenocarcinoma (PDAC), acinar cell carci-
noma (ACC), neuroendocrine carcinoma (NEC), and solid pseudopapillary neoplasm (SPN)
Marker PDAC ACC NEC SPN (Fig. 26.22)
Exocrine Glandular/ Epithelial CAM5.2, AE1/AE3, CAM5.2, AE1/ CAM5.2, AE1/
ductal CK7, CK8/18/19, AE3, CK8/18, AE3, CK19 (Positive for
pCEA, PSCA EMA -catenin,
vimentin, PR,
CD10)
MUC MUC1, MUC3,
MUC4, MUC5AC,
MUC6 (+/)
ONP CA19.9, CA125,
B72.3, DUPAN-2,
CECAM1
Acinar Trypsin, 1-antitrypsin
chymotrypsin, lipase,
amylase, elastase
Endocrine CGN, SYN CGN, SYN, NSE, CGN, SYN,
CD56, CD57 NSE, CD56
Note: CGN chromogranin, NSE neuron-specic enolase, ONP oncoprotein, PR progesterone receptor, SYN synaptophysin
a b
Fig. 26.22 Solid pseudopapillary neoplasm. Papillary projection covered by relatively bland-looking cells supported
by a hyalinized stroma (a) highlighted with vimentin (b)
26.5.9 Gastrointestinal diagnostic clues are histologic features, as well

Stromal Tumor as immunostaining for synaptophysin, chro-
mogranin, and NSE (Fig. 26.24). In addition to
Gastrointestinal stromal tumor (GIST) is a soft the mentioned markers, most of neuroendocrine
tissue tumor of the GI wall which is in the dif- tumors can express the tissue markers in which
ferential diagnosis of leiomyoma and broma- they originated which help to diagnose the ori-
tosis. Most GISTs express c-kit (>95 %), CD34, gin of metastatic neuroendocrine tumors [143,
and CD99 (Fig. 26.23). Sometimes weak posi- 146148].
tivity for S100, SMA, desmin, and synaptophy-
sin (but not chromogranin) can also be found
[135, 144, 145]. 26.6 Immunohistochemistry
of the Urinary Tract
26.5.10 Neuroendocrine Carcinomas 26.6.1 Kidney
Neuroendocrine tumors arise from different Renal cell carcinoma (RCC) is the most
organs. Most have similar morphology and common tumor of the kidney with variants of
tumor marker expression, and the most important clear renal cell carcinoma (CRCC), papillary
a b
Fig. 26.23 Gastrointestinal stromal tumor. A low-grade intestinal wall tumor shows uniform spindle cells with elon-
gated nuclei (a), with immunoreaction to c-kit (b) and CD34 (c)
a b
Fig. 26.24 Neuroendocrine carcinoma composed of atypical cells with round nuclei and dusty chromatin (a). Tumor
cells are immunostained with chromogranin (b) and synaptophysin (c)
renal cell carcinoma (PRCC), and chromophobe is 34E12+, CD10, and AMACR, in contrast to
carcinoma (CC). Commonly used immunohis- PRCC which is 34E12, CD10+, and AMACR+
tochemical Abs in the urinary system are sum- [150, 155]. Considering the histopathologic pat-
marized in Table 26.19. Immunohistochemistry tern, the following immunopanels (Tables 26.20
is an ancillary test used to distinguish variants and 26.21) compare the immunohistochemical
of RCC as well as tumors with histopathologic Abs in these tumors.
similarities including collecting duct carcinoma
and urothelial carcinoma of the renal pelvis.
Carcinomas with clear cell feature include CRCC 26.6.2 Bladder
(Fig. 26.25), papillary renal cell carcinoma, and
transitional (urothelial) cell carcinoma of the Normal urothelium exhibits a unique pattern
renal pelvis. Differential diagnoses of carci- of cytokeratin expression characterized by
noma with oncocytic appearance are chromo- coexpression of simple epithelium cytokera-
phobe carcinoma, oncocytoma, and oncocytic tin (CK7, CK20, and CAM5.2) and HMWCK
papillary RCC (Fig. 26.26) [149154]. The (CK5/6 and 34E12). While CK20 is expressed
immunophenotype of collecting duct carcinoma in umbrella cells of the normal urothelium, in
Table 26.19 Immunohistochemical markers in urinary system tumors

Marker Function Immunoreaction in tumor
AE1/AE3 Pan-CK epithelial marker RCC
CAIX Carbonic anhydrase IX: maintenance of intracellular and PRCC
extracellular pH, regulatory role in cell proliferation
CAM5.2 Intermediate cytoskeleton lament RCC, PRCC, CC, CDC
CD10 (CALLA) A zinc-dependent cell membrane metalloprotein RCC, PRCC
CD117 (c-kit) Transmembrane glycoprotein receptor tyrosine kinase CC, CDC, OC
CK7 LMWCK (simple epithelia) PRCC, CC, UC, PAC (+/)
CK20 LMWCH (simple epithelia) UC (+/), PAC (+/)
34E12 HMWCK (CK1, 5, 10, 14) CDC, UC
EGFR Receptor with tyrosine kinase activity UC (+/)
Ep-Cam Glycosylated transmembrane cell surface epithelial protein in PRCC (+/), CC, CDC
distal nephron
HMWCK Intermediate cytokeratin laments of prostate basal cell Negative marker in PAC
Ki-67 (MIB1) Nuclear protein expressed in all phases of the active cell cycle Proliferative marker
(G1, S, G2, M)
Ksp-cadherin Calcium-dependent cell adhesion molecule plays an important CC, OC
(kidney-specic) role in the maintenance of tissue integrity
p53 Tumor suppressor protein UC
p63 A member of p53 family transcription factor, marker of basal Negative marker in PAC
cells
P501S (Prostein) A 553-amino acid protein localized to the Golgi complex PAC
P504S (AMACR) Enzyme mainly localized to peroxisomal structures PRCC, PAC
PAX2/PAX8 Members of the paired box (PAX) gene family expressed in the RCC, PRCC, CC, CDC,
development of the urogenital tract OC (+/)
PSA 330-kD glycoprotein, prostate-specic antigen PAC
PSAP 100-kD glycoprotein, prostate-specic antigen PAC
PSMA 100-kD glycoprotein, prostate-specic antigen PAC
RCC 200-kD glycoprotein expressed in epithelial cells lining the RCC, PRCC
normal renal proximal tubule
Thrombomodulin 75-kD glycoprotein, to convert thrombin from a coagulant UC
protein to an anticoagulant
Uroplakin III A transmembrane protein unique to urothelium UC
Vimentin Intermediate cytoskeleton lament RCC, PRCC, CDC
Refs. [150184]
Note: CC chromophobe carcinoma, CDC collecting duct carcinoma, OC oncocytoma, PAC prostatic adenocarcinoma,
PRCC papillary renal cell carcinoma, UC urothelial carcinoma
dysplastic urothelium and carcinoma in situ, Immunohistochemistry can be helpful to differ-

it is expressed in all layers of the urothelium entiate urothelial carcinoma from direct exten-
[150154, 168, 169]. CD44 is expressed in the sion of an adjacent primary carcinoma (prostate,
basal layer of normal urothelium and shows colorectal, cervix, and uterine) as well as metas-
focal staining of basal layers of the dysplastic tasis and also to distinguish variants of urothe-
urothelium [170]. Urothelial carcinomas are lial carcinoma. Common immunohistochemistry
divided into (1) noninvasive papillary carcinoma Abs in normal urothelium, urothelial hyperpla-
and (2) invasive carcinoma which can appear sia, urothelial dysplasia, and urothelial carci-
as papillary or non-papillary itself (Fig. 26.27). noma are summarized in Table 26.22.
a b
Fig. 26.25 Renal cell carcinoma, eosinophilic to clear cells (a) is immunostained with CD10 (b) but not with CK20 (c)
26.7 Immunohistochemistry negative for vimentin and ER, whereas endome-

of Female and Male Genital trium adenocarcinomas have a reverse expression
Tumors pattern [176181].
26.7.1 Uterine Cervix

26.7.2 Vulva and Vagina
The most important and also frequent cervix
cancers are cervix SCCs and adenocarcino- As other organs, various malignancies can occur
mas. Cervix SCC markers are similar to those in these two organs, but similar to cervix, the
seen in SCCs of other origins. p16 is a unique most common cancer of these two sites is SCC,
marker expressed in tumors of the cervix which with IHC marker expression similar to cervix
can help in differentiating this lesion from the counterparts [182, 183].
same counterparts from uterine or other origins.
Adenocarcinomas of the cervix also express most
adenocarcinoma markers. One of the advantages 26.7.3 Uterine Corpus
of IHC is to differentiate adenocarcinomas of the
cervix from the endometrium. Cervix adenocar- Uterine tumors are of myometrium or endome-
cinomas usually express p16 and CEA, and are trium origin. The myometrial tumors are usually
a b
c d
Fig. 26.26 Papillary renal cell carcinoma with oncocytic feature (a). Tumor cells are positive for CK7 (b), CD10 (c),
and vimentin (d)
sarcomas and were discussed in the sarcoma sec- CK7 positive and CK20 negative (Fig. 26.28).
tion. The endometrium may develop various can- This can be used in differentiating primary
cers, but the most frequent one is endometrial ovarian carcinoma from metastatic tumors
adenocarcinoma. Endometrial adenocarcinoma [139141, 180, 188191]. The immunopheno-
has some variants in which endometrioid adeno- type of primary ovarian tumors is described in
carcinoma is the most frequent one. Endometrioid Table 26.23.
adenocarcinoma usually expresses CK7, CA125,
ER, PR, and vimentin but is negative for CEA,
CK20, and p16. Some endometrial carcinomas 26.7.5 Breast
express Her2/neu marker, which along with ER
and PR markers can be used in targeted therapies Breast cancer is one of the most common
[176181, 184187]. malignancies with various histopathological
types; however, adenocarcinomas and its two
subtypes including invasive ductal (IDC) and
26.7.4 Ovary lobular carcinomas (ILC) comprise the major-
ity. Most breast cancers including IDC and
Except the intestinal type of mucinous adeno- ILC are positive for mammaglobin, GCDFP15,
carcinoma, all primary ovarian carcinomas are ER, and PR, and some are positive for Her2/
Table 26.20 Immunoprole of kidney carcinoma with clear cell appearance: clear RCC (CRCC), papillary RCC
(PRCC), and urothelial carcinoma (UC)
Tumor CK7 CK20 Vimentin RCC CD10 PAX2/8 AMARC Uroplakin p63
CRCC + + + +
PRCC + + + + + +
UC + + + +
Refs. [150160]
Table 26.21 Immunoprole of kidney carcinoma with oncocytic cell appearance: oncocytic papillary RCC (OPRCC),
chromophobe carcinoma (CC), and oncocytoma (OC)
CAM5.2, EMA,
Tumor CK7 CK20 AE1/AE3 Vimentin RCC CAIX CD10 CD117 Ep-Cam Ksp-cadherin
OPRCC + + + + + + +
CC + + + +
OC + +
Refs. [163167]
a b
c d
Fig. 26.27 Transitional cell carcinoma, invasive, non-papillary type (a). Tumor cells exhibit immunoreaction with
CK7 (b), CK20 (c), and p63 (d)
Table 26.22 Antibody immunoprole in normal urothelium, urothelial hyperplasia, dysplasia, and carcinoma
Marker Normal urothelium Urothelial hyperplasia Urothelial dysplasia Urothelial carcinoma
CK7 + + ND +
CK20 +U + + +
34E12 +B ND ND +
CD44 +B ND /+ ND
EGFR /+ + +/ +/
p63 ND ND ND +a
UPIII +U ND ND +a
TM +U ND ND +a
p53 + +a
Refs. [168175]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %). B basal layer, TM thrombomodulin, U umbrella cell, UPIII
uroplakin III
a
Noninvasive carcinoma > invasive carcinoma
a b
Fig. 26.28 Ovarian serous carcinoma poorly differentiated (a) shows immunoreaction with CK7 (b). CA125 is high-
lighted in the luminal surface (c)
Table 26.23 Immunophenotype of ovarian cancers

Stromal tumors (almost
Epithelial tumors Germ cell tumors always negative for EMA)
Serous Embryonal Choriocar- Granulosa Sertoli-Leydig
(Fig. 26.28) Mucinous Dysgerminoma Yolk sac carcinoma cinoma cell tumor cell tumor
EMA EMA PLAP PLAP PLAP HCG Inhibin CK
CK7 CK7 CD117 (c-kit) AFP Oct-4 Inhibin CD99 CD99
CA125 CK20 Oct-4 CK AE1/AE3 CK AE1/AE3 CK WT1 WT1
DPC4 mCEA D2-40 Glypican-3 CD30 Calretinin
ER CDX2 CD56
PR MUC5A
WT1
a b
Fig. 26.29 Cytokeratin (a) stains epithelial cells and p63 (b) stains myoepithelial cells of normal breast glands
neu markers. Additionally, epithelial tumor 26.7.6 Prostate

markers, CK (especially CK7) and EMA, are
also positive in these tumors [192197]. The Prostate gland is composed of two layers,
lack of reaction with myoepithelial mark- epithelium and basal cell layer. Normal pros-
ers is in favor of an invasive carcinoma. Both tate epithelium exhibits immunoreactivity with
normal (Fig. 26.29) and proliferative glands prostate-specic antigen (PSA), prostate-specic
(Fig. 26.30) and ductal carcinoma in situ membrane antigen (PSMA), prostate-specic
(Fig. 26.31) exhibit reactivity with myoepithe- acid phosphatase (PSAP), prostein (P501S), and
lial markers. Application of p63 and calponin -methylacyl-coenzyme-A racemase (AMACR)
or p63 and SMA is a good way to evaluate the enzyme, whereas prostate basal cells display immu-
presence of myoepithelial cells [192, 198]. nostaining with HMWCK (34E12), p63, and
Immunoprole of normal breast glands and S100A6 (Fig. 26.34) [149154]. Immunolabeling
breast cancers are summarized in Tables 26.24 for basal cell markers is usually used in a mode
and 26.25 (Figs. 26.32 and 26.33). of negative diagnostic marker in order to show
a b
Fig. 26.30 Breast proliferative lesion (a). Presence of myoepithelial cells conrmed by immunoreaction to HMWCK
(b) and p63 (c) which is indicative of a benign process
the absence of basal cells in prostate carcinoma tumors are the most common type with classic
(Fig. 26.35). Basal cell cocktail is a mixture of basal seminoma subtype comprising the majority. The
cell markers (HMWCH and p63 or CK5/6 and denite diagnosis of these tumors is dependent
p63) used to highlight the presence of basal cells in on proper application of the immunohistochemis-
normal glands which differentiates benign lesions tric markers and histopathologic evaluation of the
from prostate intraepithelial neoplasia (PIN) and biopsy (Figs. 26.37, 26.38, and 26.39). Table 26.27
prostate adenocarcinoma [201]. Metastatic carci- summarized the immunophenotype of testicular
noma of prostate origin exhibits reactivity to CK 7 tumors.
and CK20 as well as PSA (Fig. 26.36). Table 26.26
summarized the immunoprole of normal prostate
glands as compared with PIN and adenocarcinoma. 26.8 Immunohistochemistry
of Lymphoma
26.7.7 Testis Immunohistochemistry is an integrated part of

diagnostic surgical pathology of Hodgkin
Testicular tumors are classied into germ cell lymphoma (HL) and non-Hodgkin lymphoma
tumors and sex cord stromal tumors. Germ cell (NHL). Various Ags, mostly CD markers, are the
a b
Fig. 26.31 Ductal carcinoma in situ (a) is immunostained with Her2neu (b) and CA15.3 (c)
targets of IHC. Neoplastic lymphoid cells express 26.9 Immunohistochemistry

the same CD Ags with some aberrancy in type of Soft Tissue and Bone
and amount. Several oncogene products are also Tumors
expressed in some lymphomas (i.e., follicular
lymphoma). These Ags have diagnostic and Soft tissue sarcomas are a diverse family with
probably prognostic value. Proliferative Ags like different histologic origins and common histo-
Ki-67 are also of great value. pathologic features. Given similar histopathologic
Morphology is the main stem of lymphoma features, immunohistochemistry is an ancillary
diagnosis; nonetheless, IHC seems mandatory method in distinguishing soft tissue tumors in order
for the diagnosis and typing of malignant lym- to attain a nal diagnosis. As soft tissue tumor
phoma. As a general rule, panels should be used classication is based on specic line tissue origin,
for immunophenotypic evaluation, and there is immunohistochemistry study by using specic
no single marker absolutely specic for one de- Abs can be valuable in distinguishing them. Soft
nite lymphoproliferative disorder. Some rou- tissue tumors are vimentin-positive and keratin-
tinely used markers are shown in Tables 26.28, negative tumors of a divergence family with het-
26.29, 26.30, 26.31, and 26.32 and Figs. 26.40, erogeneous tissue origins. Vimentin, a nonspecic
26.41, 26.42, and 26.43. marker, appears to react with all soft tissue tumors
Table 26.24 Immunoprole of normal breast gland and is considered as a control marker preserved in
tissue
the tissue [246252]. Immunohistochemistry of
Normal epithelium Immunoreactive antibodies normal mesenchymal tissues with related tumors
Luminal cells (LC) CK8/18, CK19 are summarized in Table 26.33
Myoepithelial CK5/6, CK14, CK17, p63,
cells SMA, calponin, CD10
Both LC and MC Pan-CK, AE1/AE3, CK7, S100
26.9.1 Epithelial Markers
Table 26.25 Immunoprole of invasive ductal carcinoma
Recognized as an intermediate lament protein,
(IDC) and invasive lobular carcinoma (ILC) (Figs. 26.32
and 26.33) keratin is a sensitive and specic marker in the
diagnosis of carcinomas among malignant tumors.
Marker IDC ILC
Epithelial membrane antigen (EMA), derived from
Mammaglobin /+ +/
ER +/ +
the mammary epithelium, is another epithelial
GCDFP15 /+ /+ marker expressed in most epithelial cells except
E-cadherin + squamous cells. Keratin and EMA are expressed
p120 + + exceptionally in some soft tissue tumors including
34E12 + synovial sarcoma, epithelioid sarcoma, chordoma,
Refs. [192, 197200] and myoepithelioma/myoepithelial carcinoma
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %) (previously known as parachordoma) [253].
a b
Fig. 26.32 Invasive ductal carcinoma (a) with ER (b) and PR (c) immunoreaction
a b
Fig. 26.33 Inltrating carcinoma with Indian le pattern simulating lobular carcinoma (a), revealing immunoreaction
with E-cadherin which is in favor of invasive ductal carcinoma (b)
a b
Fig. 26.34 Normal prostate tissue (a). The epithelium is immunostained with PSA (b), and basal cells are immunore-
acted with p63 (c)
a b
Fig. 26.35 Atypical prostate glands in the top of the picture which are highly suspicious of adenocarcinoma (a) show
negative reaction to p63 (b). Some normal glands at the bottom of the picture exhibit reaction with p63
a b
c d
Fig. 26.36 An undifferentiated carcinoma from the pelvis with high mitotic rate (a) demonstrates cytoplasmic reaction
with CK7 (b), CK20 (c), and PSA (d) which support the origin of this tumor as prostate
Table 26.26 Immunoprole of normal prostate (NP), high-grade prostate intraepithelial neoplasia (HGPIN), and pros-
tate adenocarcinoma (PAC)
Marker NP HGPIN PAC Application
PSA +E + + Weak reaction in HGPAC or metastatic carcinoma, to
differentiate HGPAD from other undifferentiated carcinoma
(colon, urothelium)
PSAP +E + + Similar to PSA
PSMA +E + ++ Correlated with grade and stage, more intense in HGPAC
P501S +E + + To differentiate high-grade PAC from other high-grade
adenocarcinomas (colon, urothelium)
P504S (AMACR) ++ ++ Combine with basal cell markers to differentiate HGPIN and
PAC from normal prostate
HMWCK (34E12) +B Partial loss Complete loss in PAC (negative marker)
p63 +B Partial loss More sensitive than HMWCK (negative marker)
CK5/6 +B Partial loss More sensitive than HMWCK (negative marker)
Refs. [201209]
Note: B basal cell, E epithelium
a b
Fig. 26.37 Classic seminoma with polygonal cells and abundant watery cytoplasm (a) shows immunostaining with
PLAP (b)
26.9.2 Myogenic Markers actin may react with some other cells like myo-
broblasts and myoepithelial cells [255257].
There are some Abs which react with myo- Myoglobin is exclusively seen in skeletal mus-
genic cells including desmin, actin, myoglobin, cle cytoplasm, whereas myo-D1 and myogenin
myo-D1, myogenin, caldesmon, and calponin. are nuclear transcription factors which are spe-
Desmin is an intermediate lament protein pres- cically expressed in skeletal muscle nuclei
ent in the cytoplasm of smooth and skeletal [258260]. Myogenin has technical advantages
muscles. The Ab against this protein reacts with over those of MyoD1, as the latter may cross-
myogenic tumors such as rhabdomyoma, leio- react with an unknown cytoplasmic Ag in non-
myoma, rhabdomyosarcoma, and leiomyosar- muscle cells and tumors [261, 262]. However,
coma (Fig. 26.44) [254]. Similar to desmin, actin Abs against these Ags are useful in determin-
is another myogenic protein detected in smooth ing rhabdomyosarcoma (Fig. 26.45). Calponin,
and skeletal muscles. In addition, smooth muscle a smooth muscle protein, is also expressed in
a b
c d
Fig. 26.38 Yolk sac tumor with tubuloglandular structures exhibits immunostaining with AFP (a, b) and glandular
structures with numerous hyaline globules which are positive for AFP (c, d)
a b
Fig. 26.39 Leydig cell tumor. Eosinophilic polygonal cell growth in the adjacent of seminiferous tubules (a) show
immunoreaction with inhibin A (b)
Table 26.27 Immunophenotype of testicular tumors: classic seminoma (CS), spermatocytic seminoma (SS), embryo-
nal carcinoma (EC), yolk sac tumor (YST), choriocarcinoma (CC), Sertoli cell tumor (SCT), and Leydig cell tumor
(LCT)
Germ cell tumors (PLAP+, inhibin) Sex cord stromal tumors (PLAP, inhibin+)
CS YST
(Fig. 26.37) SS EC (Fig. 26.38) CC SCT LCT (Fig. 26.39)
C-kit+ C-kit+/ C-kit+/ C-kit+/ Inhibin+ AE1/AE-/+CAM5.2+ AE1/AE-/+GAL-3+
OCT3/4+ OCT3/4+ AE1/AE+ AE1/AE3+ Vimentin+ Vimentin+
CD117+ AE1/AE3+ AFP+ Glypican-3+ SMA+ CD99+/
D2-40+ AFP+/ Glypican-3+ HCG+ SYN+
CD117+ HepPar-1+ NSE+
CD30+
Refs. [154, 210223]
Note: + (>90 %), +/ (>50 %), /+ (<50 %),(<10 %)
Table 26.28 Immunoprole of precursor lymphoid neoplasms (Fig. 26.40)

Lymphoma CD2 CD5 CD20 CD79a PAX5 CD45 CD34 CD10 CD99 Tdt CD43 CD56
B ALL/LBL +/ + + /+ + + + +
T ALL/LBL + + /+ + +/ + + + +
Refs. [224230]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
Table 26.29 Immunoprole of small B-cell lymphomas: B-cell small lymphocytic lymphoma/chronic lymphocytic
lymphoma (B SLL/CLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), mucosa-associated lym-
phoid tissue (MALT), follicular lymphoma (FL), lymphoplasmacytic lymphoma (LPL), and hairy cell leukemia (HCL)
Lymphoma CD20 CD23 CD10 CD5 BCL6 MUM1 CD43 CyclinD1 AnnexinA1 BCL2
B SLL/CLL + (weak) + + +/ + /+ +
MCL + /+ + + + +
MZL (nodal) + /+ + +/ +
MZL (MALT) + +/ +/ +
MZL (splenic) + +/ +
FL + /+ + + a +
LPL + /+ /+ +b /+ +
HCL + /+ /+ NT NT + + +
Refs. [224227, 231239]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
a
Maybe positive in grade 3
b
More intense in plasmacytoid cells
Table 26.30 Immunoprole of some aggressive mature B-cell lymphomas: diffuse large B-cell lymphoma (DLBL),
T-cell/histiocyte-rich B-cell lymphoma (TC/HRBCL), and anaplastic large cell lymphoma kinase (ALK)
Lymphoma CD20 CD10 MUM1 Bcl-2 Bcl-6 CD30 Ki-67 EMA CD45 CD138
DLBCL (NOS) (Fig. 26.41) + +a b +/ +a /a <90 % +
TC/HRBCL + /+ /+ +/ + <90 % + +
DLBCL plasmablastic a + +/ >90 % + a +
DLBCL-ALK+ (Fig. 26.42) +/ <90 % + + weak +
Burkitt lymphoma + + a + >95 % +
Refs. [224227, 236, 239242]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
a
Some cells may be weakly positive
b
Positive in non germinal centers (3565%)
Table 26.31 Immunoprole of some mature T-cell/NK-cell lymphomas: mycosis fungoides (MF), adult T-cell lym-
phoma/leukemia (ATLL), angioimmunoblastic T-cell lymphoma (AILT), anaplastic large cell lymphoma (ATCL), and
T-cell lymphoma (TCL)
Lymphoma CD3 CD5 CD4 CD8 CD30 ALK TIA1 CD56
MF + + + +b +b
ATLL + + +a a +/
AILT + + + +b
ALCL /+ + + + + (6080 %) +/c
Subcutaneous + + +
panniculitis-like TCL
Cutaneous TCL + /+ + +
Hepatosplenic TCL + /+ /+ + +
Nasal or nasal-type NK/TCL + +/ + +
(Cytoplasmic)
Enteropathy-type TCL + + +/ + +d
Refs. [224227, 243245]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
a
Most cases
b
Some large cells
c
More often ALK-positive cases
d
Subset with monomorphic small cell morphology
Table 26.32 Immunophenotypic features of classic Hodgkin lymphoma (CHL) and nodular lymphocyte predominant
Hodgkin lymphoma (NLPHL) (Fig. 26.43)
Lymphoma CD20 Pax-5 CD15 CD30 Facsin EMA ALK-1
CHL +/ +(weak) + + + /+
NLPHL + + /+ +/
Refs. [224227]
Note: + (>90 %), +/ (>50 %), /+ (<50 %), (<10 %)
a b
Fig. 26.40 Lymphoma with starry sky feature declares a highly proliferative phase (a) in which antibodies to terminal
deoxynucleotidyl transfer (TdT) marks it as a precursor lymphoid neoplasm
a b
Fig. 26.41 Diffuse large B-cell lymphoma (NOS) (a) weakly reacts with Bcl-6 and (b) indicates a high proliferative
index by Ki-67 (c)
myobroblasts and myoepithelial cells and lim- chondrocytic tumors. S100 is expressed by a
its the usefulness of diagnostic pathology [45]. A wide range of cell types including glial cells,
relatively smooth muscle-specic marker being neurons, Schwann cells, melanocytes, chondro-
expressed in cytoplasm, caldesmon is a use- cytes, lipocytes, myoepithelial cells, sustentacu-
ful Ab in distinguishing smooth muscle tumors lar cells, Langerhans histiocytes, interdigitating
from myobroblastic tumors [263]. A novel Ag reticulum cells, and various epithelia [26]. CD56
of smooth muscle differentiation, transgelin is a (neural cell adhesion molecule) and CD57
calponin-related protein found in smooth muscle (myelin-associated glycoprotein) are expressed
showing higher sensitivity and specicity than by a variety of different cell types including tis-
other markers [264]. sues of the peripheral nervous system (PNS) and
CNS, as well as natural killer (NK) cells and neu-
roendocrine cells [265267].
26.9.3 Nerve and Schwann Cell
Markers
26.9.4 Endothelial Markers
First isolated from the central nervous system
(CNS), S100 protein is known as a marker of Von Willebrand factor (vWF) is exclusively
nerve sheath tumors as well as melanocytic and expressed by endothelial cells and is principally
a b
Fig. 26.42 Diffuse large B-cell lymphoma (ALK). Large anaplastic cells intermixed with lymphoplasma cells (a) are
strongly positive for ALK (b) and EMA (c)
used to distinguish vascular neoplasms from tumors as well as ES/PNET and lymphoblastic
their morphologic mimickers. Due to low sensi- lymphoma [57].
tivity of vWF in detecting high-grade vascular
neoplasms, other endothelial markers such as
CD31, CD34, and FLI-1 have limited the rou- 26.9.5 Fibrohistiocytic Markers
tine use of vWF in the context of vascular
tumors. Given similar sensitivity to CD34, There are some nonspecic markers such as
CD31 is expressed by macrophages, being a alpha 1-antitrypsin, muramidase (lysozyme),
more specic vascular marker than CD34. alpha 1-antichymotrypsin, cathepsin B, CD68,
CD34 is expressed by bone marrow hematopoi- CD163, factor XIIIa, and the HAM 56 Ag
etic precursor cells and dendritic interstitial which are expressed in melanomas, carcinomas
cells limiting its application in vascular tumors as well as some sarcomas like MFH [272278].
[268271]. As a nuclear transcription factor, Therefore, application of these markers is limited
FLI-1 (Freund leukemia integration site) is an and should be considered after ruling out other
endothelial marker expressed in vascular sarcomas with specic line differentiation.
a b
c d
Fig. 26.43 Hodgkin lymphoma. Typical Reed-Stenberg cell with mirror binuclear feature of Owls eye (a) weakly
reacts with CD 15 (b) and CD30 (c) and strongly reacts with fascin (d)
Table 26.33 Immunohistochemical antibodies of normal mesenchymal tissues and related tumors
Soft tissue Markers of soft tissue Related tumor Immunoreactive markers
Chondrocyte S100, SOX9, vimentin Chondrosarcoma S100, vimentin, CD57, SOX9:
sensitive marker for
cartilaginous differentiation
Endothelial cells Vimentin, CD31, CD34, FLI-1 Angiosarcoma CD31, CD34, FLI-1
D2-40 (lymphatic endothelium) Lymphangiosarcoma D2-40
Fibroblasts Vimentin, CD10, CD99 Fibrosarcoma Vimentin
Fibrohistiocyte CD68, CD168, a1AT, cathepsin Malignant brous CD68
B, factor IIIA, HAM 56 histiocytoma
Lipocytes Vimentin, S100 (variable), Liposarcoma S100, MDM2, CDK4
calretinin, MDM2, CDK4, CD-34
Osteoblast CD56, osteocalcin, osteonectin, Osteosarcoma Osteocalcin, collagen IV, CK,
vimentin EMA, CD99, S100, desmin,
SMA, factor 13
Nerve/Schwann cell Vimentin, S100, CD56, CD57 MPNST S100
Skeletal muscle Desmin, myoglobin, CD56, GFAP Rhabdomyosarcoma Myogenin, myo-D1, PLAP,
WT1
Smooth muscle Desmin, NSE, SMA, MSA Leiomyosarcoma Desmin, SMA, MSA,
h-caldesmon, collagen IV
Synovial cell CD68, clusterin Synovial sarcoma CK, EMA, vimentin, CD68,
CD99, E-cadherin, collagen IV
a b
Fig. 26.44 Leiomyosarcoma. Spindle cells arranged in interlacing cross-striated fascicles (a) are immunostained with
desmin (b) and h-caldesmon (c)
26.9.6 Lipocytic Markers mesenchymal chondrosarcoma from other small

blue round cell tumors [281].
MDM2 (an inhibitor of p53 transcriptional acti-
vation) and CDK4 (a protein involved with cell
cycle progression) are markers to separate dedif- 26.9.8 Osteogenic Markers
ferentiated liposarcomas from other poorly dif-
ferentiated sarcomas [279]. Osteocalcin (a non-collagenous proteins) with
approximately 70 % sensitivity is a completely
specic marker for bone-forming tumors. In
26.9.7 Chondrocyte Markers addition, osteonectin (a bone matrix glycoprotein
participates in stromal mineralization) also has a
Chondrocytes do not display specic mark- sensitivity of 90 % and a specicity of 54 % in
ers and show reactivity with S100 and vimen- the diagnosis of osteoblastic neoplasms [282,
tin. Chondrosarcoma also exhibits reactivity 283]. These markers are rarely being used in rou-
with CD57 [280]. Being as a master regulator tine diagnosis because the diagnosis of osteosar-
of chondrogenesis, SOX9 is a sensitive marker coma is based on the presence of osteoid in the
for cartilaginous differentiation distinguishing H&E-stained slides.
a b
Fig. 26.45 Alveolar rhabdomyosarcoma. Large polygonal cells with alveolar pattern (a) are highlighted with myo-
genin (b) and desmin (c)
26.9.9 Unknown-Origin Soft Tissue by the coexpression of epithelial and mesenchymal

Tumors markers [288]. The immunohistochemistry char-
acteristics of these tumors are summarized in
Ewing sarcoma/peripheral nerve sheath tumor Table 26.34.
(ES/PNET) comprises a prototype of small round
cell neoplasms of bone and soft tissue exhibiting
neuroectodermal features. As a product of the 26.10 Immunohistochemistry
MIC2 gene, CD99 is a cell surface transmem- of the Nervous System
brane glycoprotein diffusely present in nearly all
tumors (Fig. 26.46) [284]. Clear cell sarcoma The brain tumors are classied into two major
(malignant soft part melanoma) shares markers groups: primary and metastatic. Primary brain
of malignant melanoma such as S100, MART-1, tumors are further categorized into three major
HMB45, and tyrosinase [285]. Alveolar soft part subtypes: neuroepithelial tumors (astrocytoma,
sarcoma has been evaluated by presence of myo- oligodendroglioma, ependymoma, choroid plexus
D1 and myogenin [286, 287]. Desmoplastic tumors, neuronal tumors, and pineal tumors),
small round cell tumor (DSRCT) is characterized non-neuroepithelial tumors (meningioma, nerve
a b
Fig. 26.46 Small round cell tumor (a). Immunoreaction with MIC2 (b) and NSE (c) antibodies supports the diagnosis
of PNET
Table 26.34 Immunoprole of unknown-origin soft tis- ependymoblastoma, and PNET) [289295].
sue tumors: Ewing sarcoma/peripheral neuroectodermal Primary origin of metastatic carcinoma is deter-
tumor (ES/PNET), clear cell sarcoma (CCS), alveolar soft
part sarcoma (ASPS), and desmoplastic small round cell mined by the use of immunohistochemical panel.
tumor (DSRCT) Commonly used IHC Abs in primary CNS tumors
Panel antibodies ES/PNET CCS ASPS DSRCT are demonstrated in Table 26.35.
CD99/FLI-1 +
S100/HMB45/ +
MITF/Melan-A 26.10.1 Neuroepithelial Tumors
TFE3 +
NSE + + Glial tumors (astrocytoma, oligodendroglioma,
Desmin + and ependymoma) usually react with glial bril-
CK/EMA + lary acidic protein (GFAP) [151, 152, 296].
WT1 + Oligodendroglioma variably expresses GFAP
Refs. [284288] and commonly reacts with Leu7 and S100 [297,
298]. Moreover, GFAP is present in other mixed
sheath tumors, lymphoma, chordoma, and glial and neuronal-glial tumors including oligoas-
germ cell tumors), and primitive undifferenti- trocytoma and ganglioglioma (Fig. 26.47) [296].
ated tumors (medulloblastoma, pineoblastoma, Neurocytoma and pineal tumors are GFAP negative
Table 26.35 Commonly used antibodies in primary CNS tumors

Antibody Normal brain Tumor
EMA Epithelial, perineural, Meningioma, chordoma, medulloblastoma
meningothelial cells
GFAP Glial cells Glial tumors except oligodendroglioma,
medulloepithelioma, choroid plexus tumor, ganglioglioma
Leu7 (CD57) Oligodendroglial cells, Oligodendroglioma, schwannoma, neurobroma,
Schwann cells, oligoastrocytoma
Neurolament Neuropil Ganglion cell tumors, neurocytoma, pineocytoma,
neurobroma, medulloblastoma, PNET
NSE Neuroectodermal and Neuroblastoma, hemangioblastoma, PNET,
neuroendocrine cells oligodendroglioma
S100 Glial cells, Schwann cells, dendritic Gliomas, meningioma, schwannoma, neurobroma,
and Langerhans cells, melanocytes, chordoma, craniopharyngioma, PNET,
other mesenchymal cells medulloblastoma, pineoblastoma, neuroblastoma,
melanoma, chondroid tumors
Synaptophysin Neuroendocrine cells, neuropil Neurocytoma, ganglion cell tumors, pineocytoma, choroid
plexus papilloma, medulloblastoma, pineoblastoma,
neuroblastoma, PNET, oligodendroglioma,
dysembryoblastic neuroepithelial tumor
Vimentin Meningoendothelial cells, other Meningioma, gliomas, chordoma, ependymoblastoma,
mesenchymal cells hemangiopericytoma, ganglioglioma, embryonal tumors
Collagen IV Ganglion cell, Schwann cell, other Ganglion cell tumor, schwannoma, medulloblastoma/
mesenchymal cells pineoblastoma
Refs. [151, 152, 296]
a b
Fig. 26.47 Fibrillary astrocytoma with proliferation of atypical astrocytes (a) exhibits GFAP-positive cytoplasmic
processes (b)
and synaptophysin positive. Among neuroepithelial 26.10.2 Non-neuroepithelial Tumors

tumors, choroid plexus tumors demonstrate reac-
tivity with epithelial markers such as cytokeratin, Among non-neuroepithelial tumors, meningiomas
CAM5.2, and EMA. Additionally, transthyretin, as are positive for EMA which differentiates them
a potential marker, and IGF-II, as a newer marker, from nerve sheath tumors and are negative for GFAP
are positive in choroid plexus tumors [299301]. which distinguishes meningioma from gliomas.
Pineal tumors are GFAP and epithelial-negative Schwannoma is distinct from glioma, meningioma,
tumors which exhibit reactivity with synaptophy- and neurobroma by showing reaction to collagen
sin and neurolament (Table 26.36). type IV. Neurobroma differs from schwannoma
Table 26.36 Immunopanel of neuroepithelial tumors

First-choice antibody panel Second-choice antibody panel Consistent with
GFAP+, EMA, CAM5.2 Vim+, NF+, S100+ Astrocytoma (Fig. 26.47)
Leu7+, NSE+, S100+ Oligodendroglioma
GFAP+, EMA (R), CAM5.2 (R) Vim+, S100+ Ependymoma
GFAP (S), EMA+, CAM5.2+ Laminin+, SPN+, S100+, IGF-II+ Choroid plexus papilloma
GFAP, EMA, CAM5.2 SPN+, NF+ Central neurocytoma
SPN (S), NF (S), Collagen IV+ Ganglion cell tumor
NSE+, SPN+, NF (R) Pineal tumor
Refs. [151, 152, 296298, 302310]
Note: N negative, R rare, S sometimes
a b
Fig. 26.48 Germinoma. (a) A tumor with relatively right corner). Tumor cells react with PLAP (b) and reac-
medium to large polygonal cells resembling an undiffer- tive astrocytes stain by GFAP (c) (Courtesy of Dr. Taghi
entiated tumor surrounded by reactive astrocytes (upper Ghiasi-Moghadam, Mashad, Iran)
by having neurolament-positive axons. Primary Chordoma exhibits reactivity for CK and EMA
and secondary brain lymphomas express LCA as a as well as S100, whereas chondrosarcomas lack
common marker and CD3 and CD20 as differentiat- these features (CK/EMA negative and S100 posi-
ing markers of T-cell- and B-cell-type lymphomas, tive). Primary germ cell tumors are found along the
respectively. Arising from notochord remnants, midline in the pineal and suprasellar regions which
chordomas are malignant tumors along the axial demonstrate immunostaining with placental alka-
skeleton recognized by characteristic physalipho- line phosphatase (PLAP), alpha-fetoprotein (AFP),
rous cells with large intracytoplasmic vacuoles. beta-HCG, and CEA (Fig. 26.48) (Table 26.37).
Table 26.37 Immunopanel of non-neuroepithelial tumors

First-choice antibody panel Second-choice antibody panel Consistent with
Vimentin+, S100+ EMA+ Chordoma
Vimentin+, S100 (R) EMA (S) Meningioma
Vimentin, S100+ Leu7+, collagen IV+, GFAP (R) Schwannoma
Leu7+, NF+, EMA+ Neurobroma
Vimentin, S100 LCA+, L26+ Lymphoma
PLAP+, HCG+, AFP+ Germ cell tumor (Fig. 26.48)
Refs. [151, 152, 296298, 311316]
Table 26.38 Immunopanel of primitive undifferentiated tumors

First-choice antibody Second-choice
panel antibody panel Anatomic site Consistent with
SYNP+, S100+ NF (R), GFAP (R), Posterior fossa Medulloblastoma
Collagen IV+, Vim (S), CD99 Pineal gland Pineoblastoma
NF (R), GFAP (R), Anterior fossa PNET
Collagen IV, Vim, CD99 (S)
SYNP, S100+ NF, GFAP (R), Cerebrum, cerebellum Ependymoblastoma
Collagen IV, Vim (S), CD99
Refs. [151, 152, 296298, 309, 317322]
26.10.3 Undifferentiated Tumors guishes long and short time survivals in patients
with glial tumors (Table 26.39 and Fig. 26.49).
Medulloblastoma, pineoblastoma, ependymo- p53 and EGFR overexpression can be dened
blastoma, and PNET are primitive undifferenti- immunohistochemically. Overexpression of p53
ated tumors commonly located in the posterior is associated with tumor progression in glioblas-
fossa, pineal gland, periventricular area, and toma multiforme (GBM). EGFR overexpression
anterior fossa, respectively. Medulloblastoma, correlates with poor prognosis in gliomas and is
pineoblastoma, and ependymoblastoma differen- not present in low-grade gliomas. As a new thera-
tiate from PNET by negative reaction for CD99. peutic target, EGFR tyrosine kinase inhibitors are
Ependymoblastoma can be distinguished from used for the treatment of GBM.
meduloblastoma/pineoblastoma/PNET by the
absence of reactivity to synaptophysin and neuro-
lament (Table 26.38). 26.11 Immunohistochemistry
of Pediatric Tumors
26.10.4 Proliferative Markers Solid pediatric tumors comprise a heterogenic

group of variable entities with morphologies
MIB1 (Ki-67) is an Ab that detects proliferat- including small round cells, spindle cells, and
ing cells in various phases of the cell cycle and polygonal cells. Small round cell tumors include
is important in the grading of CNS tumors. It neuroblastoma, rhabdomyosarcoma, Ewing sar-
is used to predict patient outcome and distin- coma/PNET, desmoplastic small round cell tumor,
Table 26.39 Proliferative factor of MIB1 in some CNS tumors and correlation with survival (Fig. 26.49)
Tumor MIB1 % Survival
Astrocytoma <2 80 %
>2 20 %
Anaplastic astrocytomas 510
Glioblastoma multiforme >10
Oligodendroglioma <5 Longer survival
>5 Shorter survival
Ependymal tumor >5 Shorter survival
Choroid plexus papilloma 3.7 <6 % nonaggressive
Choroid plexus carcinoma 14 >6 % aggressive
Meningioma Ozen study Abramovich study Lanzafame study
Benign (grade 1) 1.2 1 <1 % no recurrence
Anaplastic (grade 2) 2.3 5.5 >1 % recurrence
Malignant (grade 3) 6.7 12
Medulloblastoma 50 %
Refs. [151, 323330]
a b
Fig. 26.49 Proliferating marker of Ki-67 is nonreactive in normal brain (a), 30 % reactive in astrocytoma (b), and
80 % reactive in germinoma (c)
a b
c d
Fig. 26.50 Wilms tumor. Epithelial component with tubuloglandular structures (a) showing immunoreaction with
CKAE1/AE3 (b), EMA (c), and WT1 (d)
Wilms tumor (Fig. 26.50), small cell osteosarcoma, ric soft tissue sarcoma subclassied into embryo-
lymphoma, and melanoma. Rhabdomyosarcoma, nal, botryoid, alveolar, and spindle cell subtypes.
Wilms tumor, and melanoma also display spindle Embryonal rhabdomyosarcoma (including botry-
cell components or present as pure spindle cell oid), the most common type in childhood, usu-
tumor. Polygonal cell tumors of childhood com- ally displays small cell morphology, whereas
prise of rhabdomyosarcoma, malignant rhabdoid the alveolar variant usually exhibits features of
tumor, osteosarcoma, and melanoma [331, 332]. polygonal cells [337340].
Frequently confused with primitive neuroec- Initially regarded as an undifferentiated sar-
todermal tumors (PNETs), neuroblastoma is the coma of the bone and soft tissue, Ewing
most common malignant tumor of the posterior sarcoma/primitive neuroectodermal tumor (ES/
mediastinum in pediatric patients with morphol- PNET) is now being classied as a small round
ogy of small round cell tumor. Neuroblastoma cell tumor with varying degrees of neuroecto-
has a predilection for adrenal glands and sym- dermal differentiation with pseudorosette for-
pathetic ganglia, whereas PNETs are choliner- mation [341]. Desmoplastic small round cell
gic tumors [333, 334]. Expression of CD44s and tumor is an aggressive, malignant tumor usually
c-kit receptor correlates with favorable prog- involving the abdominal or pelvic cavity of chil-
nosis in a subset of neuroblastoma [335, 336]. dren or young adults with the morphology of
Rhabdomyosarcoma is the most common pediat- small round cells arranged in nests and sepa-
Table 26.40 Immunopanel of pediatric tumors

First-choice Second-choice antibody Additional antibody/histopathologic
antibody panel panel feature Consistent with
AE1/AE3+, DES+, WT1+, EMA+ SYN+, CHG+, NSE+/ Wilms tumor
CAM5.2+, VIM+ Small round cell
SYN+, CHG+, NSE+/ Malignant rhabdoid
Polygonal cell tumor
SYN, CHG, NSE+/ Desmoplastic small
Small round cell round cell tumor
AE1/AE3, DES+, MYOG+, MyoD1+ MSA+, CD99, CK/ Rhabdomyosarcoma
CAM5.2, VIM+ Small round/spindle/polygonal cell
DES, MYOG, MyoD1 CD45+/ Lymphoma
Small round cell
CD99+, S100+/ Osteosarcoma
Small round/polygonal cell + osteoid
CD99+/ ES/PNET
Small round cell
S100+, SYN+, CHG+, NSE+/ Neuroblastoma
Small round cell
S100+, HMB45+, MART1+/ Melanoma
Small round/polygonal cell
Refs. [55, 348362]
rated by a dense collagenized and desmoplastic with abundant eosinophilic cytoplasm and large
stroma [288]. eccentric nuclei containing prominent eosino-
Wilms tumor (WT) or nephroblastoma is the philic nucleoli [346, 347]. Table 26.40 displays
most common pediatric neoplasm of the kidney an immunopanel to the diagnosis of common
derived from nephrogenic rests displaying diver- pediatric tumors.
gent differentiation. The classic histopathologic
pattern of WT consists of triphasic elements of
blastemal, epithelial, and stromal components. 26.12 Immunosurveillance,
Blastemal component is composed of small Immune Editing, Immune
round cells exhibiting reactivity with vimentin Constant of Rejection,
and desmin. Epithelial component shows stain- Immune Contexture, and
ing with cytokeratin, whereas stromal compo- Immune Scoring of Cancers
nent demonstrates variable reactivity based on
its differentiation pattern [342, 343]. Lacking a Cancer is a complex disease involving cellular
characteristic immunohistochemical prole, the and molecular interactions between the tumor
diagnostic feature of osteosarcoma is the pres- and the immune system [363]. The concept of
ence of osteoid which can be distinguished from immunosurveillance, rst described by Lewis
other undifferentiated small round cell tumors Thomas and Macfarlane Burnet, refers to the
[344, 345]. Originally described in the kidney detection and destruction of tumor cells by the
and CNS, malignant rhabdoid tumor is a highly immune system [363365]. This theory has been
aggressive neoplasm of the childhood with a supported by the analysis of experimental and
tendency of widespread metastases. Malignant clinical tumor microenvironment data. The stron-
rhabdoid tumor is a densely cellular tumor com- gest argument for the existence of immuno-
prised of cords and sheets of polygonal cells surveillance is that immunodecient hosts are
Tumor advancement
Immune Tumor Tumor Tumor Genetic

strength elimination equilibrium escape instability
Immune coordination
Fig. 26.51 Cancer-immune spectrum. The immunoediting theory describes how a tumor can evade from immune
destruction and how the immune system restrains the tumor
associated with increased frequency of cancers. neutrophils, mast cells, NK cells, and immature
In addition, regression of primary and metastatic dendritic cells (DC)] and the adaptive immune
tumors has been attributed to immunologic cells [mature DC, B lymphocytes, T lymphocyte,
mechanisms, but many other factors may have and regulatory T cells (Tregs)]. Initially mediated
been responsible (e.g., hormonal, nutritional, or by innate immunity, interaction between tumor
vascular). Tumor microenvironment is a complex cells and immune system develops and the tumor
milieu comprised of extracellular matrix and host is eliminated through adaptive immune system
cells, including mesenchymal, endothelial, and activation [373, 374]. The immune-mediated tis-
immune cells. During carcinogenesis process, the sue destruction process described by the concept
neoplastic cells constantly interact with the host of immunologic constant of rejection (ICR)
cells, extracellular matrix, and bioactive mole- includes the coordination of interferon-stimulated
cules, which constitute the tumor microenviron- genes (ISGs) pathway and immune effector func-
ment [366368]. tions (IEFs) pathway. This constant demonstrates
The concept of cancer immunoediting, pro- the activation of ISGs, recruitment of cytotoxic
posed by a series of mouse model publications immune cells (primarily through CXCR3/CCR5
that immune deciencies are associated with ligand pathways), and activation of the IEFs
tumor aggressiveness, describes how the immune pathway (IEF genes; granzymes A/B, perforin)
system encounters with tumor cells during [375, 376].
tumorigenesis [369372]. Immune cells engage The immune contexture is characterized as the
to combat with cancer cells in three sequential density, type, location, and functional orientation
phases: cancer elimination, cancer equilibrium, of adaptive immune cells within the tumor which
and cancer escape. In the elimination phase, the is essential to accurately dene the impact of can-
immune system clears most tumor cells; a popu- cer prognosis [377379]. Parameters of the
lation of immune-resistant tumor cells appears in immune contexture comprise of CD3+ density,
the equilibrium phase; and nally, in the escape cytotoxic CD8+ and memory CD45RO+ T cells,
phase, the tumor develops strategies to evade their location at the tumor center (CT) and inva-
immune destruction. The last phase is a conse- sive margin (IM), combined with the quality of
quence of immune exhaustion and inhibition or tertiary lymphoid structures (TLS) (Fig. 26.52).
results from the emergence of tumor cell variants Evaluation of immune contexture in the clinical
(Fig. 26.51). setting will provide prognostic and predictive
It is now well known that innate and adaptive benets [377, 378].
immune systems can promote tumor develop- In human, the presence of tumor inltrating
ment and progression through immunosurveil- lymphocytes (TILs) has been reported as a favor-
lance. However, there are many interactions able prognostic factor in many primary tumors.
between the innate immune cells [macrophages, The high density of TILs associated with good
Immune contexture
Immunoscore Immunologic
constant
of rejection
- Type - Th1
- Density Functional orientation - Cytotoxicity
- Location - Chemokine
- Adhesion
Fig. 26.52 The immune contexture at the background bution of immune cells within the tumor. The
is dened by combination of immune variables associat- Immunoscore and the immunologic constant of rejec-
ing the nature, density, functional orientation, and distri- tion are overlapped by functional orientation
prognosis has been well documented, not only to [384]. The purpose of the Immunoscore interna-
various organs of cancer origin (such as breast, tional task force was: (1) to validate the feasibil-
colon, lung, head and neck, kidney, bladder, ovary, ity and reproducibility of the Immunoscore and
prostate) but also to various cancer cell types (ade- (2) to validate the major prognostic and predic-
nocarcinoma, squamous cell carcinoma, large cell tive power of the Immunoscore in colon cancer
cancer, melanoma, etc.) [reviewed in 379381] patients. In order to become globally applica-
(Fig. 26.53). The quantication of TILs allowed ble in routine clinical setting, evaluation of the
dening a novel scoring system based on the den- Immunoscore must be pathology based, feasible
sities of two lymphocyte populations (CD3+ and in routine settings, simple, inexpensive, rapid,
CD8+), both in CT and in IM of tumors. Based on robust, reproducible, quantitative, standardized,
the immune contexture, a standardized, simple, and powerful [29, 384].
powerful immune scoring system (Immunoscore) Multiple laboratory variables inuence the
was determinate. Immune classication of can- validity and reliability of immunoscoring in the
cers provides a scoring system ranging from clinical setting which need to coordinate with
Immunoscore 0 to 4 and low to high densities of distinct criteria. They are included in the com-
both lymphocyte populations in CT and IM of plexity of quantitative IHC assay, variable proto-
tumors (Table 26.41). The Immunoscore system cols across laboratories, and immune cell
has shown to have a prognostic signicance supe- analysis accompanied by uneven region selec-
rior to AJCC/UICC-TNM staging systems. Thus, tion criteria and variable ways to quantify TILs.
incorporating the Immunoscore into traditional An effort for harmonization and reproducibility
staging systems has an essential prognostic and of IHC method recommends laboratories to test
predictive value [382, 383]. the prognostic value of Immunoscore using
In 2012, an international task force was ini- the initial guidelines [383, 384]. It is also
tiated to promote the Immunoscore in routine acknowledged that additional markers may be
clinical settings as a new component of cancer used to further rene the prognostic value of the
classication, designated TNM-I (TNM-Immune) Immunoscore.
a b
Fig. 26.53 (a) Colon adenocarcinoma and (b) skin SCC with surrounding TILs, immunostained with CD45RO, CD3, and CD8
Table 26.41 The characteristics of immune contexture, immunoscore, and immunologic constant of rejection
Concepts Characteristic
Immune contexture Type, density, location, and functional orientation of adaptive immune cells (Th1 cell,
cytotoxicity, chemokine, adhesion)
Immunoscore Standardized, simple, quantitative, routine test derived from the immune contexture
Type CD3+ T cell, CD8+ T cell
Density Quantication (cells/mm2)
Location Tumor center, invasive margin, tertiary lymphoid islets
Immunologic Immune-mediated, tissue destruction processes
constant of rejection (A) Interferon-stimulated genes pathway
(B) Cytotoxic immune cells (primarily through CXCR3/CCR5 ligand pathways)
(C) Immune effector functions pathway (IEF genes; granzymes A/B, perforin)
skin tumors: assessment of the specicity and sensi-

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Pathol. 2001;54(3):196200.
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should be used in an appropriate setting by an expression of langerin in Langerhans cell histiocyto-
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Index
238 G>A (rs361525), 326, 329 Activated B-cell (ABC)-like subtypes, 219
238 G>A (rs361525) polymorphism, 326 Activation-induced cell death (AICD), 66, 69
+252G allele (rs909253), 326 Activator protein-2 (AP-2), 325
+277 G>A (rs568408), 323 Active cancer immunotherapy, 471
308G>A (rs1800629), 325, 326, 329 Active immunotherapy, 473
308GA/GG genotypes, 326 Acute ER stress, 228
308G>A (rs1800629) polymorphism, 326, 329 Acute inammation, 385
376 G>A (rs1800750), 326 Acute lymphoblastic leukemia (ALL), 229, 260,
509 C>T (rs1800469), 333 262, 289, 361
592 A>C (rs1800872), 323 Acute lymphocytic leukemia, 410
857 C>T (rs1799724), 326, 329 Acute myeloid leukemia (AML), 36, 109, 226,
863 C>A (rs1800630), 326 291, 351353
+874 T>A (rs2430561), 330 Acute myeloid leukemia (AML) cells, 109
1,031 C>T (rs1799964) polymorphisms, 326 Acute neutropenia, 443
1082 A>G (rs1800896), 321, 323 Acute promyelocytic leukemia (APL), 251
+1188 A>C (rs3212227), 323 Acute respiratory distress syndrome (ARDS), 155
Acute respiratory distress syndrome bronchoalveolar
lavage (ARDS BALs), 155
A Acute T-cell leukemia, 360
A1/B-1, 217 Acylation, 387
A2a receptor, 179 Adaptive immune cells, 280, 386
A7 clone, 165 Adaptive immune mechanisms, 186
A*0201, 307 Adaptive immune reaction, 411
A549 lung cancer cell line, 291 Adaptive immune responses, 32, 123, 189, 198,
A*1101, 307 381385, 387, 411, 435
AA genotype, 319, 326, 329, 330 Adaptive immune system, 177, 278, 279, 378,
A allele, 323, 326, 329 379, 385, 386
ABC-DLBCL. See Activated B-cell-diffuse large Adaptive immunity, 177, 186, 306
B-cell lymphomas (ABC-DLBCL) Adaptive mechanism, 386
Ab-dependent cell-mediated cytotoxicity (ADCC), 199 Adaptive response, 385
Abscess, 351 Adaptor proteins, 144
Absolute neutrophil counts (ANCs), 351 ADCC. See Ab-dependent cell-mediated
ABT-263, 226 cytotoxicity; Antibody-dependent
ABT-737, 226 cellular cytotoxicity (ADCC)
AC genotypes, 329 ADCP. See Antibody-dependent cellular
-chemokine, 318 phagocytosis (ADCP)
Acidity, 203 Addisons disease, 356
Acid sphingomyelinase (ASM), 151 Adenine nucleotide translocase (ANT), 259
Acinic cell carcinoma, 502, 503 Adenocarcinoma, 15, 167, 247, 329, 410, 509,
Acinic cell tumor, 503 511, 521, 543
Acoustic neuroma, 360, 413, 414 Adenoid cystic carcinoma, 502, 503
Acquired immune deciency syndrome (AIDS), Adenomas, 79, 291
198, 300 Adenosine, 179
Actin, 526 Adenosine 5-(, -methylene) diphosphate (APCP), 52
Activated B-cell-diffuse large B-cell lymphomas Adenosine triphosphate (ATP), 33, 244
(ABC-DLBCL), 219 Adhesion molecules, 412

DOI 10.1007/978-3-662-44006-3, Springer-Verlag Berlin Heidelberg 2015
562 Index
Adipose tumors, 496 All-trans-retinoic acid (ATRA)-treated

ADP. See Anti-adipophilin (ADP) neuroblastoma, 287
Adrenal leiomyoma, 348 Alopecia, 356
Adrenocortical tumors, 217 ALP. See Alkaline phosphatase (ALP)
Adult T-cell leukemia, 80 47, 82
Advanced bone, 249 Alpha chain, 305
Advanced hepatocellular carcinoma, 220 Alpha-fetoprotein (AFP), 3, 537
Advanced NSCLC, 249, 251 Alpha-galactosylceramide (alphaGalCer), 50
Advanced pancreatic neuroendocrine tumors, 249 ALPS. See Autoimmune lymphoproliferative
Advanced RCC, 249 syndrome (ALPS)
Advanced solid malignancies, 250 Alveolar soft part sarcoma, 534
AEG35156, 230 Alzheimers disease, 143, 248, 383
AEG40730, 230 Ambra1. See Autophagy/beclin-1 regulator 1 (Ambra1)
Aerobic respiration, 386 AML. See Acute myeloid leukemia (AML)
Afferent lymphatics, 177 AML1/ETO fusion, 291
Afnity chromatography, 452 Anabolism, 244
AFP. See Alpha-fetoprotein (AFP) Anaerobic glycolysis, 203
Age-associated diseases, 385, 386 Anal SCC, 511
Age-related chronic inammatory processes, 387 Anaphylaxis, 409
Age-related proinammatory milieu, 385 Anaplastic astrocytoma, 251
Aggressive melanoma, 249 Anaplastic B-cell lymphoma, 348
Aging, 377, 380, 382, 384, 387 Ancestral haplotype 8.1, 312
Aging/geriatric environment, 388 ANCs. See Absolute neutrophil counts (ANCs)
Ag presentation, 202, 305 Anergic molecules, 202
Ag-specic DNA vaccine, 439 Anergic T cells, 63
AHR. See Aryl hydrocarbon receptor (AHR) Anergy, 63, 64
AICD. See Activation-induced cell death (AICD) Ang-2. See Angiopoietin-2 (Ang-2)
AIDS. See Acquired immune deciency Ang-2/Tie2 axis, 13
syndrome (AIDS) Angiogenesis, 10, 11, 14, 15, 19, 30, 32, 39, 80, 94, 97,
AIF. See Apoptosis-inducing factor (AIF) 98, 100, 101, 103105, 109, 123, 126, 130,
AIM2, 379 132, 137, 188, 204, 205, 217, 250, 297, 312,
Airway constriction, 409 318, 323, 324, 330, 331, 386, 387, 414, 415,
Airways, 409 431, 442, 443
AKR-derived B cell lymphomas (H-2k), 163 Angiogenic activity, 107
Akt, 245, 246, 248 Angiogenic dormancy, 200
Akt genes, 248 Angiogenic factors, 97
Akt signaling, 444 Angiogenic proteins, 93
Alarmins, 385 Angiopoietin-2 (Ang-2), 11, 13
Albumin-bound paclitaxel, 227 Angiostatic chemokine, 107
Alkaline phosphatase (ALP), 454, 492 ANN. See Articial neural networks (ANN)
ALL. See Acute lymphoblastic leukemia (ALL) ANT. See Adenine nucleotide translocase (ANT)
Allele, 300, 326 AntagomiRs, 292
Allergenicity, 408 Antagonists, 216
Allergens, 408, 409, 413 Anthropogenic, 424
Allergen-specic IgE, 414 Anti-adipophilin (ADP), 496
Allergic asthma, 409 Anti-adipophilin/adenosine triphosphate
Allergic diseases, 408 (ADP/ATP), 259
Allergic disorders, 346, 414 Antiangiogenic factors, 97, 98
Allergic inammation, 409 Antiangiogenic program, 98
Allergic mediators, 408 Antiangiogenic properties, 101
Allergic reactions, 407409, 412, 414, 415 Anti-apoptotic molecules, 225
Allergic response, 408 Anti-apoptotic proteins, 217
Allergic rhinitis, 409, 413, 414 Antiapoptotic signal transducer, 346
Allergies, 348, 407, 408, 411414, 416 Anti-BDCA-2, 181, 185
Allogeneic bone marrow transplantation, 5 Anti-BDCA-2 Ab, 182
Allogeneic MHC class I genes, 170 Antibodies, 3
Allogenic transplant, 153 Antibody array technology, 466468
Allograft, 2 Antibody-dependent cellular cytotoxicity (ADCC),
Allograft rejection, 423 50, 55, 100, 411, 414, 415, 435
All-trans retinoic acid (ATRA), 80, 251 Antibody-dependent cellular phagocytosis (ADCP), 414
Index 563
Antibody-dye conjugate, 476 Apogossypolone (ApoG2), 226

Antibody microarrays, 465468 Apomab, 220
Antibody response, 423 Apoptose-inducing ligands, 297
Anticancer immune response, 205 Apoptosis, 3, 127, 128, 143145, 148, 152, 154, 179,
Anticancer immunotherapies, 477 202, 210213, 215217, 219, 225, 228230,
Anticancer mechanism, 377 251, 252, 261263, 348, 349, 351, 355357,
Anticancer therapies, 251, 431 378, 386, 387, 410, 415, 421, 441, 444, 460
Anticancer vaccines, 435 regulation, 217
Anti-CD95 mAbs, 146 resistance, 250
Anti-CSF1R-neutralizing antibody (AFS98), 39 Apoptosis-inducing factor (AIF), 215, 216, 259
Anti-CTLA-4, 4, 5 Apoptosis-resistant cancers, 250
Antiestrogen therapy, 248 Apoptotic, 153
Antigen-encoding gene, 440 bodies, 211
Antigenicity, 203 cell death, 212, 217
Antigen-independent immune responses, 411 pathway, 217
Antigen presentation machinery (APM), 162164 signals, 152
Antigen-presenting cells (APCs), 29, 32, 3739, 48, APS-1. See Autoimmune polyendocrine
62, 64, 66, 7982, 95, 100, 128, 129, 177, syndrome type I (APS-1)
185, 189, 199, 202, 312, 381, 382, 385, 408, APT-1, 148
435, 445, 511 ARB. See Average relative binding (ARB)
Antigens, 3, 4 ARDS. See Acute respiratory distress syndrome (ARDS)
Antihistamines, 413 Arginase 1 (Arg-1), 11, 12, 79
Antiinammatory, 316, 321 Argonaute family (Ago1-4 in humans), 286
antibodies, 203 Aromatase inhibitor-resistant metastatic
cytokines, 313, 385 breast cancer, 249
functions, 184 Arsenic trioxide (ATO), 251
Antimalarials, 252 Artemis deciency, 357
Anti-melanoma TIL repertoire, 484 Arthritis, 355
Antimetastatic treatments, 170 Articial neural networks (ANN), 437
Anti-MFG-E8, 34 Aryl hydrocarbon receptor (AHR), 81
Antimicrobial peptides, 12 Ascorbic acid, 251
Antineoplastic reaction, 304 ASM. See Acid sphingomyelinase (ASM)
Antinuclear Abs, 185 Assay harmonization, 477
Antioxidant response, 387 Asthma, 188, 348, 407, 411, 412, 414, 416
Anti-PD1, 5, 188 Astrocytoma, 361, 535
Anti-PD1 monoclonal antibody (mAb), 4 AT-101, 226
Anti-PDL1, 5 ATA haplotype, 321
Anti-Ri, 456 Ataxia diplegia, 347
Antitumor activities, 388, 414 Ataxiatelangiectasia, 357
Antitumor cells, 186 Ataxia telangiectasia mutated/ataxia telangiectasia/
Antitumor cytotoxicity, 415 Rad3-related kinase (ATM/ATR), 36
Antitumor immune response, 205 Atg1-Atg13-Atg17 kinase complex, 244
Antitumor immunity, 179, 203, 205, 411, 415 Atg1/ULK1, 245
AP-1 signaling, 387 Atg4, 244
AP-12009, 105 Atg5, 244, 245, 252
AP23573, 249 Atg5-Atg12, 251
Apaf-1, 212, 215 Atg5-Atg12-Atg16 complex, 245
APCP. See Adenosine 5-(, -methylene) Atg5-Atg12 complex, 244
diphosphate (APCP) Atg8, 244
APCs. See Antigen-presenting cells (APCs) Atg9, 244, 245
APECED. See Autoimmune polyendocrinopathy Atg12, 244, 245
with candidiasis and ectodermal Atg13, 244
dystrophy (APECED) Atg14L, 244
APL. See Acute promyelocytic leukemia (APL) Atg16, 244
Aplastic anemia, 355 Atg16L, 244, 245
APM. See Antigen presentation machinery (APM) AT genotype, 330
Apo2L/TRAIL, 263, 264 Atherosclerosis, 383
Apocrine glands, 493 ATO. See Arsenic trioxide (ATO)
Apocrine tumors, 495 Atopic dermatitis, 409, 412, 413, 416
ApoG2. See Apogossypolone (ApoG2) Atopy, 408, 409
564 Index
ATP. See Adenosine triphosphate (ATP) B

Atypical broxanthoma, 498 B2M, 423
Autoantibodies, 356, 384 B2 microglobulin, 305
Autocrine activation, 202 2-microglobulin (2m), 4, 161, 162, 164
Autocrine loops, 432, 434 B7-1 molecule, 37
Autocrine manner, 183, 324 B7-2 molecule, 37
Autouorescence, 475 B7 clones, 165
Autoimmune, 77, 356 B7 family, 37
Autoimmune diseases, 63, 80, 185, 186, 307, 344, B7 brosarcom, 170
345, 356, 361 B7-H1, 18
Autoimmune disorders, 154, 347 B7-H1 (PD-L1), 37
Autoimmune hemolytic anemia, 347, 348 B7-H3, 37
Autoimmune lymphoproliferative syndrome (ALPS), B7-H4, 37
148150, 154, 347, 355, 356 B7 molecule, 64
Autoimmune lymphoproliferative syndrome B8, 346
(ALPS)-like disorders, 355 B11, B7, C5 clones, 164
Autoimmune lymphoproliferative syndrome B16 melanoma, 107, 163, 167
(ALPS)-related conditions, 355 Bacillus Calmette-Gurin (BCG), 32
Autoimmune manifestations, 355 Bacterial infections, 344, 345, 347, 348, 351, 357,
Autoimmune nephritis, 355 387, 443
Autoimmune neutropenia, 347 Bacterial invasion, 80
Autoimmune polyendocrine syndrome BAFF. See B-cell activating factor (BAFF)
type I (APS-1), 356 BAFF-R, 345
Autoimmune polyendocrinopathy with candidiasis and Balomycin A1, 252
ectodermal dystrophy (APECED), 356 Bak, 215, 220, 226
Autoimmune regulator (AIRE) BALB/c S49 lymphoma, 163
gene (OMIM*607358), 356 BALs. See Bronchoalveolar lavage (BALs)
Autoimmunity, 4, 54, 65, 98, 108, 143, 145, 153, 184, B-and T-lymphocyte attenuator (BTLA, CD272),
305, 307, 344, 346, 355, 360 63, 67, 68
Autolysosome, 245 Barcoding method, 464
Automated analysis, 481 Basal cell adenocarcinoma, 502
Automated ow analysis, 482 Basal cell carcinoma (BCC), 189, 219, 353, 410,
Automation systems, 461 494, 495
Autophagic activity, 247 Basal cells, 501
Autophagic cell deaths, 244, 245, 248, 261, 263 Basal inammatory state, 387
Autophagic index, 248 Basic ELISAs, 468
Autophagic pathway, 245, 248, 251 Basic broblast growth factor (bFGF), 12, 15
Autophagosomal membrane, 245 Basic research, 466
Autophagosomal structure, 244 Basophils, 408, 415
Autophagosome fusion, 252 Bax, 215, 219, 220, 226, 259
Autophagosomes, 244, 245 Bax/Bak, 225, 226
Autophagy, 244, 246249, 251, 252, 261263, 386 Bax gene, 219
Autophagy activities, 251 BCC. See Basal cell carcinoma (BCC)
Autophagy/Beclin-1 regulator 1 (Ambra1), 244, B-cell activating factor (BAFF), 345
245, 261 B-cell acute lymphoblastic leukemia, 98
Autophagy-dependent cell death, 262 B-cell antigen receptor (BCR), 459
Autophagy inhibitors, 251, 252 cross-linking, 345
Autophagy modulators, 245 protein, 459
Autophagy promotes necroptosis, 262 B-cell lymphoma, 360, 361
Autoradiography, 455 B-cell lymphoma 2 (Bcl-2), 148, 212, 215, 217, 219,
Autoreactive cells, 360 225228, 259, 261, 262, 351, 497
Autosomal dominant, 346, 353, 357 antagonist, 260
Autosomal recessive disease, 346, 347, 351, 352 family, 219, 225, 226
Autosomal recessive hyper-IgE syndrome, 348 family proteins, 225, 226
Autosomal recessive pattern, 353 homology, 225
Autosomal recessive SCN, 351 B-cells, 132, 344346
Average relative binding (ARB), 437 precursors, 346
5-Aza-2-deoxycytidine (5-AZA), 290292 progenitors, 345
5-Azacytidine (5AC), 169 B-cell-type lymphomas, 537
Azurocidin, 32 BCG. See Bacillus Calmette-Gurin (BCG)
Index 565
Bcl-2 gene, 219, 285 Bistability, 433, 440

Bcl-2 homolog (BH)3-only subfamily proteins, 215 Bladder, 219
Bcl-2 homolog Bcl-XL, 261 Bladder cancer, 289, 329
BCL6/LAZ3, 350 Blood, 12
Bcl-XL, 215, 226 circulation, 166
BCR. See B-cell antigen receptor (BCR) pre-pDCs, 178
BCR/ABL, 98 vessels, 166
BCR-ABL1, 251 Bloom syndrome, 357
BCR-coupled calcium signaling cascade, 345346 B lymphocytes, 348, 422
BDCA-2, 179, 182, 184, 185 B lymphopenia, 354
BDCA2-DTR, 179 BN472 mammary carcinoma, 250
BDCA-4, 179, 182 Bnip3, 262
BDCA-4 (neuropilin-1), 182 Bone-forming tumors, 533
Bead-based assay, 464 Bone marrows, 4, 11, 12, 354
Beclin-1, 244, 245, 248, 261, 262 failure, 353
Benign cancer, 2 stroma, 352
Benign thyroid nodules, 457 Bone metastasis, 127
Benign tumor, 424 Bortezomib, 228, 229, 251, 263
BerEP4, 495 Bortezomib-induced cytotoxicity, 228
2m. See 2-microglobulin (2m) Bortezomib resistance, 229
2m gene, 163 Bowen carcinoma in situ, 353
-actin, 52 BRAF, 506
-defensins, 13 BRAF inhibitors, 497
Beta-catenin, 497 BRAF p.V600E, 497
Beta-HCG, 537 Brain, 357
Bevacizumab, 137, 225, 250 cancer, 410
bFGF. See Basic broblast growth factor (bFGF) tumors, 250, 534
FGF, 132 BRCA1, 290
BH1-BH4, 225 BRCA1-decient, 290
BH3, 225, 226 BRCA1-mutant human tumors, 290
BH3 domain, 261 Breast, 67, 344, 357
BH3-only proteins, 148, 215, 226, 261 cancer, 2, 12, 18, 19, 52, 125, 127, 131, 133, 135,
Bid cleavage, 148, 152, 215, 219, 220 154, 186, 187, 217, 248, 250, 317, 319,
Bif-1, 261 323, 326, 333, 410, 445, 456, 492
Bimodal response, 441 cancer cell lines, 290
Biochemical interactions, 432, 433 carcinomas, 54, 248, 280
Biochemical networks, 430432, 440, 443 tumor progression, 189
Biochemical systems, 440, 441 Breathlessness, 409
Bioinformatics, 301, 303, 430, 432, 439 Bronchial constriction, 409
algorithms, 437, 440 Bronchiectasis, 357
biology, 432 Bronchoalveolar carcinomas, 15
Biological data-based mathematical modeling, 431 Bronchoalveolar lavage (BALs), 155
Biological mechanisms, 431 Bronchoalveolar lavage uids, 15
Biological relevance, 280 Bronchus, 2
Biological scales, 432 Brutons tyrosine kinase (BTK; OMIM*300300),
Biological systems, 429, 434 345, 346
Biology, 429 BST-2, 180, 187
Biomarkers, 430 BTLA. See B-and T-lymphocyte attenuator (BTLA)
Biomechanical forces, 432 Buffy-coat material, 476
Biomedical context, 433 Burkitts lymphoma, 80, 288, 307, 347350, 355
Biomedical data, 430, 432 BW T lymphoma, 167
Biomedical knowledge, 432
Biomedical scenarios, 432
Biomedicine, 429, 430 C
Biometric data, 432 CA72.4, 496
Biomolecules, 430 CAFs. See Cancer-associated broblasts (CAFs)
Biotin, 466 Calcitonin, 505
Biphasic tumors, 503 Calcium mobilization, 384
BIR, 216 Calcium modulator, 346
-islets, 153 Caldesmon, 498
566 Index
Calibration, 464 Carcinogen MCA, 203

C allele, 317, 323, 324, 329, 331, 333 Carcinogens, 356, 409
Calpains, 259 Carcinomas, 79, 109, 425
Calreticulin (CRT), 33 of the cervix, 307
Cancer, 145, 184, 200, 247, 263, 277, 303, 312, of the lung, 307
344, 385, 407409, 414, 416, 419 CARD. See Caspase-recruitment domain (CARD)
biomarkers, 436 Cardiac anomalies, 361
cells, 260, 261, 292, 410 Cardiac defects, 361
chronotherapy, 444 Cardiac disorders, 352
elimination, 542 Cardiac subtype, 323
epidemiology, 411 Cardia gastric cancer, 319
equilibrium, 542 Cardiovascular diseases, 381, 385, 388, 407
escape, 542 Carlzomib, 229
evolution, 426 Cartilage hair hypoplasia, 357
genotypes, 432 CASP8, 355
growth, 210 CASP10, 356
immune equilibrium, 200 CASP10 (OMIM*601762), 356
immunity, 195, 198, 201, 306 Caspase-1, 379
immunoediting, 186, 196, 197, 203, 205, 206, Caspase-3, 144, 148, 212, 213, 215, 230
276, 542 Caspase-5, 379
immunosurveillance, 410 Caspase-6, 212, 213
incidence, 414 Caspase-7, 212, 213, 215, 230
microenvironment, 248, 281 Caspase-8, 144, 145, 148, 151153, 212, 213, 219,
multi-scale models, 431, 442 220, 256, 262
phenotypes, 432 Caspase-8 gene, 219
progression, 210, 276, 430, 432 Caspase-9, 144, 212, 213, 215, 220, 230
signaling, 431 Caspase-9-dependent mechanism, 212
vaccination, 473 Caspase-10, 144, 213, 220
vaccines, 205, 434436 Caspase-dependent apoptosis, 211, 213, 262
Cancer-associated broblasts (CAFs), 186 Caspase-independent apoptosis, 212, 213
Cancer-associated genetic signatures, 432 Caspase-independent cell death, 259
Cancer immunotherapy (CIMT), 188, 206, 388, Caspase-independent signaling pathway, 217
425, 471, 476 Caspase-mediated cleavage, 213
Cancer Immunotherapy Consortium (CIC), 478 Caspase-recruitment domain (CARD), 33
Cancer-inducing infectious, 303304 Caspases, 143, 144, 152, 230
Cancerous, 423 Catabolism, 244
Cancerous inhibitor of PP2A (CIP2A), 212 Cathepsin B, 259
Cancer-related inammation, 412 Cathepsin G, 32
Cancer-related pathways, 441 Cathepsins (CTS), 17, 245
Cancer Research Institute in the USA (CIC/CRI), 477 Causal perspective, 432
Cancer-specic CpG island, 291 CBA. See Cytometric bead array (CBA)
Cancer-specic point mutations, 436 CCAT/enhancer-binding protein b (C/EBPb), 13
Cancer-testis antigens, 4, 306 CC chemokine 2 (CCL2), 12, 13, 19, 39, 103, 130,
Candida albicans, 356 133, 186, 278
Canine species, 420 CC chemokine receptor 2 (CCR2), 12, 135, 280
Canine transmissible venereal tumor (CTVT), 419426 CC chemokines, 13
genomes, 420 C cells, 505
growth phase, 421 CCL-2. See CC chemokine 2 (CCL2)
Canonical multiparameter assay, 472 CCL2 (MCP-1), 39
Capture Ab, 458 CCL2 (MCP1-1), 31
16-Carbon fatty acid (palmitic acid), 151 CCL2-CCR2, 39
Carboplatin, 227 CCL3, 13
6-Carboxyuorescein diacetate succinimidyl ester CCL3 (MIP-), 39
(CFSE), 473 CCL4 (MIP-), 39
Carcinoembryonic antigen (CEA), 205, 493, 495, CCL5, 12, 13, 39, 131
496, 516, 517, 537 CCL5 (RANTES), 31
Carcinogenesis, 154, 203, 275, 354, 357, 387, CCL7, 12, 13, 16
409412, 415 CCL8, 12, 13
Carcinogen-induced sarcoma, 186 CCL17, 11, 17, 18, 131, 135, 136
Carcinogen-induced tumors, 195 CCL18, 18
Index 567
CCL19, 178 CD56bright NK cells, 381, 382

CCL20, 13, 106, 127 CD56bright subset, 353
CCL21, 178 CD56dim NK cells, 381, 382
CCL22, 17, 18, 131 CD57, 381383
CCR1, 39, 130 CD62L, 178, 380
CCR2. See CC chemokine receptor 2 (CCR2) CD64, 380
CCR2+, 39 CD68+ TAMs, 14, 279
CCR2-V64I polymorphism, 135 CD70, 184
CCR4, 131 CD80, 14
CCR4+, 17 CD80 (B7-1), 62
CCR5, 13, 128, 134137 CD80/86, 188
CCR532, 135 CD83+, 12
CCR6, 13, 127 CD86, 14
CCR7, 178, 179, 188 CD86 (B7-2), 62
CCR7, 383 CD86 signaling, 381
CCR9, 82, 127 CD94, 382
CCR10, 127 CD94/NKG2 lectin-like receptors, 305
CD1d, 31, 382 CD95, 144, 146, 147, 149154
CD3CD56bright NK cells, 382 CD95/CD95L, 154
CD4:CD8 ratio, 345 CD95-DD, 149, 150, 152, 154
CD4+CD25+ regulatory T cells, 184 CD95/FADD/caspase-8/caspase-10, 147
CD4+ T cells, 5, 15, 179, 345, 384, 408 CD95L, 146, 152155
CD4+ T-helper lymphocytes, 279 CD112, 37
CD4+ T lymphocytes, 279 CD155, 37
CD8 T cells, 383 CD161, 31
CD8+ T cells, 5, 278, 345, 381, 382, 384, 389, 472 CD178/FasL, 152
CD8+ TILs, 280 CD200R, 184
CD11b+, 11, 12, 17, 388 CD204+ TAM, 14
CD11clow/cells, 179 CD276 (B7H3), 5
CD14, 12 CD326, 472
CD14+, 380 CDC. See Complement-dependent cytotoxicity (CDC)
CD14++(high) CD16+, 380 Cdc42, 348
CD14++(high) CD16, 380 Cdc42-specic GEF, 348
CD14+(low) CD16+, 380 CDK6, 289
CD14HLADR, 388 CDK6-Rb pathway, 289
CD15, 12 C/EBPb. See CCAT/enhancer-binding protein b
CD16, 382 (C/EBPb)
CD16+, 380 Celecoxib, 136
CD19, 6 Celiac disease, 346
CD20, 436 Cell adhesion molecules, 410
CD23, 415 Cell-autonomous process, 296
CD25+Foxp3+ T-regulatory cells, 202 Cell biology, 462
CD27, 383 Cell cycle, 444
CD28, 37, 62, 63, 384, 385 Cell death, 210, 211, 220, 225, 357
CD28, 383 Cell division, 209, 444
CD28/CTLA-4, 63 Cell environment, 439
CD31, 16 Cell growth, 210
CD33+, 388 Cell-mediated immunity, 354, 421
CD34, 495, 498 Cell migration, 122
CD34+, 182, 388 Cell phenotypes, 432
CD34+ precursor cells, 414 Cell proliferation, 410, 419
CD40, 20, 188, 408 Cell rearrangements, 209
CD40 ligand (CD40L), 188 Cell recruitment, 123
CD45, 386 Cell survival, 225, 386
CD45RA, 383 Cell-to-cell communication circuits, 434
CD45RA+, 179 Cellular, 200, 281
CD45RA, 383 apoptosis, 216
CD45RA+ CCR7+, 383 autouorescence, 475
CD45RA+ T cells, 347 disintegration phases, 257
CD47, 34 environment, 386
568 Index
Cellular (cont.) Chromogranin, 513

immune responses, 385, 440 Chromophobe carcinoma (CC), 514
immunodeciency, 360 Chromosomal aberrations, 345, 436
mediators, 279 Chromosome 6, 325
phenotypes, 441 Chromosome 8p21-22, 217
radiosensitivity, 357 Chromosome 15, 305
RNAses, 292 Chromosome 22q11.2 deletion syndrome, 361
senescence, 127, 377 Chronic diarrhea, 350
Cellular FLICE-inhibitory protein (c-FLIP), 145, 256 Chronic eczema, 357
Central memory TCM (CD45RA CCR7+), 383 Chronic enteroviral infections, 345
Central nervous system (CNS) Chronic immune stimulation, 307
malignancies, 250251 Chronic infections, 277, 307, 349
vasculitis, 347 Chronic inammation, 146, 312, 408
Cerebral palsy, 347 Chronic inammatory diseases, 145, 307, 345, 386,
Cerebrospinal uid, 457 409, 441
CERVARIX, 436 Chronic inammatory state, 384
Cervical cancer, 52, 106, 311, 323, 324, 326, 419, Chronic leukemia, 128
434, 436 Chronic low-grade inammation, 385, 387
Cervical carcinoma, 169 Chronic lymphocytic leukemia (CLL) cells, 68, 109,
Cervical intraepithelial neoplasia, 3 133, 219, 226, 227, 285, 436
Cervical squamous cell carcinoma, 312 Chronic mucocutaneous candidiasis (CMC), 356
Cervix, 67, 326, 436 Chronic myeloid/myelogenous leukemia (CML),
adenocarcinomas, 516 227, 251, 308, 312, 459
cancers, 516 Chronic pancreatitis, 410
SCC, 516 Chronic viral hepatitis, 184
c-FLIP. See Cellular FLICE-inhibitory protein (c-FLIP) Chronotherapy, 444
Chaperone-mediated autophagy (CMA), 244 cIAP1(s), 145, 148, 215, 217, 219, 230, 252, 256, 260
Chemical irritants, 410 cIAP2, 145, 148, 215, 217, 252, 256, 260
Chemically induced cancers, 415 CIC. See Cancer Immunotherapy Consortium (CIC)
Chemiluminescent substrate, 455 Cigarette smoke, 188, 410
Chemoattractants, 12, 153, 278 CIMT. See Cancer immunotherapy (CIMT);
Chemoimmunotherapy, 6, 170 European Cancer Immunotherapy (CIMT)
Chemokine receptors, 178 Cinogenesis, 153
Chemokines (CKs), 10, 12, 17, 29, 32, 39, 79, 82, CIP2A, 217
83, 94, 104, 106, 121, 123, 125, 127129, Circulating tumor cells (CTC), 17
131133, 135137, 178, 181, 186, 205, Circulation, 186, 202
278, 354, 386, 387, 412 9-cis RA, 80
CCL20, 106 Cixutumumab, 249
central roles, 125 c-Jun N-terminal kinase (JNK), 257
CXCL12, 106 CK5/6, 495
Chemoradiotherapy, 200, 205 CK14/15/19, 495
Chemoresistance, 217, 441 CK20, 495
Chemoresistant tumor cells, 442, 445 CK/CKR, 130
Chemotactic, 129, 130 CK/CKR network, 123
receptors, 186 CK/EMA, 537
responsiveness, 354 CKRs. See Corresponding/cognate receptors (CKRs)
Chemotaxis, 107, 126, 133, 379, 380 CKs. See Chemokines (CKs)
Chemotherapeutic agents, 445 CLA. See Cutaneous lymphocyte-associated
Chemotherapy(ies), 5, 6, 72, 137, 170, 225 antigen (CLA)
ChemR23, 186 Class 1 genes, 305
Childhood acute lymphoblastic leukemia, 308 Classical (class Ia), 161
Chimeric, 453 Class III phosphoinositide-3-kinase (PI3K), 244, 248
Chloroquine (CQ), 252 Class II-restricted epitopes, 482
Cholangiocarcinoma, 287, 508, 509 Class switch recombination (CSR), 307
Cholangiocellular carcinoma cells, 15 clc-DNA Workbench 5.0.1., 440
Cholinergic tumors, 540 Clear cell cholangiocarcinoma, 509
Chondrosarcomas, 537 Clear cell sarcoma, 534
Chordoma, 535 Clear renal cell carcinoma (CRCC), 513, 514
Choriocarcinomas, 426 Cleavage motifs, 439
Choroid plexus tumors, 534 Clinical benet response (CBR), 249, 250
Index 569
Clinical immunodeciency, 347 Conventional experimentation, 433

Clinical trials (ICH-GCP), 479 Conventional therapy, 443
CLL cells. See Chronic lymphocytic Coombs and Gell classication, 408
leukemia (CLL) cells Corneal epithelia, 152
Clonality, 422 Corresponding/cognate receptors (CKRs), 121, 122,
Clonally transmissible cancers, 424 124, 128, 131, 136
Clonal transmission, 420 expression, 127
CLRs. See C-type lectin receptors (CLRs) polymorphisms, 137
ClustalX software, 437 Costimulatory molecules, 181
Clusters of Differentiation (CD), 472 Co-stimulatory signals, 381
CMC. See Chronic mucocutaneous candidiasis (CMC) Coughing, 409
CML. See Chronic myeloid/myelogenous Counterintuitive behavior, 431
leukemia (CML) COX-2. See Cyclooxygenase-2 (COX-2)
CMV, 383385, 387, 389 COX-2 inhibitors, 20
c-Myc gene, 288, 420 Coyotes, 420
c-Myc-induced tumorigenesis, 288 CpG, 181, 187189, 290, 437
Coding RNAs, 431 CpG-A, 181
Codon optimization, 437, 439 CpG-activated pDCs, 184
Codons, 437, 439 CpG-A oligodeoxynucleotide (CpG-A ODN), 51
Cold abscesses, 357 CpG-B, 181
Cold protein, 457 CpG generated inhibition, 169
Colitis, 355 CpG motifs (CpG-ODNs), 169
Collecting duct carcinoma, 514 CpG motifs optimization, 437
Colon cancer(s), 12, 67, 79, 132, 153, 200, 248, CpG-ODN, 169, 181
344, 413, 507 CpG-ODN 1585, 169
Colon carcinogenesis, 291 CpG-ODNs immunotherapies, 169
Colon carcinoma, 109, 219 CpG optimization, 439
Colony-stimulating factor (CSF), 13 cPLA2, 259
Colony-stimulating factor 1 (CSF-1), 12, 13, 17, 18, 39 CR. See Complete remission (CR)
Colonystimulating factor 3 receptor (CSF3R) Craniofacial, 357
gene (OMIM*138971), 351 CRC. See Colorectal cancer (CRC)
Colorectal adenocarcinomas, 506 CRCC. See Clear renal cell carcinoma (CRCC)
Colorectal cancer (CRC), 2, 13, 52, 83, 99, 103, CRDs. See Cysteine-rich domains (CRDs)
135137, 220, 280, 287, 289, 291, 319, Criopreserved, 164
333, 346, 413, 416, 444 Cross reactivity, 454, 468
cells, 248 CRT. See Calreticulin (CRT)
disease progression, 280 Cryopreservation, 480
prognosis, 280 CSF-1 receptor (CSF-1R), 13
Colorectal carcinoma, 317, 346 CSF-1R inhibitor (Ki20227), 39
Combinatorial encoding, 482 CSF3R mutations, 351, 352
Combined immunodeciency disorder, 347, 348 CSF-R1, 18
Commercial software analysis packages, 482 CSR. See Class switch recombination (CSR)
Common myeloid progenitor cells, 178 CTC. See Circulating tumor cells (CTC)
Common variable immunodeciency (CVID), 344346 C-terminal disulde, 453
Complement-dependent cytotoxicity (CDC), 50, 55, 411 CTL. See Cytotoxic T lymphocytes/cytotoxic
Complete remission (CR), 5 T-cell (CTL)
Complex ow cytometry assays, 472 CTLA-4. See Cytotoxic T-lymphocyte-associated
Complex I, 256 antigen 4 (CTLA-4)
Complex mathematical models, 430 CTVT. See Canine transmissible venereal
Complex multifactorial diseases, 430 tumour (CTVT)
Complex nonintuitive relations, 431 C-type lectin and lectin-like receptors (CLRs), 34
Computational algorithms, 432 C-type lectin domain family 9A (CLEC9A), 34
Computational biology, 430, 432, 434 C-type lectin receptors (CLRs), 181, 182
Computational methods, 444 C-type lectins, 381, 382
Computational publications, 432 C-type lectin transmembrane glycoprotein, 179
Computer-based image analysis systems, 461 Cutaneous anergy, 354
Conatumumab, 220, 225 Cutaneous bacterial infections, 354
Confocal microscope, 461 Cutaneous infection, 353
Contagious pathogens, 419 Cutaneous lymphocyte-associated antigen (CLA), 179
Conventional chemotherapy, 445 Cutaneous melanoma, 52, 198
570 Index
Cutaneous T-cell lymphoma, 137 Cytokine network, 312, 313

Cutaneous viral infections, 348 Cytokine response modier A (CrmA), 256
CVID. See Common variable immunodeciency (CVID) Cytokines, 3, 6, 1013, 15, 2932, 3739, 53, 62, 68,
CX3C chemokine, 135 79, 80, 94, 95, 97, 99101, 103, 105, 107109,
CX3CR1, 130, 135 123, 130, 162, 178, 186, 197, 204, 205, 252,
CXC, 12, 39, 132, 135 278, 297, 304, 312, 357, 379382, 384386,
CXC chemokine 1 (CXCL1), 12 408, 412, 423, 432, 434, 441, 471, 474, 477,
CXC chemokine receptors, 130 484, 485
CXC chemokine stromal cell-derived factor 1 Cytokinesis, 360
(SDF-1), 354 Cytokine-specic receptors, 313
CXCL1, 12, 16, 127 Cytolysis, 204
CXCL2, 12 Cytomegalovirus infections, 345, 348
CXCL3L1, 30 Cytometer(s), 464, 465, 472, 475
CXCL4, 16 Cytometer calibration, 474
CXCL5, 12 Cytometer Setup and Tracking (CS&T), 476
CXCL5-CXCR2, 18 Cytometric bead array (CBA), 464, 465
CXCL6, 12, 16 assay, 464, 465
CXCL7, 16 technologies, 464
CXCL8, 12, 13, 15, 16, 135, 181 Cytometry (CyTOF), 474, 485
CXCL8 (IL-8), 39 Cytopenias, 353
CXCL8 polymorphism, 135 Cytoplasmic proteins, 243
CXCL10, 131, 136, 186 Cytoplasmic tyrosine kinase of the Btk/Tec, 345
CXCL12, 12, 13, 30, 106, 126, 128, 132, 133, 135, Cytosolic PLA2 (cPLA2), 258
137, 186, 354 Cytosolic state (c-state), 259
CXCL12 (SDF1), 31 Cytostasis, 204
CXCL12-CXCR4, 18 Cytostatic drugs, 442, 443
CXCL12-CXCR4 homing axis, 13 Cytotoxic activity, 382
CXCL chemokines, 79 Cytotoxic agents, 226
CXCR1, 127, 380 Cytotoxic capability, 11
CXCR2, 18, 127 Cytotoxic capacity, 382
CXCR3, 131, 178, 179 Cytotoxic CD8+ T lymphocytes, 350
CXCR3 ligands, 179 Cytotoxic CD95L, 153
CXCR4, 18, 128, 186, 188, 354 Cytotoxic chemotherapy, 220
CXCR4 (OMIM*162643), 354 Cytotoxic drugs, 5, 451
CXCR4 antagonist, 137 Cytotoxic granules, 474
CXCR6, 127 Cytotoxicity, 109, 169, 347, 414, 421, 473, 485
CXCR7, 126, 128, 132 Cytotoxicity receptor family, 382
Cyclic neutropenia, 351 Cytotoxic potentiality, 202
Cyclin-D1, 497 Cytotoxic potentials, 414
Cyclin-D3, 497 Cytotoxic radiation, 226
Cyclin-dependent kinase 5 (CDK5), 228 Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4),
Cyclooxygenase-2 (COX-2), 15, 20, 38, 80, 81, 386 5, 6, 37, 63, 105, 188, 202, 346, 383, 436
Cyclophilin A, 259 Cytotoxic T lymphocytes/cytotoxic T-cell (CTL), 3, 50,
Cyclophilin D (CYPD), 259 55, 94, 103, 108, 129, 162164, 167, 169, 179,
Cyclophilin ligand interactor (TACI) gene 181, 189, 199, 279, 304, 388, 435, 444, 445
(TNFRSF13B; OMIM*604907), 346
Cyclophosphamide, 227, 229
Cylindromatosis (CYLD), 145, 152, 256 D
Cysteine 199 (C183), 151 D1 region, 163
Cysteine-rich domains (CRDs), 144, 212 Dacarbazine, 227, 250
Cys-thiol, 151 Daclizumab, 105
Cytarabine, 230 Damage/danger-associated molecular patterns (DAMPs),
Cyt c. See Cytochrome c (Cyt c) 32, 77, 80, 178, 189, 259, 260
Cytochrome c (Cyt c), 144, 212, 215, 217, 220, 257, Danger signals, 177
259, 348 DAP10, 37
Cytogenetic abnormalities, 352, 353 DAP12, 36, 37
Cytokeratin, 514, 541 DAPK1, 212
Cytokine, 19, 97, 100, 101, 103, 105, 107, 108, 153, DARC. See Duffy antigen receptor for CK (DARC)
185, 187, 230, 261, 313, 348, 411, 414 Data analysis, 432
Cytokine (cl-TNF), 145 Data-based ODE model, 443
Index 571
Databases, 432 DFF45, 213

Data-driven mathematical models, 431, 445 DFTD. See Devil facial tumor disease (DFTD)
Data-driven modeling, 443 dGTP, 348
Data-driven perspective, 432 Diabetes mellitus, 385, 410
Data engineering, 430 Diabetes mellitus type 2, 383
Daughter B cells, 452 Diamindichloridoplatin (DDP), 262
DCIR, 184 Diarrhea, 345, 347, 409
DC-LAMP+DC, 12 Dietary habits, 410
DCs. See Dendritic cells (DCs) Differential equations, 433
DC-SIGN. See Dendritic cell-specic ICAM-3 Differentiation, 410
grabbing non-integrin (DC-SIGN) Differentiation Ags, 306
DC-SIGN (CD209), 181 Diffuse intrinsic pontine glioma, 251
DC subsets, 181 Diffuse large B-cell lymphomas (DLBCL), 136, 219
DD. See Death domain (DD) Diffuse large cell lymphoma cells, 226
Dd molecule, 163 Diffuse-type cancer, 323
DD mutations, 150 DiGeorge syndrome critical region gene 8,
DDP-induced cell death, 262 or Pasha (DGCR8), 286
Dd promoter, 163 Digoxigenin, 466
Death-associated protein kinase 1 (DAPK1), 212 Diphtheria toxin (DT), 179
Death domain (DD), 144, 146, 151, 153, 212 Diphtheria toxin receptor (DTR), 179
Death effector domain (DED), 148 Direct Ab arrays, 467
Death-inducing signaling complex (DISC), 144, 145, Direct array, 467
147, 148, 150152, 212, 256 Direct data analysis, 431
Death receptors, 212 Direct ELISA, 458
DEC-205, 34, 182 DISC. See Death-inducing signaling complex (DISC)
DEC-205 (CD205), 181 DISC-like complex, 262
Dectin-1, 182 Disease-free period, 472
DED. See Death effector domain (DED) Disease-free survival, 289
Dedicator of cytokinesis 8 (DOCK8) deciency, 348 Disequilibrium, 347
Deforolimus, 249 DKC1, 352
Deleted in colorectal cancer (DCC), 212, 219 Dk molecules, 163
Deletions, 355 DKO. See Double knockout (DKO)
Dementia, 385 DLEU1, 352
Demethylating agents, 290 DMB, 424
Dendritic cell-based vaccines, 169 DMSO, 163
Dendritic cells (DCs), 12, 32, 110, 123, 202, 277, 381, DNA, 357, 379, 386, 387
382, 385, 386, 388, 408, 421, 422, 435 damage, 312, 386
mDC, 177 demethylating agent, 289
pDCs, 177 fragments, 308
Dendritic cell-specic ICAM-3 grabbing non-integrin ligase IV deciency, 357
(DC-SIGN), 34, 182 methylation, 289, 290
Dental, 357 methyltransferase 1 gene, 425
DEP, 246 microarray, 465, 466
Dependence receptors, 212 promoter methylation and chromatin histone
Deptor, 246 modications, 286
Dermatobrosarcoma, 498 sequence, 308, 439
Desmin, 498, 513, 526, 541 vaccines, 436, 437, 439, 440
Desmoplastic small round cell tumor (DSRCT), 534, 538 vaccine vector, 439, 440
Detection Ab, 458 DNA fragmentation factor 45 (DFF45), 213
Detergent-resistant microdomains (DRMs), 150, 151 DNAM. See DNAX-accessory molecule (DNAM)
Deubiquitinases (DUBs), 228, 260 DNAM-1, 103
Devil facial tumor disease (DFTD), 419, 422426 DNAM-1 (CD226), 37
karyotype, 422 DNA methyltransferases (DNMTs), 286, 287, 290
Devil karyotype, 422 DNAX-accessory molecule (DNAM), 30
Devils, 422, 423, 425, 426 DNAX-activating protein-10 (DAP10), 36
Devils biting behavior, 424 DNAX-activating protein-12 (DAP12), 36
Devils immune system, 423 DNMT1, 287, 289, 290
Dexamethasone, 227, 260 DNMT1 null HCT-116, 290
Dextran sulfate sodium (DSS), 33 DNMT3A, 286
DFF40, 213 DNMT3B, 286, 289
572 Index
DNMTs. See DNA methyltransferases (DNMTs) Eccrine glands, 493

Docetaxel, 226 Eccrine tumors, 495
DOCK8 deciency, 348, 349, 357 ECL. See Electrochemiluminescent (ECL)
DOCK8 gene (OMIM*611432), 348 ECM. See Extracellular matrix (ECM)
Docking based techniques, 434 ECP. See Eosinophil cationic protein (ECP)
Dogs, 420, 424, 426 Ecroptosis, 260
Dominant autosomal, 351 Ectodermal dystrophies, 356
Donor-derived malignancies, 200 Ectopic lymphoid structures, 279
Dormant state, 200, 201 Eczema, 409
Dormant tumor cells, 201 Effective immune response, 201
Dot plots, 480, 481 Effector CD4+, 472
Double knockout (DKO), 289 Effector function, 122
Double-stranded oligonucleotide, 301 Effector memory (EM), 383
Downregulation, 205 Efciency, 306
DP, 305 EGF. See Endothelial growth factor (EGF)
DQ, 305 EGFL7, 291
DQ2 (8.1), 346 EGFR-driven phosphorylation, 153
DQB1*0301, 307 EGFR overexpression, 538
DR, 305 Ehrlich, Paul, 195
DR3, 346 EIAs. See Enzymatic immunoassays (EIAs)
DR3 death receptor, 144 eIF4E genes, 248
DR4 death receptor, 144 EL4 lymphoma, 164
DR5 death receptor, 144 Elastase (ELA2) gene (OMIM*130130), 351
DR6 death receptor, 144 Electrochemiluminescent (ECL), 459
DRAL/FHL2, 212 Electrophoresis, 301
DRMs. See Detergent-resistant microdomains (DRMs) Electrophoretic mobility shift assay (EMSA), 301
Drosha, 286 Elimination, 197
Drug discovery, 434 Elimination stage, 378
DSRCT. See Desmoplastic small round cell ELISA. See Enzyme-linked immunosorbent
tumor (DSRCT) assays (ELISA)
DSS. See Dextran sulfate sodium (DSS) ELISPOT, 477, 478
DTFD, 423 Elliptical/polygonal gates, 480481
DTR. See Diphtheria toxin receptor (DTR) ELR, 132
DTR models, 180 EM. See Effector memory (EM)
Dual PI3K/mTOR modulator, 250 EMA. See Epithelial membrane antigen (EMA)
Duffy antigen receptor for CK (DARC), 132 Emberger syndrome, 353
Dulanermin, 263 Embryonal rhabdomyosarcoma, 540
Duncan disease, 354 Emission, 461
DyNAVacS server, 439 EMRAlike CD4, 383
Dysgammaglobulinemia, 354 EMRA T cells, 383
Dysplasia, 312 EMSA. See Electrophoretic mobility shift assay (EMSA)
Dysplastic changes, 354 EMT. See Epithelial-mesenchymal transition (EMT)
Dysplastic urothelium and carcinoma in situ, 515 Ena/Vasp-like (EVL) gene, 291
Ena/Vasp-like (EVL) promoter, 291
Encephalomyelitis, 456
E Endocrine system, 1
E22 transcription factor, 178 EndoG, 216
E2F1, 441 Endogenous antioxidant species, 386
E2F1-centered network, 442 Endogenous nucleic acids, 185
E2F1/p73/miR-205 networks, 442 Endothelia, 152
E3 ubiquitin, 260 Endothelial barrier, 123
4E-BP1, 245, 246 Endothelial cells, 415
EBV. See Epstein-Barr virus (EBV) Endothelial cells (CD31), 472
EBV-associated HLH, 355 Endothelial growth factor (EGF), 15, 18
EBV-associated lymphoma, 306 Enhancer of Zeste homolog 2 (EZH2), 288, 290
EBV-associated lymphoproliferative disease, 354 Enigmatic, 154
EBV-directed cytotoxicity, 355 Environment, 296
EBV-infected B cells, 355 Environmental background, 300, 301
EBV infection, 354 Environmental carcinogens, 4
E-cadherin, 17, 497, 501 Environmental factors, 296, 300, 311, 313, 408, 409
Index 573
Environmental risk factors, 301 E-selectin, 179

Enzymatic immunoassays (EIAs), 457 Esophageal, 67
Enzyme activation, 386 Esophageal cancers, 127, 509
Enzyme-linked immunosorbent assays (ELISA), Esophageal SCC, 356, 509
457460, 464, 466, 474 Esophagus cancer, 329
Eosinophil cationic protein (ECP), 414 ES/PNET. See Ewing sarcoma/primitive
Eosinophilia, 348, 357, 414 neuroectodermal tumor (ES/PNET)
Eosinophil peroxidase, 414 Estrogen receptor-positive (ER+)
Eosinophils, 408, 414, 415 breast cancer, 248
Ependymoblastoma, 535, 538 Estrogen receptor protein (ER), 461
Ependymoma, 534, 535 Etoposide, 227
Epidermal growth factor (EGF), 216 Eukaryotic proteasomes, 439
Epidermodysplasia verruciformis (EV), 349, 353, 354 Eukaryotic translation initiation factor 4E
Epidermoid lung carcinoma cells, 248 (4E-BP1), 245
Epigenetic changes, 441 Eukemic transformation, 353
Epigenetic deregulation, 387 Eumesodermin, 280
Epigenetic drugs, 289 European Cancer Immunotherapy (CIMT), 477
Epigenetic effectors, 291 Eutherian immune systems, 423
Epigenetic features, 206 Eutherians, 423
Epigenetic machinery, 287289 EV. See Epidermodysplasia verruciformis (EV)
Epigenetic mechanisms, 169, 291, 349 EV1, 353
Epigenetic modications, 200 EV2, 353
Epigenetic regulation, 289, 291 Evasive mechanism, 163
Epigenetics, 63, 286, 291, 306, 425 EVB-associated immune dysregulation, 346
Epigenetic silencing, 290, 291 EVER1 (OMIM*605828) genes, 353
Epigenome, 289 EVER2 (OMIM*605829) genes, 353
Epilepsy, 351 EVER genes, 354
Epi-miRNAs, 287, 288 Everolimus (RAD001), 249, 263
Epithelial carcinoma cells, 281 EVER proteins, 353
Epithelial cells (EpCAM), 472 EV-HPV. See EV-specic HPV (EV-HPV)
Epithelial membrane antigen (EMA), 494496, Evolutionary pressure, 297
523, 536 EV-specic HPV (EV-HPV), 349, 353
Epithelial-mesenchymal transition (EMT), 102, 132 Ewing sarcoma/peripheral nerve sheath
Epithelial-myoepithelial carcinoma, 502 tumor, 534, 538
Epithelial ovarian cancers, 200, 289 Ewing sarcoma/primitive neuroectodermal
Epithelial surfaces, 356 tumor (ES/PNET), 540
Epithelial-to-mesenchymal transition (EMT), 126, 289 Excitation, 461
Epitope-based vaccines, 436 Exhausted T cells, 66
Epitope mapping, 437 Exome sequence, 204
Epitopes, 437, 452 Exonic polymorphisms, 298
Epitope screening, 485 Exons 2, 305, 306
Epitopic sequences, 437 Exons 3, 305, 306
Epstein-Barr virus (EBV), 2, 3, 80, 306, 307, 347, Extended haplotypes, 311
348, 350, 355, 434, 501 External genital mucosa, 420
Epstein-Barr virus nuclear antigen (EBNA-4 and Extracellular encapsulated bacteria, 345
EBNA-6), 307 Extracellular matrix (ECM), 15, 132
Equilibrium, 197, 206 Extracellular proteases, 412
Equilibrium phase, 200, 204, 205, 276, 410, 411, 542 Extracellular-regulated kinase 1 (ERK1), 107
Eradication, 205 Extracellular stress signals, 212
ErbB3 receptor, 444 Extranodal, 344
ErbB family, 444 Extranodal sites, 355
ErbB/PI3K signaling network, 444 Extravasation, 166
ER+ breast cancer, 248 Extrinsic apoptosis, 211, 212, 262
ERK1. See Extracellular-regulated kinase 1 (ERK1) Extrinsic apoptotic pathway, 212, 261
ERK1/2 MAPKs, 380 Extrinsic apoptotic signals, 212
ERK pathways, 288 Extrinsic pathway, 220
Erlotinib, 249, 250, 252 Extrinsic signaling pathways, 217
ER stress, 228 Eye drops, 413
Escape, 197, 206 EZH2. See Enhancer of Zeste homolog 2 (EZH2)
Escape phase, 201, 205 EZH2-mediated histone methylation, 290
574 Index
F Fluorescent, 461
F(ab)2 fragment, 453 dyes, 464, 473475
Fab fragment, 453 probe, 475
Facial dysmorphism, 361 reporters, 461
Factor-alpha, 131 Fluorochrome conjugates, 475
FADD. See Fas-associated death domain adapter Fluorochromes, 454, 475, 482, 485
protein (FADD) Fluorophore, 454, 461, 464, 465
FADD-DD, 150 FMLP, 380
Familial adenomatous polyposis, 296 FMO. See Fluorescence minus one (FMO)
FANCD2, 352 FMS, 178
3Fas, 146, 252, 352 FMS like tyrosine kinase 3 ligand (Flt3L), 178
Fas-associated death domain adapter protein (FADD), Focal adhesion kinase (FAK), 213
212, 256, 257, 262 Focal adhesion kinase family interacting protein of 200
Fas-associating protein with a death domain, 144153 kDa (FIP200), 244
Fas/CD95, 144, 212 FOLFIRI, 225
Fas death receptor, 144 FOLFOX6, 225
FAS gene (TNFRSF6, or CD95; OMIM*134637), 356 Follicular cells, 505
FAS gene mutations, 356 Follicular lymphoma, 522
Fas-induced apoptosis, 213 Food allergies, 409
FAS ligand (FASL) (TNFSF6 or Food and Drug Administration (FDA), 5
CD95L (OMIM*134638)), 356 Foreign cells, 423
Fas ligand (FasL), 3, 50, 69, 104, 146, 202, 212, 297 Foreign tissue, 421
Fas-mediated lymphocyte apoptosis, 355 Forkhead/winged helix transcription factor (FoxP3)+ T
Fatigue, 229 lymphocytes, 277
FcRI, 408, 415 Forward scatter light (FSC), 472
Fc, 380 Foxes, 420
Fc receptor II (FcRII), 185 FOXO, 386
Feedback loop-like structures, 432 FoxP3, 81, 108, 279
Feedback loop-regulated pathways, 434 FoxP3+, 5, 55
Feedforward loops, 440 FoxP3+ T cells, 77, 78, 8085
FGF-2, 12 Foxp3+ Tregs, 100
Fhit, 165 Free radicals, 386, 387, 408
Fibroblastic morphology, 278 Free T4, 457
Fibroblasts, 133, 205, 384 Freund leukemia integration site (FLI-1), 531
Fibroblasts (ER-TR7), 472 FSC. See Forward scatter light (FSC)
Fibrogenesis, 126 5-FU, 287
Fibrohistiocytic tumor, 498 Full-edged tumors, 200
Fibromatosis, 513 Fulminant hemophagocytosis, 347
Fibrosarcoma cell line, 168 Functional polymorphisms, 301
Fibrosarcoma clones, 167, 170 Fungal infections, 348, 352, 357
Fibrosarcoma tumor, 169 Fungi, 356
Fibrosis, 132
FIP200, 244, 246
FIST, 51 G
Five methylcho-lanthrene-induced sarcomas, 167 G0 stage, 474
FK506-binding protein (FKBP12), 246 GA genotypes, 329
FKBP-12, 246, 249 Galaxies, 430
FKBP12-rapamycin-binding domain (FRB), 246 G allele, 321, 326, 330
Flexible hinge region, 454 -T cells, 31, 32, 37, 103, 199, 200, 387
FLI-1. See Freund leukemia integration site (FLI-1) Gamma radioisotope of iodine, 457
FLICE-inhibitory protein (FLIP), 147, 256 Ganglioglioma, 535
Flow cytometric assay, 480 Gangliosides, 20
Flow cytometry/cytometer (FCS), 461, 462, 464, GARDASIL, 436
472476, 478482, 485 Gastric, 67
Flow operator, 482 adenocarcinoma, 346, 509
Flt3L. See FMS like tyrosine kinase 3 ligand (Flt3L) cancer, 79, 99, 127, 133, 135, 288, 289, 312, 319,
Fludarabine, 227 323, 329, 344
Fluorescence, 464 carcinogenesis, 289
Fluorescence minus one (FMO), 476 carcinoma, 319, 326, 346
Index 575
Gastrointestinal (GI), 354, 507 Genome sequence analysis, 290

diseases, 346 Genome-wide association studies (GWAS),
infections, 346 299301, 303, 304
manifestations, 361 Genome-wide linkage studies, 353
symptoms, 229 Genomic instability, 201, 387
systems, 346 Genomic instability syndromes, 357
tract, 357 Genomic loss, 290
tumors, 507 Genotoxic chemotherapy, 445
Gastrointestinal stromal tumor (GIST), 513 Genotoxic drugs, 442, 445
GATA, 353 Genotype-phenotype mapping, 441
GATA2 deciency, 353 Genotyping, 299
GATA2 gene (OMIM*137295), 353 Germ cell tumors, 521, 535, 537
Gating, 481 Germinal center (GC) risk, 329
Gating strategy, 481 Germ-line mutations, 356
GL, 246 GFAP. See Glial brillary acidic protein (GFAP)
GCC haplotype, 321 GG genotypes, 329
GCDFP15, 493, 495, 496, 517 Gingival hyperplasia, 351
GCLP, 474 Gingivostomatitis, 349
GCP regulation, 480 GIST. See Gastrointestinal stromal tumor (GIST)
G-CSF, 352 GITR. See Glucocorticoid-induced tumor necrosis
GEFs. See Guanine nucleotide exchange factors (GEFs) factor receptor-related protein (GITR)
Gemcitabine, 225 Glial brillary acidic protein (GFAP), 535
GEMM. See Genetically engineered mouse Glial tumors, 496, 535
models (GEMM) Glioblastoma, 99, 135, 219, 250, 251, 288
Gene(s), 295, 296, 299, 313, 431 Glioblastoma multiforme (GBM), 250, 252, 538
chips, 299 Gliomas, 127, 137, 360, 410, 413, 414, 416, 536
expression, 432 Gliosarcoma, 250
fusion, 459 Glucocorticoid dexamethasone, 262
product, 301 Glucocorticoid-induced tumor necrosis factor receptor-
regulation, 298 related protein (GITR), 105
silencing, 425 Glucocorticoid resistance, 260, 262
therapy, 360 Glucose-6-phosphatase catalytic subunit 3 (G6PC3)
Geneenvironment interactions, 301 gene (OMIM*611045), 351
Genegene interactions, 301 Glutamate-ammonia ligase (GLUL), 257
Genetic(s), 298, 299, 301 Glutamate dehydrogenase 1 (GLUD1), 257
aberrations, 357 Glutaredoxin 1 (Grx1), 151
background, 300, 301 Glutathione (GSH), 151
changes, 441 Glycogen phosphorylase (PYGL), 257
component, 299, 300 Glycosylation, 387, 408
defect, 355 GM-CSF. See Granulocyte-macrophage colony-
disease, 409 stimulating factor (GM-CSF)
diversity, 423 Good clinical laboratory practice (GCLP), 474, 479
engineering, 453, 454 G-protein-coupled receptor superfamily, 354
factors, 4, 296, 300, 311, 313, 321 GR9 cells, 168
modications, 200 cell lines, 164, 165
mutations, 217 brosarcoma, 170
polymorphisms, 133, 297, 298 brosarcoma tumor, 164
regulation, 210 murine tumor model, 170
risk factors, 301 tumor model, 164
signatures, 430, 432, 442 GR9-derived clones, 164
variation, 299 Granule proteins, 414
Genetically engineered mouse models (GEMM), Granulocyte-macrophage colony-stimulating factor
166, 301 (GM-CSF), 5, 13, 16, 32, 38, 39, 79, 199
Genetically transplantable tumor model systems Granulocytes, 12, 32, 443
(GRAFT), 166 Granulocytic MDSC (G-MDSC), 12
Genetic and epigenetic events, 196 Granulomatous lung disease, 344, 349
Genital system, 1 Granzyme B (GrB), 50, 100, 181, 186, 279
Genital warts, 354 Granzyme-dependent mechanisms, 199
Genome, 296, 410, 423 Graphical processing units (GPU), 481
576 Index
GRB7.1, 165 Head and neck carcinoma, 178

GRB7.2, 165 Head and neck squamous cell carcinoma (SCC), 5, 99,
GRIR5, 165 500, 501
Growth arrest-specic 6 (Gas-6), 33 Head cancers, 68, 186, 217
Growth factors, 12, 32, 79, 123, 130, 178, 205, 261, Heat-sensitive factor, 421
307, 357 Heat shock proteins (HSPs), 304, 311, 439
GSH. See Glutathione (GSH) Heat shock protein/tumor antigen complexes, 94
GTPase-activating protein (GAP), 246 Heavy chains, 162, 453
Guanine nucleotide exchange factors (GEFs), 348 Helicobacter pylori, 344, 345
Guanosine, 181 Helminthic infections, 408
Guanosine triphosphatase (GTPase), 360 Helminths, 415
decient, 349 Hematologic(al), 344
GWAS. See Genome-wide association studies (GWAS) malignancies, 99, 200, 229, 307, 308, 360, 464
GX15-070, 226 tumors, 123
Gynecological cancers, 456 Hematopoiesis, 15, 352, 353
Hematopoietic cells, 122, 353, 360
lines, 261
H transplantation, 344
H2AX, 259 Hematopoietic progenitor cells, 349
H-2b haplotype, 167 Hematopoietic stem cells (HSCs), 9, 17, 353, 426
H-2 class I, 165 Hematopoietic stem-cell transplantation (HSCT),
H-2 class I D, 163 352, 360
H-2 class I heavy chains, 164 Hematopoietic tumors, 107
H-2 class I K, 163 Hemochromatosis gene, 312
H-2 class I Kd, Dd, and Ld molecules, 164 Hemodynamic forces, 166
H-2 class I L, 163 Hemophagocytic lymphohistiocytosis (HLH), 347, 354
H-2 class I molecules, 164 Hemopoietic system, 313
H-2 class I phenotype, 164 Hepatic cancer, 508
H-2 D Ags, 167 Hepatic disorders, 352
H-2 Dk-Ags, 167 Hepatitis, 355, 356
H-2 D surface expression, 167 Hepatitis B virus (HBV), 3, 65, 80, 434
H-2 K and H-2 D Ags, 163 Hepatitis C virus (HCV), 65, 184, 307, 434
H-2 K-decient primary tumors, 170 Hepatoblastoma, 361
H-2 K gene, 167, 170 Hepatocellular, 280
H-2k haplotype, 167 Hepatocellular cancer, 133, 135
H-2 K-low decient primary tumors, 170 Hepatocellular carcinoma (HCC), 68, 80, 99, 135,
H-2 K tumor cell, 170 262, 312, 316, 323, 329, 434, 508, 509
H-2 Ld heavy chain, calreticulin, 164 Hepatocellular carcinoma cells, 15
H-2 phenotype, 164 Hepatocyte growth factor (HGF), 13, 15
H-2-positive wild-type cell line (RMA), 164 Hepatoma, 128
H-2 surface expression, 167 HepG2, 288
H3 lysine 27 (H3K27me3), 288 Her2, 506, 517
Haplo-insufcient tumor suppressor, 261 HER2 ELISA, 460
Haplotype, 299, 300 HER2-negative breast cancer, 249
Hay fever, 409, 411, 413 HER2/neu, 459
HBV. See Hepatitis B virus (HBV) Hereditary cancers, 295
HCC. See HCV-associated hepatocellular carcinoma Herpes simplex (HS) virus, 348, 354
(HCC); Hepatocellular carcinoma (HCC) Herpesviridae viruses, 354
HC class-II epitope, 439 Herpes virus, 80
HCT-116, 289 Herpes zoster, 354
HCV. See Hepatitis C virus (HCV) Heterogeneous, 11
HCV-associated hepatocellular carcinoma (HCC), 307 Heterogeneous tumors, 442
HCV-associated liver cirrhosis, 307 Heterozygous Fas mutation, 355
HCV infection, 307 Heterozygous mutations, 353
HCV-related B-cell lymphoma, 307 HEVs. See High endothelial venules (HEVs)
HCV-related NHL, 307 HEYGAEALERAG motif, 439
HDAC1, 287 HGF. See Hepatocyte growth factor (HGF)
HDAC4, 287, 288 HGS1029, 230
HDAC5, 288 HIES. See Hyper-IgE syndrome (HIES)
HDACs. See Histone deacetylases (HDACs) HIF-1. See Hypoxia-induced factor 1 (HIF-1)
Index 577
HIFs. See Hypoxia-inducible factors (HIFs) Hormone therapies, 461

High endothelial venules (HEVs), 178 Horseradish peroxidase (HRP), 454
High heterochromatic markers, 289 Host defence/defense, 324, 421
High HLA polymorphisms, 308 Host hematopoietic cells, 186
High mobility group box 1 (HMGB1), 32, 185, 385 Host immune response, 419, 421, 423, 425
High-resolution digital image, 461 Host immune system, 422, 423, 425, 434
Histamine, 408, 414 Host immunosurveillance, 4
Histiocytic, 496 Hot protein, 457
Histiocytic origin, 420 Hotspots, 300
Histocompatibility complex class II (MHC-II), 181 HPV. See Human papilloma virus (HPV)
Histograms, 480 HPV-associated SCC, 353
Histone deacetylases (HDACs), 287, 291 HPV-mediated carcinogenesis, 353
inhibitors, 289, 290 H. pylori infection, 289
inhibitor trichostatin A, 291 HRP. See Horseradish peroxidase (HRP)
Histone H2AX, 259 HS-1-associated protein X (HAX1)
Histone modications, 291 gene (OMIM*605998), 351
HIV. See Human immunodeciency virus (HIV) HSCs. See Hematopoietic stem cells (HSCs)
HIV infection, 183 HSCT. See Hematopoietic stem-cell
HL. See Hodgkin lymphoma (HL) transplantation (HSCT)
HLA, 304, 307, 308, 311 HSP70, 36
HLA-A, 161, 305 HSPs. See Heat shock proteins (HSPs)
HLA-A1, 346 HSV-1 infections, 350
HLA-A2, 307, 311 HtrA2/Omi, 215, 216, 229, 230
HLA-A*02:07, 311 Hu, 456
HLA-A*03, 312 Hu antineuronal nuclear, 456
HLA-A*0201, 189, 306 Human aging, 380
HLA-A*0201+, 189 Human beta-defensin-3, 12
HLA-A*0207, 306, 311 Human breast cancer cell, 290
HLA allele, 306, 308, 311 Human genome, 297, 299
HLA-B, 161, 305 Human Genome Project, 298
HLA-B*0801 HLA-DRB1*0101, 307 Human herpesvirus, 345
HLA-B*4405, 307 Human immunodeciency virus (HIV), 2, 65, 143,
HLA-B*4601, 311 184, 185
HLA-C, 161, 305 Human intuition, 430, 431
HLA class 1, 30, 162, 304, 305, 308, 482 Human leukocyte antigen (HLA) class I, 297
HLA class 2, 304, 305, 308 Human leukocyte antigens (HLAs), 304, 306
HLA-DR, 110 Human malignancies, 260
HLA-DR4, 307 Human monoclonal antibody, 444
HLA-DR6, 307 Human papilloma virus (HPV), 2, 3, 68, 164, 311,
HLA-DRB1*0301, 307 323, 348, 349, 353, 354, 419, 434, 436, 500
HLA-DRB1*0401, 307 infections, 352, 354
HLA-E, 305, 307 L2 capsid, 437
HLA-F, 305 vaccine(s), 436, 437
HLA-G, 305, 307, 425 Human papillomavirus (HPV)-associated
HLA/H-2 class I, 161162 cervical cancer, 306
HLA haplotypes, 307 Human T-cell leukemia virus, 80, 434
HLA loci, 304 Humoral immune responses, 440
HLAs, Human leukocyte antigens (HLAs) Humoral immune system, 203
HLH. See Hemophagocytic lymphohistiocytosis (HLH) Humoral immunity, 347, 421, 422
H/L heterodimers, 453 Humoral immunodeciency, 345
HMGB1. See High mobility group box 1 (HMGB1) Huntingtons disease, 248
Hodgkin lymphoma (HL), 109, 135, 149, 279, Hybrid models, 433
306308, 344, 347, 356, 357, 434, 521 Hybridomas, 452
Hodgkin lymphoma cells, 109 Hydroxychloroquine, 252
Homeostasis, 101, 177, 210, 217, 324, 385 4-Hydroxytamoxifen (4-OHT), 248
Homogenous, 300 Hygiene, 408
Homozygous deletions, 349 Hyperactive immune system, 414
Homozygous loss-of-expression mutations (Y38X), 349 Hyperautophagic conditions, 262
Hormonal therapy, 5 Hyper-IgE syndrome (HIES), 357
Hormones, 5, 410, 457 Hyperinammatory, 252
578 Index
Hypermethylation, 290 IL-2, 38, 49, 202, 204, 408, 414

Hyperreactivity, 409 IL-2-inducible T-cell kinase (ITK), 346
Hypersensitive immune system, 414 deciency, 347
Hypersensitivity, 415 gene (OMIM*186973), 347
Hypocalcemia, 361 protein, 347
Hypogammaglobulinemia, 344, 346, 354 IL-2 toxicity, 307
Hypoparathyroidism, 356 IL-3, 79, 188
Hypotension, 409 IL-3R (CD123)+, 179
Hypothyroidism, 356 IL-4. See Interleukin-4 (IL-4)
Hypoxia, 203, 431 IL-4 gene, 316
Hypoxia-induced factor 1 (HIF-1), 104 IL-5, 31, 408, 414, 415
Hypoxia-inducible factors (HIFs), 131 IL-6, 13, 183, 188, 205, 317, 318, 380,
Hypoxia-mediated apoptosis, 431 381, 385387, 408, 412, 421
Hypoxic factors, 131 IL-7, 204
IL-8, 181, 188, 205, 318, 319, 380, 382, 412
IL-9, 408
I IL-10, 3, 11, 13, 1820, 30, 37, 38, 54, 55, 63, 81,
IAP-like protein-2 (ILP-2), 215 103, 108111, 181, 184, 187189, 202,
ICAM-1. See Intercellular adhesion molecule (ICAM)-1 205, 319, 321, 355, 385, 388
ICAM-3. See intercellular adhesion molecule 3 (ICAM-3) IL10+-CD68+ TAMs, 279
ICC. See Immunocytochemistry (ICC) IL-10 polymorphisms, 321
ICC/IHC, 461 IL-12, 3, 5, 18, 32, 37, 38, 103, 108110, 169,
ICOS. See Inducible costimulator (ICOS) 204, 205, 323, 381, 382
ICOS-L. See Inducible costimulator ligand (ICOS-L) genes, 323
ICR. See Immunologic constant of rejection (ICR) receptor, 5
ICS. See Intracellular cytokine staining (ICS) and TNF-, 11
ICS assays, 473 IL-12p70, 183
Idarubicin, 230 IL-13, 11, 15, 19, 31, 408, 415
IDC. See Invasive ductal (IDC) IL-15, 38, 381
iDC. See Immature phenotype (iDC) IL15Ra/IL15, 382
Idiopathic thrombocytopenia, 347 IL-17, 38, 39, 79, 105108, 381, 383
IDO. See Indoleamine 2,3-dioxygenase (IDO) IL-17A, 356
IEFs. See Immune effector functions (IEFs) IL-17F, 356
IFB-, 188 IL-17-VEGF, 106
IFN-, 110, 179, 181, 184, 187, 189 IL-17-VEGF loop, 106
IFN-/IFN-, 181183, 186 IL-18, 33, 38, 355, 381
IFNAR1, 186 IL-21, 38, 49
IFN-, 104 IL-21/, 38
IFN-. See Interferon gamma (IFN-) IL-22, 356
IFNGR/, 37 IL-23, 38, 39, 108
IFN-inducible protein 10 (IP-10), 98, 101, 103 IL-23/, 38
IFNs. See Interferons (IFNs) IL-23-IL-17, 38
IgA, 344, 357 IL-27, 100, 101, 109
IgA deciency (IgAD), 346 IL-35, 109
Ig E. See Immunoglobulin (Ig) E ILC. See Iobular carcinomas (ILC)
IgG, 344, 346, 357 Ileocecal region, 355
IgG2b, 52 ILT-1low/, 179
IgM, 344, 346, 349, 357 ILT7, 187, 188
IHC. See Immunohistochemistry (IHC) ILT7L, 188
IkB, 228, 256 ILT7L-ILT7, 188
IB kinase (IKK)-, 53 Imatinib, 251, 252
IB kinases (IKKs), 256 Imatinib-induced autophagy, 251252
IKK-, 53 Imatinib-induced cytotoxicity, 252
IL-1, 313, 316, 385, 412 Imiquimod, 186, 187
IL-1, 313, 314 Imiquimod (TLR7 ligand), 189
IL-1, 13, 20, 33, 313, 314, 379, 387 Immature mDCs, 189
gene, 314 Immature pDCs, 179
gene polymorphisms, 314 Immature phenotype (iDC), 12, 13, 16
IL-1RA, 316 Immature thymocytes, 348
IL-1RN, 316 Immediate-type hypersensitivity, 408
Index 579
Immediate-type reactions, 408 Immunity, 4, 125, 195, 200, 276, 313, 378
Immune abnormality, 348 Immunization, 164, 357
Immune aging, 378 Immunization memory cells, 440
Immune assay, 478 Immunoblindness, 162
Immune-based therapies, 304 Immunoblot, 456, 464
Immune cascade, 202 Immunoblotting, 455, 456
Immune cells, 102, 186, 202, 278, 386, 388, Immunocompetent, 165
411, 414, 472 Immunocompetent hosts, 168
anergy, 79 Immunocompromised mice, 106
migration, 82 Immunocompromised patients, 20
Immune checkpoint molecules, 276 Immunocytochemistry (ICC), 460, 461
Immune complex(es), 185 Immunodeciency, 2, 185, 297, 345, 352, 353, 355, 356
Immune contexture, 542 Immunodeciency syndromes, 344
Immune deciencies, 542 Immunodecient, 163, 195, 198
Immune dormancy, 200 Immunodecient host, 168
Immune-driven apoptosis, 431 Immunodominant, 307
Immune dysregulation, 385 Immunoediting process, 153, 195197, 201, 204206,
Immune effector cells, 201 276, 378, 410, 411
Immune effector functions (IEFs), 542 Immunoedition process, 162, 296297
Immune elimination phase, 197 Immunouorescence, 461
Immune environment, 381, 387 Immunogen, 452
Immune equilibrium, 205 Immunogenetics, 298, 303
Immune escape, 188, 201 information, 304
Immune evasion, 425 studies, 311
Immune exhaustion, 384 Immunogenic, 131, 165
Immune insult, 200 Immunogenicity, 105, 163, 198, 200, 203, 411,
Immune-modulating cytokines, 425 415, 437, 439, 472
Immune molecules, 386 Immunogenic mutants, 411
Immune monitoring, 485 Immunogenic peptide, 437
Immune network, 388 Immunogenic regions, 437
Immune polymorphism, 296, 298, 299, 303, 304, 333 Immunogenomics, 303
Immune-privileged, 152 Immunoglobulin, 183, 454
Immune process, 201 engineering, 451
Immune reactions, 281, 306, 411 protein, 451
Immune recognition, 425 superfamily, 412
Immune rejection, 425426 Immunoglobulin (Ig) E, 348, 357, 408, 414, 415
Immune resistance, 123 Immunoglobulin-like transcript factor (ILT)-3+, 179
Immune response-associated genes, 297 Immunoguiding Program (CIP), 476478
Immune responses, 36, 19, 32, 33, 38, 39, 166, Immunohistochemistry (IHC), 460, 461, 472, 492,
168, 182, 189, 196198, 200, 205, 306, 495, 507, 514, 515, 543, 545
378, 384, 385, 387389, 411, 421, 422, Immunoinformatics, 434
432, 434, 437, 439, 445, 452, 454, 471, 472 Immunological parameters, 479
Immune Risk Prole, 384 Immunological synapses, 128
Immune score, 280 Immunologic constant of rejection (ICR), 542
Immune scoring system (Immunoscore), 543 Immunologic disorders, 352
Immune selection, 123, 125 Immunologic reaction, 408
Immune-stimulatory molecules, 437 Immunology, 298, 426
Immune-sufcient, 200 Immunomodulatory, 54, 55
Immune suppression, 205 factors, 188
Immune-suppressive cytokines, 276 NK cell, 382
Immune suppressor, 378 Immunomonitoring, 472, 476, 477
Immune surveillance, 102, 276, 347, 378 Immunopharmacology, 304
Immune synapse, 360 Immunophenotyping, 472
Immune system, 26, 30, 153, 162, 166, 195198, 200, Immunoprecipitation (IP), 454, 455, 464
201, 203, 206, 260, 276, 296, 297, 304, 324, Immunoproteomics, 303
357, 361, 378, 380, 384, 388, 389, 407, 408, Immunoreceptor tyrosine-based activation
410, 411, 422, 423, 426, 434, 451, 452, 468, motif (ITAM), 36
471, 482 Immunoreceptor tyrosine-based inhibition
Immune systemcancer interactions, 304 motif (ITIM), 36
Immune tolerance, 38, 48, 146 Immunoregulation, 63, 65
580 Index
Immunoregulatory, 109 Inltrating leukocytes, 277

Immunoregulatory cytokine, 109, 319 Inammaging, 378, 380, 381, 386, 387, 389
Immunoscore, 543 Inammasome, 379, 386
Immunosenescence, 378, 385, 387, 389 Inammation, 911, 5355, 78, 101, 107, 108, 123,
Immunostaining, 493 125, 145, 152154, 179, 211, 252, 260,
Immunostimulation, 168 262, 276, 277, 297, 312, 313, 318, 324,
Immunostimulatory, 11, 205 346, 357, 378, 379, 381, 387, 410, 413
Immunostimulatory complexes, 185 Inammation-related diseases, 389
Immunosuppressed dogs, 421 Inammatory, 3, 155, 312
Immunosuppressed patients, 93 caspases, 379
Immunosuppressed transplant, 198 cell, 187
Immunosuppressed transplant recipients, 200 cytokines, 11, 80
Immunosuppression, 3, 11, 12, 18, 65, 101, 103, 104, diseases, 383
129, 168170, 184, 188, 202, 203, 426 environment, 379, 412
Immunosuppressive, 5, 65, 67, 131, 385 genes, 262
activity, 110, 187 leukocytes, 408
cell, 104 mediators, 408
cell types, 104 microenvironment, 277, 412
cytokines, 4, 79, 99, 184, 421, 423 processes, 408, 412
environment, 99, 187 reactions, 408, 412, 415
factors, 3, 94, 186, 423 responses, 80, 252, 387, 412
immune cells, 388 sites, 178
mechanisms, 169 Inuenza virus, 182
microenvironment, 99 Inhaled allergens, 409
molecules, 4 Inhaled carcinogens, 412
nature, 188 Inherited immunodeciency, 352
network, 94 Inhibitor 1 (B7x), 5
pDCs, 189 Inhibitor of caspase-activated DNase (ICAD), 213
phenotype, 13, 18 Inhibitors of apoptosis proteins (IAPs), 148, 215,
property, 249 216, 219, 229, 230
treatment, 426 family, 217
Immunosurveillance, 3, 4, 30, 36, 38, 39, 162, 195, family genes, 217
197, 344, 351, 357, 378, 379, 411, 414, Initiation codon (ATG), 440
416, 421, 425, 541, 542 Innate cellular components, 278
Immunotherapeutic agents, 206 Innate immune cells, 130, 280, 386
Immunotherapeutic strategies, 205 Innate immune response, 3, 181, 382, 383, 385, 387, 411
Immunotherapy(ies), 123, 131, 170, 304, 388 Innate immune systems, 29, 30, 378, 379, 381, 411, 423
Immunotherapy strategies, 5 Innate immunity, 177, 183, 186
Immunotoxin, 443 Innate T lymphocyte, 382
Implantation, 127 Inner mitochondrial membrane (IM), 215
IMS. See Intermembrane space (IMS) Inner mitochondrial transmembrane potential (m),
Imunoglobulin, 451 215, 217, 259
Imunosuppression, 188 iNOS. See Inducible nitric oxide synthase (iNOS)
Imunosuppressive microenvironment, 187 InScape system, 461
Indoleamine 2,3-dioxygenase (IDO), 18, 32, 99, Insulin, 457
184, 188, 203, 297, 378, 388 Integration, 432
Inducible costimulator (ICOS; OMIM*604558), Integrin activation, 82
246, 344 Integrin v3, 33
Inducible costimulator ligand (ICOS-L), 184 Interacting proteins, 431
Inducible nitric oxide synthase (iNOS), 79, 386 Intercellular adhesion molecule (ICAM)-1, 106
Infancy, 357 Intercellular adhesion molecule 3 (ICAM-3), 34
Infected keratinocytes, 354 Interferon- (type I IFN), 177
Infection(s), 2, 36, 61, 63, 6567, 72, 80, 81, Interferon gamma (IFN-), 13, 3032, 37, 38, 48, 79,
93, 184, 185, 344, 361, 381 106, 110, 163, 164, 169, 170, 188, 199, 200,
Infection-induced death, 185 202, 204, 205, 257, 323, 330, 381, 382, 389,
Infectious, 2 408, 411, 415, 421
agents, 306 ELISPOT, 478
diseases, 408, 435, 441 gene, 330
mononucleosis, 354 Interferon regulatory-factor 7 (IRF-7), 181, 187
tumor Ags, 306 Interferon regulatory transcription factor (IRF), 29
Index 581
Interferons (IFNs), 3, 33, 37, 100, 136, 185 ITK. See IL-2-inducible T-cell kinase (ITK)
Interferon-stimulated genes (ISGs), 542 ITP, 356
Interleukin-1 (IL-1), 216 iTreg, 441
Interleukin-2 (IL-2), 382, 384 IVD. See In vitro diagnostic (IVD)
interleukin-2 receptor- (IL-2R), 3
Interleukin-4 (IL-4), 11, 15, 31, 199, 316, 385,
408, 415 J
Interleukin-10 (IL-10), 3 Jackals, 420
Interleukin-12 (IL-12), 323
Interleukins, 3
Intermediate stage, 200 K
Intermembrane space (IMS), 216, 217 K63, 256
Interpretation, 430 K63-linked polyubiquitin chains, 256
Intestinal-type, 323 K63-linked ubiquitination, 256
Intestinal type of gastric cancer, 329 Kaposi sarcoma-associated herpesvirus (KSHV), 287
Intestine, 80, 82 Kaposis sarcoma, 80, 131, 308, 360, 434, 497
Intestine metastasis, 127 KARs. See Killer activation receptors (KARs)
Intracellular cytokine staining (ICS), 473, 477, Karyotype, 422, 425
478, 482 Kd, Dd, Ld surface molecules, 163
Intracellular mechanisms, 432 Keratin, 523
Intracellular pathways, 441 Keratinocyte carcinoma, 412
Intracellular staining protocols, 464 Keratinocytes, 353, 494
Intrinsic apoptosis, 211, 213, 217 Keratosis-like lesions, 353
Intrinsic pathway, 220 Ki-67, 522
Intrinsic signaling pathways, 217 Kidney cancer, 12
Intronic SNP (+874 T>A (rs2430561)), 330 Killer activation receptors (KARs), 30
Invasive carcinoma, 515 Killer-cell immunoglobulin-like receptors (KIRs),
Invasive ductal (IDC), 517 36, 304
Invasive margin (IM), 542 Killer inhibitory receptors, 30
Invasive phenotype, 166 Kinases, 432
Invasive tumor cells, 166 KIRs. See Killer-cell immunoglobulin-like receptors
In vitro diagnostic (IVD), 479 (KIRs); Killer inhibitory receptors
In vitro diagnostic (IVD)-certied reagents, 479 KIT ligand, 12
Iobular carcinomas (ILC), 517 Kk molecules, 163
Ionizing radiation, 357 KLRG1, 382, 383
IP. See Immunoprecipitation (IP) KO, 145
IP3/Akt pathway, 386 Kozak, 440
IP-10. See IFN-inducible protein 10 (IP-10) KSHV. See Kaposi sarcoma-associated
Ipilimumab, 436 herpesvirus (KSHV)
Ipilimumab (anti-CTLA-4), 206
IRF. See Interferon regulatory transcription factor (IRF)
IRF-7. See Interferon regulatory-factor 7 (IRF-7) L
IRF7-dependent mechanism, 184 L-1, 15
IRF-mediated inammatory signaling pathways, 33 L1 capsid proteins, 437
Iridocyclitis, 356 L2 capsid proteins, 437
Irradiation, 444 Laboratory of genetics and physiology-2
Irradiation protocols, 444 (LGP2/DHX58), 33
Irreversible defects, 162 Laboratory-specic protocols, 478
Irreversible proteasome inhibitor, 229 LAG-3, 65, 67, 68
ISCs, 154 Laminin superfamily, 212
ISGs. See Interferon-stimulated genes (ISGs) LAMP2, 245
Isoforms, 442, 458 Langerhans cells (LCs), 182
Isolation, 480 Langerin (CD207), 182
Isolation membrane, 244 Langerin+ DC, 12
Isotype switch, 408 LARG, 352
ITAM. See Immunoreceptor tyrosinebased Large cross-talked biochemical networks, 431
activation motif (ITAM) Large granular lymphocytic leukemia, 154
Iterative cycles, 433 Laryngeal cancer, 226
ITIM. See Immunoreceptor tyrosine-based Larynx cancer, 67, 329
inhibition motif (ITIM) Lasers, 461
582 Index
Latent membrane protein-1 (LMP-1), 306, 307 Low-grade inammation, 385389

Latent membrane proteins-2 (LMP-2), 306 Low-light camera-based systems, 455
Late phase, 408 Low-penetrance genetic factors, 296
LC3. See Light chain 3 (LC3) LOX. See Lysyl oxidase (LOX)
LC3 complex, 245 LPDs, 347, 357
LC3-I, 245 LPS. See Lipopolysaccharide (LPS)
LC3-II, 245, 251, 262 LTA-252 A>G, 326
LCL161, 230 LUBAC. See Linear ubiquitin chain assembly
LCs. See Langerhans cells (LCs) complex (LUBAC)
LD. See Linkage disequilibrium (LD) Luciferase gene, 314
LD with non-HLA genes, 307 Luminal cells, 501
Leiomyoma, 498, 513 Luminex, 464, 465
Leiomyosarcoma, 498, 526 Luminex technology, 465
Leishmania infantum, 420 Lung, 2, 67, 127, 326, 347, 357
Let-7a-3 promoter, 289 adenocarcinomas, 83, 136, 289, 507
Leukapheresis, 476 cancer cells, 109
Leukemia, 6, 137, 226, 227, 351353, 357, cancers, 12, 52, 69, 70, 72, 79, 109, 135, 186,
410, 425, 464 219, 285, 287, 290, 316, 321, 410412,
Leukemia cancer, 133 414, 416, 506
Leukemogenesis, 352 carcinomas, 200, 219
Leukocyte function antigen-1 (LFA1), 48 non-neuroendocrine carcinomas, 506
Leukocyte inltrate, 10 tumor progression, 189
Leukocyte recruitment, 130 tumors, 505
Lewis lung carcinoma (3LL), 167 Lupus, 185, 186, 347
Lexatumumab, 220 Ly6C, 12
Light chain, 453 Ly6Chigh, 12
Light chain 3 (LC3), 244, 245, 248, 261, 262 Ly6G, 12
Light microscope, 461 Ly49, 36
LINE, 420 Ly49C, 30
Linear ubiquitin chain assembly complex (LUBAC), 145 Ly49H, 36, 37
Linkage disequilibrium (LD), 299, 300, 311, 312 LY2181308, 230
Lipase, 220 Lymphadenopathy, 148, 355
Lipid mediators, 408 Lymphatic and hematopoietic cancers
Lipid peroxidation, 259 Hodgkins lymphoma, 410
Lipid peroxides, 386 leukemia, 410
Lipoarabinomannan, 182 non-Hodgkins lymphoma, 410
Lipopolysaccharides (LPSs), 51, 100, 182, 216, 380 Lymphatic vessels, 166
Liposarcomas, 533 Lymphedema, 353
Lipoxygenase (LOX), 258 Lymph nodes (LNs), 177, 178, 420
Liver, 357 Lymphoblastic lymphoma, 531
Liver metastasis, 127 Lymphocyte activation gene-3 (LAG-3, CD223), 63
Livin, 219 Lymphocyte-derived growth factors, 3
LKB1, 248 Lymphocytes, 152
LL-37, 185 Lymphocytic cell lines, 441
LMO2, 360 Lymphocytic inltration, 277, 421
LMP-1. See Latent membrane protein-1 (LMP-1) Lymphocytic vasculitis, 355
LMP-2, 164 Lymphoid, 227
LNA anti-miRNAs, 292 aggregates, 279
LNA-modied anti-miRNAs, 287 islets, 277
LNs. See Lymph nodes (LNs) organs, 177, 178, 185
Local immune response, 186 system, 3
Local immune system, 297 Lymphomagenesis, 350, 355
Local inammatory processes, 386 Lymphomas, 1, 219, 226, 344347, 350, 355, 361,
Local maturation, 179 425, 464, 492, 499, 522, 535, 537, 540
Locked nucleic acid (LNA)-modied anti-miRNAs, 287 Lymphoproliferative diseases, 355
Locus-specic regulation, 163 Lymphoproliferative disorders, 350, 522
LOH. See Loss of heterozygosity (LOH) Lymphoreticular origin, 360
Longevity, 386 Lymphoreticular systems, 346
Losing immunogenicity, 297 Lymphosarcoma, 347
Loss of heterozygosity (LOH), 150 Lymphotoxin, 53
Index 583
Lymphotoxin- (LTA), 325 signaling network, 248

Lymphotoxin-, 53 signaling pathway, 248
Lyophilized reagents (lyoplates), 476 Mammary adenocarcinoma, 126
Lysosomal-associated membrane protein 1 (LAMP1), 245 Mammary carcinoma, 79
Lysosomal cleavage machineries, 439 MAMPs. See Microbe-associated molecular
Lysosomal membrane permeability (LMP), 259 pattern (MAMP)
Lysosomal protein, 243 Mannose receptor (CD206), 182
Lysosomes, 243, 439 Mantle cell lymphoma, 228, 229
Lysyl oxidase (LOX), 17 Mapatumumab, 220, 264
MAP kinase phosphatases (MKPs), 257
Maravirok, 136
M Marrow failure syndromes, 352
M1 cells, 276, 279 MART-1. See Melanoma antigen recognized by
M1/M2 classication, 278 T-cells-1 (MART-1)
M2 macrophages, 17, 203, 276, 278, 279 Mass spectrometry, 485
M2-polarized TAMs, 278 Mast cells, 408, 415
m157, 37 mAtg13, 246
mAbs. See Monoclonal antibodies (mAbs) Mathematical equations, 432
Machine algorithms, 481 Mathematical model-based approaches, 431
Macroautophagy, 244 Mathematical models(ing), 430432, 434, 443, 445
Macroenvironment, 388 Matrix metalloproteinase-2 (MMP-2), 16, 17, 133, 278
Macromolecule functions, 386 Matrix metalloproteinase-3 (MMP-3)
Macrophage colony-stimulating factor (M-CSF), metalloproteases, 153
13, 19, 39, 79 Matrix metalloproteinase-7 (MMP-7), 15, 154, 155
Macrophage/granulocyte progenitors, 9 metalloproteases, 153
Macrophages, 1214, 31, 130, 199, 276, 278, 280, Matrix metalloproteinase-9 (MMP-9), 1517, 38,
380382, 385, 386, 388, 415, 420 101, 132, 278
MAGE, 306 metalloproteases, 153
Magnesium, 350 Matrix metalloproteinase-12 (MMP-12), 15
Magnesium transporter 1 (MAGT1) deciency, 350 Matrix metalloproteinase-13 (MMP-13), 16
MAGT1 deciency (OMIM*300715), 350 Matrix metalloproteinase-14 (MMP-14), 16
MAGT1-decient B cells, 350 Matrix metalloproteinases (MMPs), 16, 17, 130, 132, 386
MAGT1-decient T cells, 350 Matrix state (m-state), 259
Maintenance DNMT1, 286 Mature B lymphocytes, 345
Major basic protein, 414 Mature DCs, 177
Major histocompatibility complex (MHC), 36, 161, MC. See Molluscum contagiosum (MC)
162, 164, 184, 202, 304, 311, 346, 421426, MCA-induced tumors, 165, 200
435, 437 MCF-7 cell, 445
antigens (Ags), 421 Mcl-1, 262
class I, 48 MCMV. See Mouse cytomegalovirus (MCMV)
class II, 408 MCRPC. See Metastatic castrate-resistant prostate
Malignancy(ies), 2, 195, 296, 346, 426 cancer (MCRPC)
Malignant, 2, 3, 11, 457 M-CSF. See Macrophage colony-stimulating
cancer, 436 factor (M-CSF)
cell line, 420 mDC. See Myeloid dendritic cell (mDC)
glioma cells, 251, 252 mDC-based vaccines, 189
lymphoma, 347, 354, 443, 444 MDS. See Myelodysplasia (MDS)
masses, 186 MDSCs. See Myeloid-derived suppressor cells (MDSCs)
melanoma cells, 178, 250, 410, 412 Mean uorescence intensity (MFI), 464, 465
rhabdoid tumor, 540 Mechanistic interpretation, 430, 431
transformation, 296, 303, 306307 Mechanistic perspective, 432
Mammalian Atg13 (mAtg13), 244 Mediastinal lymph nodes, 347
Mammalian lethal with SEC13 protein 8 (mLST8), 246 Medulloblastoma, 219, 535, 538
Mammalian stress-activated map kinase-interacting Meduloblastoma/pineoblastoma/PNET, 538
protein 1, 246 Melan-A, 306
Mammalian target of rapamycin (mTOR), 245, 246, Melanocytic and chondrocytic tumors, 530
262, 386 Melanoma, 5, 37, 38, 52, 54, 67, 68, 72, 79, 99101,
inhibitors, 249, 263 109, 111, 127, 133, 135, 136, 166, 187,
kinase, 246, 250 189, 200, 227, 249, 306, 307, 312, 415,
pathway, 248 425, 484, 499, 540
584 Index
Melanoma antigen recognized by T-cells-1 MHC class I chain-related A (MIC-A), 311

(MART-1), 496 MHC class II (MHC-II), 48, 54, 103, 415, 421,
Melanoma inhibitor of apoptosis protein (ML-IAP), 215 423, 424, 439
Melanomas, 496, 497, 531 MHC class IIA, 423
cell lines, 127, 290 MHC class IIB RNA transcripts, 423
system, 130 MHC class I-related stress-inducible surface
Melphalan, 251 glycoprotein A (MICA), 36, 94
Memory B cells (CD27+ IgD), 354 MHC class I-related stress-inducible surface
Memory TEM (CD45RA-CCR7), 383 glycoprotein B (MICB), 36, 94
Meningioma, 410, 413, 414, 536 MHC-decient tumors, 169
Mental retardation, 351 MHC-I-decient phenotype, 163
Mesenchymal chondrosarcoma, 533 MHC-I-decient tumor cells, 169
Mesenchymal stem cell (MSC), 133 MHC molecules, 162, 163, 165
Mesenchymal tumors, 497 MHC-positive tumors, 169
Mesotheliomas, 507 MIATA label, 479
Metabolites, 430, 431 MIB-1 (Ki-67), 497
Metalloproteases, 16, 153 Microarrays, 299, 466
Metalloprotease TACE, 145 data, 478
Metalloproteinases, 102, 131, 205 Microautophagy, 244
Metastases, 127, 130, 132, 166168, 170, 409, 410, Microbead assays, 465
420, 422, 424 Microbe-associated molecular pattern (MAMP), 80, 82
Metastasis, 4, 9, 11, 17, 32, 51, 98, 101103, 126, Microbial elimination, 386
133, 153, 167, 203, 312, 430, 501, 507 Microbial infection, 179
assays, 166, 167 Microbial stimuli/CD40, 178
disease, 167, 169 Microcystic adnexal carcinoma, 348, 495
formation, 388 Microenvironment, 3, 16, 39, 204, 388, 389, 431
Metastasized lesions, 97 Microorganisms, 379
Metastasize/metastasization, 19, 200, 426 MicroRNAs (miRNAs), 285, 286, 288291,
Metastatic, 506 298, 432, 436, 442, 468
Metastatic adrenocortical carcinoma, 249 binding sites, 301
Metastatic breast cancer, 252 promoter, 291
Metastatic capacity, 170 Microsatellite, 330
Metastatic carcinoma, 495 Microsatellite instability (MSI), 50
Metastatic cascade, 166 Microvascular density (MVD), 15, 16
Metastatic castrate-resistant prostate cancer (MCRPC), 136 MIG. See Monokine induced by interferon- (MIG)
Metastatic colonies, 166 Migration, 166, 357
Metastatic colorectal cancer, 200, 225, 249 Migration-stimulating factor (MSF), 17
Metastatic lesion, 99 Milk-fat globule-EGF factor 8 (MFG-E8), 33
Metastatic melanoma, 5, 99 Minimal information about microarray
Metastatic melanomas, 497 experiments (MIAME), 478
Metastatic pancreatic cancer, 225 Minimal information about T-cell assays (MIATA), 479
Metastatic phenotype, 167 Minimal information for cellular assays (MIACA), 478
Metastatic progression, 166, 168 Minimal information on biological and biomedical
Metastatic tumors, 517 investigations (MIBBI), 478
3-Methyladenine (3-MA), 252, 262 Minor histocompatibility Ags, 423
Methylcholanthrene (MCA), 167 MI projects, 478
brosarcom, 164, 415 MiR-9, 287, 288
3-Methylcholanthrene (MCA), 196 MiR-26a, 288
MFI. See Mean uorescence intensity (MFI) MiR-29 family, 286
MGAT1, 350 MiR-29s, 286
MHC. See Major histocompatibility complex (MHC) MiR-31 in melanoma, 290
MHC class I (MHC-I), 36, 64, 103, 161164, 166, 167, MiR-34b/c, 291
169, 199, 202, 421, 424, 425, 439 MiR-101, 288
Ags, 163 MiR-101-mediated suppression, 288
downregulation, 169 MiR-107, 289
epitope, 439 MiR-124a, 289
expressions, 170 MiR-126, 291
heavy chains, 163, 165 MiR-127, 289
molecules, 4 MiR-140, 287
RNA transcripts, 423 MiR-148a, 287
Index 585
MiR-148a/b-152, 287 Monocytes, 12, 13, 130, 131, 278, 351, 380,
MiR-148b, 287 381, 385, 386, 421
MiR-152, 287, 290 stimulation, 385
MiR-155, 290 Monocytic MDSC (M-MDSC), 12
MiR-182, 290 Monocytopenia, 352
MiR-200c/141 CpG, 290 Monofunctional alkylating agent, 250
MiR-200 family members, 289 Monokine induced by interferon- (MIG), 98, 101
miR-205, 442 Mononucleosis, 347
MiR-214, 288 Monophasic salivary gland, 503
MiR-290 cluster, 287 Monophyletic origin, 420
MiR-342, 287, 291 Monosomy 7, 351
MiR-370, 290 Monotherapy, 38
MiR-449a, 287 Mortality, 2
MiR-K12-4-5p, 287 Most polyclonal Ab, 452
miRNA-101, 288 Mother to foetus transmissions, 425
miRNA-200 family, 289 Motifs, 439, 452
miRNAs. See MicroRNAs (miRNAs) Motif XCGY, 439
miRNome, 289, 292 Motility inducing signaling complex (MISC), 153
MISC. See Motility inducing signaling complex (MISC) Mouse cytomegalovirus (MCMV), 37
Missing self hypothesis, 164 mPDCA-1, 179
Mitochondria, 143 MSC. See Mesenchymal stem cell (MSC)
Mitochondrial dGTP, 348 MSF. See Migration-stimulating factor (MSF)
Mitochondrial outer membrane MTLn3, 251
permeabilization (MOMP), 215, 217 mTORC1, 246
Mitochondrial permeability transition (MPT), 259 mTORC2, 246
Mitochondrial permeability transition pore (MPTP), 259 MUC4, 503
Mitochondrion, 259 Mucociliary clearance, 412
Mitogen, 423 Mucoepidermoid carcinoma (MUCI), 502, 503
Mitogen-activated protein kinase (MAPK), 154, 251, Mucosa-associated lymphoid tissue, 409
256, 380, 386 Mucosal addressin cell adhesion
non-apoptotic signals, 150 molecule-1 (MAdCAM-1), 82
Mitosis, 360 Multifocal glioblastoma, 250
Mitozolomide, 250 Multiple myeloma (MM), 101, 137, 227, 229, 251, 252
Mixed glial, 535 Multiplexed ELISA, 459
MK16 (MHC-I-negative), 164 Multiplexing, 452, 465
ML-IAP, 219 Multiplicity of disciplines, 430
MLL, 352 Multi-reagent assays, 474
mLST8, 246 Multi-scale models, 432, 443
M-MDSC. See Monocytic MDSC (M-MDSC) Multi-spot ELISAs, 465
MMP-9. See Matrix metalloproteinase-9 (MMP-9) MuLV-induced AKR tumor, 163
MMPs. See Matrix metalloproteinases (MMPs) Musculoskeletal, 357
Model calibration, 433 Mutagenization, 164
Modeling formalisms, 433 Mutations, 2, 210, 219, 307, 355, 386, 409,
Model-obtained potential drugs, 444 410, 451, 461
Model simulations, 443 Myc/Bcl-2, 219
Molecular assays, 468 MYCN, 287
Molecular mechanisms, 162, 430 Mycobacteria, 182
Molecular mediators, 281 Mycobacterial infection (MonoMAC) syndrome, 352353
Molecular memory, 441 Mycobacterium avium complex (MAC), 352
Molecular oncology, 431, 434 MyD88. See Myeloid differentiation primary response
Molecular signatures, 201 gene 88 (MyD88)
Molluscum contagiosum (MC), 348350 Myelodysplasia (MDS), 226, 351353, 361
infections, 354 Myeloid, 11, 13
Monoclonal Ab, 105, 188 Myeloid cell-inltrating, 10
Monoclonal antibodies (mAbs), 4, 5, 34, 48, 66, 205, Myeloid cells, 911, 15
388, 436, 452, 453, 472, 496 Myeloid cells like myeloid-derived suppressor cells
Monoclonal gammopathy of unknown (MDSCs), 203
signicance (MGUS), 200 Myeloid DCs-based vaccines, 189
Monocyte chemotactic protein-1 (MCP-1), 12, 106 Myeloid dendritic cell (mDC), 18, 64, 177, 178,
Monocyte-derived DCs, 182 180, 181, 183, 184, 189, 381
586 Index
Myeloid-derived suppressor cells (MDSCs), 1113, Neoangiogenesis, 98

15, 16, 1820, 38, 39, 64, 79, 81, 82, 94, Neoplasia, 219
103, 169, 205, 378, 387, 388, 472, 473 Neoplasms, 186, 281, 409, 423
Myeloid differentiation antigen-5 (MDA5), 33 Neoplastic cells, 276
Myeloid differentiation primary response gene 88 Neoplastic process, 201
(MyD88), 181, 379 Neovascularization, 15, 97, 98, 203
signaling, 80 Nephroblastoma, 541
Myeloid leukemias, 135, 352 Nerve sheath tumors, 496, 530
Myeloid recruitment, 12 Netrin-1, 212
Myeloma, 79, 137, 200, 229 Netrins, 212
Myelopoiesis, 351, 360 Network biology, 432
Myelosuppression, 251 Neural cell differentiation, 287
Myoepithelioma/myoepithelial carcinoma, 502, 503, 523 Neuroblastoma, 37, 361, 538, 540
Myobroblastic tumors, 530 Neurocytoma, 535
Myoglobin, 526 Neurodegenerative disease, 388
Myometrial tumors, 516 Neurodegenerative disorders, 346
Neuroendocrine carcinoma, 506, 509
Neuroendocrine tumors (NET), 511, 513
N Neuroepithelial tumors, 534, 536
N2 phenotype, 278 Neurobroma, 536
NADPH oxidases, 257 Neurologic abnormalities, 347
Naive B cells, 384 Neurological symptoms, 226
Nave CD4+ T, 408 Neuronal apoptosis inhibitory protein (NAIP), 215
Nave T cells, 354 Neuronal-glial tumors, 535
Nalp3 inammasome, 386 Neuronal tumors, 534
Nasal mucosa, 409 Neurons, 152
Nasal spray, 413 Neurothekeoma (NTKs), 498
Nasopharyngeal cancer (NPC), 288, 307, 311, Neutralizing monoclonal antibodies (mAbs), 145
323, 324, 501 Neutropenia, 220, 225, 352, 354, 360, 443, 444
Nasopharyngeal carcinoma (NPC), 288 Neutrophils, 12, 15, 351, 352, 379382, 385, 386
Nasopharynx, 326 apoptosis, 354
Natural cytotoxicity, 414 migration, 12
Natural cytotoxicity receptor (NCR), 36 NF-AT, 384
Natural killer (NK) cells, 30, 123, 131, 146, 152, NF-B-mediated inammatory signaling pathways, 33
162164, 169, 183, 205, 304, 306, 349, NHL. See Non-Hodgkin lymphoma (NHL)
355, 381, 382, 389, 411, 414, 421 Niche environment, 204
cytotoxicity, 349 Nickel (Ni) compounds, 290
lymphoma, 154 Nickel sulde (NiS)-transformed human bronchial
mediated lysis, 169 epithelial (16HBE) cells, 290
numbers, 347 Nicotinamide adenine dinucleotide phosphate
response, 423 oxidases (NADPH), 257
Natural killer group two member D (NKG2D), 30 NIH, 227, 230
Natural killer T (NKT) cells, 31, 199, 200, 347, Nijmegen syndrome, 357
349, 355, 360, 382 NiS-induced lung carcinogenesis, 291
Natural regulatory T cells (nTregs), 473 Nitric oxide synthase (iNOS), 12
Navitoclax, 226 Nitrogen, 205
NCBI, 437 4-Nitroquinoline-1-oxide, 356
NCR. See Natural cytotoxicity receptor (NCR) Nitrosamine N-nitrosobenzylmethylamine, 356
ncRNA. See Noncoding RNAs (ncRNAs) NK-cell-mediated cytotoxicity, 347
Neck cancers, 68, 186, 217 NK cell-mediated rejection, 164
Necroptosis, 145, 152, 252, 256, 257, 259263 NKG2D, 30, 36, 38, 94, 103, 109, 350
signaling, 262 NK-mediated destruction, 424
Necroptotic signals, 152 NKp30, 30, 36, 38, 103
Necroptotic stress, 262 NKp44, 36
Necrosis, 98, 252, 257, 259, 324, 431 NKp46, 36
Necrostatin, 152 NKR-P1, 31
Necrostatin-1 (Nec-1), 262 NLCLC, 127
Necrotic cells, 152, 252 NM23, 497
Necrotic tumor, 77 NMC. See NUT midline carcinoma (NMC)
Negative feedback loop, 440 N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 259
Index 587
NO, 37, 378, 388 NVPBEZ235, 250

NO2, 199 NY-ESO-1, 203
NOD-like receptor family pyrin domain
containing 3 (NLRP3), 33
NOD-like receptors (NLRs), 33 O
Nomenclature Committee on Cell Death (NCCD), O2, 199
210, 211, 217, 245 Obatoclax, 226
Non-apoptotic signals, 152 Obatoclax mesylate, 226, 260
Non-bead-based ow cytometry, 465 Oblimersen, 227, 263
Noncardia cancers, 323, 329 Oblimersen Melanoma Study Group, 227
Nonclassical HLA, 307 Oblimersen sodium, 227
Noncoding RNAs (ncRNAs), 285, 291, 431 ODE-based mathematical model, 444
Non-hematological malignancies, 308 ODEs. See Ordinary differential equations (ODEs)
Non-hematopoietic cells, 122 Ofatumumab, 436
Non-HLA genes of class 3, 307, 311 O-glycosyltransferase GaLNT14, 263
Non-Hodgkin lymphoma (NHL), 6, 99, 109, 137, 149, 21-OH hydroxylase, 304
219, 220, 226, 307, 308, 321, 326, 344, 347, Olfactory neuroblastoma (ONB), 499, 500
350, 356, 357, 521 Oligoastrocytoma, 535
Nonimmunological treatments, 388 Oligodendroglioma, 534, 535
Noninvasive papillary carcinoma, 515 Oligonucleotides, 292
Nonlinear behavior, 431 Omics paradigm, 430
Nonlinear posttranscriptional networks, 433 OMIM*607444, 352
Nonlinear properties, 440 ONB. See Olfactory neuroblastoma (ONB)
Nonlinear transcriptional networks, 433 Oncocytic papillary RCC, 514
Nonlymphoid tumors, 357 Oncocytoma, 514
Nonmelanoma skin cancer, 412 Oncogenes, 122, 306, 307, 352, 410
Non-neuroepithelial tumors, 534, 536 growth factors, 410
Non-pathogenic cancer, 434 transcription factors, 410
Nonpathogenic regions, 437 Oncogenesis, 410
Non-resected pancreatic cancer, 413 Oncogenic modications, 125
Non-self, 304 Oncogenic mutations, 412
Non-small cell lung cancer (NSCLC), 69, 217, 249, 286 Oncogenic phenotype, 291
Non-small cell lung carcinoma (NSCLC), 5, 105, Oncomine, 436
106, 219, 220, 225, 226, 230, 251, 252, Oncoproteins, 436
263, 264, 288 Oncostatin M, 15
Non-small lung cancer, 127 One highly polymorphic beta chain (2628 kDa), 305
Nonsteroidal anti-inammatory drugs, 20 OPN. See Osteopontin (OPN)
Nox1 recruitment, 257 Opportunistic infections, 347, 360
NPC. See Nasopharyngeal cancer (NPC) Optical lters, 461
NPI-0052, 229 Optimized Multicolor Immunouorescence
NRAS, 355 Panels (OMIPs), 475
Nrf2, 387 Oral cancer, 135, 344
NSAID, 330 Oral carcinogenesis, 356
NSE, 513 Oral squamous cancer, 133
Nuclear factor of B (NF-B), 80, 107, 145, 153, 154, Oral squamous cell carcinoma, 109, 312
184, 216, 217, 228, 256, 257, 313, 326, 379, Ordinary differential equations (ODEs), 441
381, 384, 386, 387 Organ transplantations, 426
non-apoptotic signals, 150 Oropharynx cancer, 329
pathway, 261 Osteoblastic neoplasms, 533
Nuclear protein in testis (NUT), 499 Osteonectin, 533
Nucleotide-binding domain and leucine-rich-repeat- Osteopontin (OPN), 101, 497
containing proteins (NLRs), 379 Osteoporosis, 385
Nucleotide-binding oligomerization domain (NOD), 260 Osteosarcoma, 39, 198, 533, 540, 541
Nucleotide-binding oligomerization domain-containing Ovarian cancers, 5, 52, 68, 79, 127, 128, 132,
protein 1 (NOD1), 33 186, 251, 280
NUT. See Nuclear protein in testis (NUT) Ovarian carcinoma cells, 178, 251, 415
NUT midline carcinoma (NMC), 499 Ovary cancers, 200
Nutrients, 431 Overall survival, 249, 263, 289
Nutrient starvation, 261 Overexpression, 410
Nutrition, 4 OX40, 13
588 Index
Oxidation, 387 Pasmacytoid monocytes, 178

Oxidatively modied proteins, 386 Patched dependence receptor (Ptc), 212, 219, 262
Oxidative stress, 259, 261, 377, 385388 Pathogen-associated molecular patterns (PAMPs),
32, 177, 178, 379
Pathogen-derived products, 177
P Pathogen-specic immunity, 437
p14 protein deciency, 351 Pathognomonic, 356
p16, 497 Pattern recognition receptors (PRRs), 30, 260, 379
p21-activated kinase (PAK), 213 PBMCs. See Peripheral blood mononuclear cells
p38 MAPK signaling, 380, 383, 384 (PBMCs)
p53, 219, 220, 306, 497, 538 PDGF. See Platelet-derived growth factor (PDGF)
p53 gene, 219 PEA-15, 148
p53 protein, 219 Pediatric neoplasm, 541
p62, 261, 262 Pediatric patients, 250
p62/SQSTM1, 261 Pediatric soft tissue sarcoma, 540
p63, 494, 495, 509 Pediatric tumors, 541
p70 ribosomal S6 kinase (p70S6k), 245, 248 PED/PEA-15, 147
p73, 442 Pepsin, 454
p150, 244 Peptide-based vaccination, 473
Paget disease, 495 Peptides (antigens), 435
Paired box (PAX), 505 Peptide transporters (TAPs), 163
p-Akt, 485 Perforins, 30, 100, 186, 199, 279, 280
Palatal dysfunction, 361 Perianal Paget diseases, 495
PAMPs. See Pathogen-associated molecular patterns Periodontitis, 351
(PAMPs) Peripheral blood, 177
Pancreas, 410 Peripheral blood mononuclear cells (PBMCs), 51, 189,
Pancreatic adenoma, 247 472, 476, 477, 480, 484, 485
Pancreatic cancer cells, 278 Peripheral lymph node addressin (PNAd), 178
Pancreatic cancers, 2, 125, 127, 248, 250, 252, Peripheral neutropenia, 354
279, 312, 413, 415, 416 Peripheral tissues, 177
Pancreatic carcinogenesis, 247 Permeability transition pore complex (PTPC), 215
Pancreatic neoplasms, 511 Pernicious anemia, 344, 356
Pancreatic tumors, 217, 511 Personalized peptide vaccination, 388
Pan-keratin, 492 Peyers patches, 82
Papillary/non-papillary carcinoma, 515 PGD2. See Prostaglandin D2 (PGD2)
Papillary renal cell carcinoma (PRCC), 513514 Ph+ acute lymphoblastic leukemia (ALL), 251
Papillary thyroid cancer, 125 Phage display, 453
Papillary thyroid carcinoma, 12 Phagocytes, 33
Papillomavirus, 80 Phagocytic cells, 351
Papules, 353 Phagocytize tumor cells, 199
Parachordoma, 523 Phagocytosis, 360, 379, 380, 415, 435
Paracrine activation, 202 Phagophore, 244
Paracrine loop, 126 Pharyngeal tumors, 347
Paracrine mediators, 277 Phase contrast microscope, 164
Parabromin, 505 Phase III, 187
Paraneoplastic cerebellar degeneration, 456 Phenotypic responses, 441, 442
Paraneoplastic myoclonus/opsoclonus syndrome, 456 Philadelphia chromosome, 312, 459
Paraneoplastic sensory neuropathy, 456 Phorbol myristate acetate (PMA), 484
Paraneoplastic syndromes, 456 Phosphatase and tensin homologue on
Parasites, 409, 419, 420 chromosome 10 (PTEN), 148, 245, 248
Parathyroid adenomas, 505 Phosphatidylethanolamine (PE), 245
Parathyroid carcinomas, 505 Phosphatidylinositol-3,4,5-triphosphate (PIP3), 245
Parathyroid hormone (PTH), 505 Phosphatidylinositol-3,4-bisphosphate (PIP2), 245
Parathyroid tumors, 505 Phosphatidylinositol 3-kinase (PI3K), 148, 150, 153,
Parental tumor cells, 170 154, 245, 250, 381, 386
Parkinsons disease, 143, 248 Phosphatidylinositol 3-kinase (PI3K) genes, 248
PARP. See Poly (ADP-ribose) polymerase (PARP) Phosphatidylinositol-dependent kinase 1 (PDK1), 245
PARP-1. See Poly ADP-ribose polymerase1 (PARP-1) Phosphatidylserine (PS), 33, 205
PARP-2. See Poly ADP-ribose polymerase2 (PARP-2) Phosphoinositide 3-kinase (PI3K), 148
Pasmacytoid cells, 178 Phospholipase A2 (PLA2), 258, 259
Index 589
Phosphorylated (p)-Erk, 484, 485 PR65, 217

Photomultipliers, 461 Pracrine loops, 432
Photomultiplier tube (PMT), 476 Practical treatments, 426
Phytohemagglutinin (PHA), 484 PRAS40, 246
Phytohemagglutinin-activated mononuclear cells, 326 PRCC. See Papillary renal cell carcinoma (PRCC)
PI3K-Akt, 245, 251 Pre-B-cell, 345
PI3K-Akt-mTOR pathway, 245, 250 Precancerous lesions, 436
Pineal tumors, 534, 535 Precursor of pDCs (Pre-pDCs), 178
Pineoblastoma, 535, 538 Predisposition, 296
PIP2. See Phosphatidylinositol-3,4-bisphosphate (PIP2) Prednisone, 229
PIP3. See Phosphatidylinositol-3,4,5-triphosphate (PIP3) Pregnancy, 425, 426
Pituitary adenomas, 346 Pre-ligand binding assembly domain (PLAD), 144
Pityriasis versicolor-like plaques, 353 pre-miR-101, 288
PLAD. See Pre-ligand binding assembly domain (PLAD) pre-miR-223, 291
Plasma cells, 183, 199, 345, 408, 422, 453, 455 Primary antibodies, 454, 455
Plasma-cytoid DCs (pDCs), 64, 177189, 354 Primary bladder, 291
induced lung tumor progression, 187 Primary immunodeciency (PID) diseases, 304, 344,
Plasmacytoid dendritic cells (pDCs), 180, 381 346, 349, 351, 354, 357, 361
Plastics, 277 Primary immunodeciency syndromes, 3
Platelet-derived growth factor (PDGF), 15 Primary melanoma, 178
Pleckstrin homology (PH), 245 Primary ovarian carcinomas, 517
Pleiotropic cytokine, 316, 319 Primary ovarian tumors, 517
PMA. See Phorbol myristate acetate (PMA) Primary pulmonary adenocarcinomas, 505
PML-RAR-negative PBSC graft, 251 Primary tumors, 164, 167, 170, 186
PMS2 deciency, 357 Primitive neuroectodermal tumors (PNETs), 535,
PNAd. See Peripheral lymph node addressin (PNAd) 538, 540
PNETs. See Primitive neuroectodermal tumors (PNETs) Proangiogenic factors, 130, 132
Pneumatocele, 357 Pro-apoptotic AIF form (57 kDa), 259
p-NF-B, 484, 485 Pro-apoptotic molecules, 225
PNP gene (OMIM*164010), 347 Pro-apoptotic proteins, 215
Poly ADP-ribose polymerase1 (PARP-1), 259, 321 Pro-apoptotic signaling, 256
Poly ADP-ribose polymerase2 (PARP-2), 213, 259 Pro-autophagic cytotoxic drug, 250
Polychromatic analysis, 485 Pro-B-cell, 345
Polychromatic ow cytometry, 472, 473, 475, 479 Pro-caspase-8, 212
Polychromatic panels, 475 Pro-caspase-10, 212
Polyclonal antibody, 452 Procaspases-8, 147
Polycomb repressive complex 2 (PRC2), 288 Professional antigen-presenting cells (APC), 304
Polycomb repressive complex (PRC) genes, 287 Prociency panels, 477, 478, 481
Polygenes, 300 Progesterone receptor (PR), 461
Polygonal cell tumors, 540 Prognosis, 11, 67, 217, 279, 280, 503
Polymerization, 360 Prognostic application, 492
Polymorphic genes, 297, 305, 313 Prognostication, 205
Polymorphic heavy chain of HLA class 1, 305 Prognostic factor, 289
Polymorphisms, 135, 136, 296299, 301, 306, Programmed cell death (PCD), 210
312, 313, 316, 420 Programmed cell death ligand 1 (PD-L1), 5, 6, 50,
Polymorphisms of cytokine genes, 313 6769, 72, 81, 99, 188, 202, 383
Polymorphous low-grade adenocarcinoma, 503 Programmed cell death ligand-2 (PD-L2), 6, 50, 63, 81
Poly (ADP-ribose) polymerase (PARP), 213, 259 Programmed cell death protein 1 (PDCD1/PD1), 5
Polystyrene microspheres, 465 Programmed death-1 (PD-1), 6, 63, 6572, 188, 383, 388
Polyubiquitinated proteins, 252 Programmed necrosis, 252
Position-specic scoring matrix (PSSM)-based Progression, 281
SYFPEITHI, 437 Progression-free survival (PFS), 225, 226, 263
Positive feedback loop, 440, 441 Progressive growth, 421
Post-germinal center (GC) lymphoma, 149 Progressive phase, 421
Posttranslationally modied macromolecules, 387 Proinammatory activity, 386
Post translational modication (PTM), 298, 301, Proinammatory cytokines, 177, 179, 181183,
458, 461, 468 313, 317, 323, 324, 330, 381, 383388
PP2Ac, 262 Pro-inammatory cytokines, 216, 252, 276
PPARc, 441 Proinammatory effects, 145
PR65, 217 Proinammatory factors, 413
590 Index
Proinammatory functions, 184 Q

Proinammatory immune response, 316 Quantitative experimental data, 430
Proinammatory molecules, 386 Quantitative experimental techniques, 430
Proinammatory state, 384, 387 Quiescent state, 379, 381
Prokineticine 2 (PROK2), 12
Proliferation, 130, 360, 377, 386, 473
Promoter SNP, 316 R
Promoter variant in (179 T>G (rs2069707)), 330 Rabaptin-5, 213
Promyelocyte/myelocyte, 351 Rac1, 348
Prooncogenic E6, 353 Rac GTPase, 349
Prooncogenic E7, 353 Radiation, 2, 6, 410
Prophylactic immunization, 2 therapy, 251
Prostaglandin D2 (PGD2), 81 Radioactive isotopes, 451
Prostaglandin E2 (PGE2), 13, 18, 99, 102, 106, Radioactivity, 457, 473
108, 188, 205, 378 Radioimmunoassay (RIA), 457
Prostate cancer (CaP), 2, 52, 53, 127, 133, 287, Radioimmunotherapy, 6
288, 312, 317, 326, 330, 410, 436 Radiolabeled protein, 457
cell lines, 288 Radiotherapy, 72, 251, 388, 444
risk, 329 Raf-Ras-MAP kinase pathway, 384
Prostate carcinoma, 521 RAGE. See Receptor for advanced glycation end
Prostate intraepithelial neoplasia (PIN), 521 products (RAGE)
Prostate-specic acid phosphatase (PSAP), 520 RANTES. See Regulated on activation, normal T cell
Prostate-specic antigen (PSA), 520 expressed and secreted (RANTES)
Prostate-specic membrane antigen (PSMA), 520 Rapamycin, 246, 249, 260, 262
Prostate tumors, 291 analogs, 249
PROSTVAC, 436 derivative (001), 249
Protagonists, 133 Rapamycin-insensitive companion of mTOR (Rictor), 246
Proteases, 10 Raptor, 246
Proteasome, 227, 228 Raptor-independent pathway, 246
Proteasome inhibitors, 228, 229 RAS, 306, 386
Proteinases, 412 RasGTP, 441
Protein coding gene (PCG), 286, 288, 289, 291, 292 Ras homolog enriched in brain (Rheb), 246
Protein G, 454 Ras homolog family member H (RHOH) deciency,
Protein kinase C (PKC), 288, 384 349, 350
Protein-labeling process, 466 RAS mutations, 351
Protein phosphatase 2A (PP2A), 212, 217, 219 RAS oncogene, 285
Proteins, 387 RAS oncogene family (Rab7), 245
Protein-tyrosine phosphorylation, 182, 384 Ras proteins, 440
Proteosomal cleavage motif, 439 Ratio of M1/M2 macrophages, 279
Proto-oncogene growth factor independent 1 (GFI1) RBL2/Rbl2, 287
gene (OMIM*600871), 351 RBL-5 lymphoma (RMA-S), 164
Pro-tumoral effect, 277 Reactivation-induced cell death (RICD), 355
Proximal, 453 Reactive lymph nodes, 178
PRRs. See Pattern recognition receptors (PRRs) Reactive oxygen species (ROS), 11, 12, 15, 32, 37, 131,
PS. See Phosphatidylserine (PS) 205, 215, 228, 257, 259, 384, 386, 412
Psoriasis, 185, 186, 355 Reagents, 485
Psoriatic lesions, 349 Receptor-driven signaling pathway, 385
PTGS2, 15 Receptor for advanced glycation end
PTH. See Parathyroid hormone (PTH) products (RAGE), 77
PTM. See Post translational modication (PTM) Receptor interacting protein (RIP), 213, 252, 257
Pulmonary adenocarcinoma, 507 Receptor-interacting protein kinase 1 (RIP1), 145, 152,
Pulmonary alveolar proteinosis, 352 252, 256, 257, 259, 260, 262
Pulmonary lymphoid granulomatosis, 355 activity, 260
Pulmonary metastasis, 49 dependent necroptosis, 262
Pulmonary obstruction, 412 dependent necroptotic pathway, 262
Pulmonary tumors, 419 polyubiquitination, 260
Purine nucleoside phosphorylase (PNP) deciency, RIP3, 152
347, 348 RIP3 pro-necrotic complex, 256
Purkinje cell, 456 TAG2, 16
pVAX1 vector, 440 ubiquitination, 260
Index 591
Receptor-interacting protein kinase 3 (RIP3), Retinoic acid-inducible gene-I (RIG-I), 33

145, 152, 256, 257 Retinoic acid-inducible gene I (RIG-1)-like
decient cells, 257 receptors (RLR), 260
gene, 257 Retinoic acids, 80
phosphorylation, 256 Retinol (vitamin A), 80
Receptor Tie2, 11 Reverse-phase Ab array, 468
Recombinant GSCF, 443 Reverse T3 hormone, 457
Recombinant immunotoxins, 443 Reversible defects, 162
Recombinant protein, 452 RFLPs. See Restriction fragment length
Recombinant vaccinia virus, 436 polymorphisms (RFLPs)
Recruitment, 123 Rhabdoid tumors, 361, 541
Rectum cancer, 413 Rhabdomyoma, 526
Recurrent sinopulmonary infections, 348 Rhabdomyosarcoma, 499, 526, 538, 540
Reductionist approach, 275 rhApo2L, 220, 225
ReedSternberg cells, 356 Rheumatoid arthritis, 389
Reference samples (RS), 476, 477 Rho GTPase (RHOH), 349
Refractory, 229 Rho GTPases Cdc42, 348
Refractory AML, 230 RHOH gene (OMIM*602037), 349
Refractory colorectal cancer, 220 RhoH/TTF gene, 350
Refractory hematologic malignancies, 250 RHOJ, 348
Regression, 421 RHOQ, 348
Regulated on activation, normal T cell expressed and RIA. See Radioimmunoassay (RIA)
secreted (RANTES), 106 Ri Abs, 456
Regulation, 432 Riboavin kinase (RFK), 257
Regulatory cytokines, 184 Ribonucleic acid (RNA), 430, 432, 476
Regulatory elements, 436 Ribosome, 228
Regulatory loops, 441 RICD. See Reactivation-induced cell death (RICD)
Regulatory motifs, 301 Rictor (PROTOR), 246
Regulatory T cells, 131 Rictor. See Rapamycin-insensitive
Relapse, 200 companion of mTOR (Rictor)
Relapsed, 250 Ridaforolimus, 249, 250, 263
Relapsed AML, 229 Ri immunoblot, 456
Remission, 200 Rintatolimod, 136
Remodeling, 11 RIP3. See Receptor-interacting protein kinase 3 (RIP3)
Renal angiomyolipoma, 249 RIP homotypic interaction motif (RHIMs), 256
Renal anomalies, 361 Rituximab, 99, 220
Renal cancer, 99, 125, 127 RLRs. See Retinoic acid-inducible gene I (RIG-1)-like
Renal carcinomas, 67, 383 receptors (RLR)
Renal cell, 280 RMA-S. See RBL-5 lymphoma (RMA-S)
Renal cell cancer, 99 RNA-induced silencing complex (RISC), 286
Renal cell carcinoma (RCC), 5, 38, 99, 109, 252, RORt, 81, 106
361, 513, 514 ROS. See Reactive oxygen species (ROS)
Renca (renal cell cancer), 51 RTA 203, 252
Repair, 125 RUNX1 gene, 352
Reporter-conjugated specic Abs, 468
Reporter-labeled primary Ab, 458
Reporter molecules, 454 S
Reprogramming, 196 S100, 495, 496, 513
Research use only (RUO), 476 S100A8, 12, 17, 33
grade reagents, 479 S100A9, 12, 17, 18
Residual tumor disease, 169 SAA-3. See Serum amyloid A 3 (SAA-3)
Resistance mechanisms, 229, 248, 249, 251, 430, 445 SAHA, 291
Resistant colon cancer, 287 Salivary duct carcinoma, 502
Respiratory, 354 Salivary gland carcinoma, 501
Respiratory drugs, 411 Salivary glands, 501, 502
Restriction fragment length polymorphisms (RFLPs), 299 Salvage pathway, 262
Retinoblastoma, 198 Sandwich approach, 467468
Retinoblastoma protein (Rb), 213 Sandwich ELISA, 458460, 464
Retinoic acid-inducible gene 1 protein (RIG-1)-like Sandwich microarray, 468
helicases (RLHs), 33, 379 Sarcomas, 424, 517, 531
592 Index
S. aureus, 357 Siglec-H-DTR models, 179

SCC. See Squamous cell carcinoma (SCC) Signaling lymphocytic activation molecule (SLAM), 355
SCF. See Stem cell factor (SCF) Signaling pathways, 441
Schwann cell originator cells, 422 Signal-regulatory protein- (SIRP-), 34
Schwannoma, 536 Signal transducer and activator of
SCID, 347 transcription-1 (STAT1), 213
SCLC. See Small cell lung cancer (SCLC) Signal transducers and activators of
SCN. See Severe congenital neutropenia (SCN) transcription (STATs), 484
SDF-1. See Stromal cell-derived factor 1 (SDF-1) Signature, 465
SDS. See ShwachmanDiamond syndrome (SDS); Silico analysis, 204
Sodium dodecyl sulfate (SDS) Simulation, 432
SDS-PAGE. See Sodium dodecyl sulfate Single array, 467
polyacrylamide gel electrophoresis Single-cell network proling (SCNP), 485
Sebaceous adenoma, 496 Single hybridomas, 452
Sebaceous carcinoma, 496 Single nucleotide polymorphism (SNP), 133, 219,
Sebaceous tumors, 496 297301, 313, 314, 318, 319, 323, 326, 330
Secondary Ab, 455 Sinonasal undifferentiated carcinoma, 499
Secondary antibodies, 455 Sinopulmonary infections, 346, 357
Secondary lymphoid tissues, 178 siRNA. See Small interfering RNA (siRNA)
SED. See Subepithelial cell dome (SED) SIRP-. See Signal-regulatory protein- (SIRP-)
Selective IgA deciency (IgAD), 344, 346 Sjgrens syndrome, 186
Self-antigens, 4, 356 SkBr3 breast cancer cell line, 291
Self-limiting infection, 307 Skeletal disorders, 352
Self MHC-I molecules, 164 Skin, 354, 357
Self-recognition, 436 allergies, 413
Self-sustained oscillations, 433 cancer, 1, 125, 187, 410, 412
Self-tolerance, 65 grafts, 423
Semi-allogeneic pDC vaccine, 189 infections, 345, 357
Semiconductor nanocrystals, 485 metastasis, 127
Seminoma, 521 tumors, 186
Senescent mechanisms, 127 SLAM-associated protein (SAP), 355
Sensory neuropathy, 229 SLAMSAP, 355
Sentinel lymph node metastasis, 280 SLE. See Systemic lupus erythematosus (SLE)
Sepsis, 443 SMA, 498
Sequence-specic primers (SSP), 308 Smac, 230
Sequence-specic probes (SSO), 308 Smac/Diablo, 148, 215, 216, 229, 230
Sequestosome-1 (SQSTM1), 261 Smac/Diablo-N7, 230
Serologic typing, 311 Small blue round cell tumors, 533
Serotype, 311 Small cell carcinomas, 499
Serum amyloid A 3 (SAA-3), 17 Small cell lung cancer (SCLC), 226, 227, 456
Severe atopy, 348 Small cell melanoma, 496
Severe congenital neutropenia (SCN), 351, 352 Small cell neuroendocrine carcinoma, 499
Sex cord stromal tumors, 521 Small cell osteosarcoma, 540
sFASL, 355(INITIAL SMALL CASE) Small cell squamous carcinoma (SSCC), 501
S-Glutathionylation, 151 Small interfering RNA (siRNA), 3, 245, 252
SH2D1A, 355 Small-molecule kinase suppressors, 5
Shannon entropy, 437 Small ncRNAs, 285
Sheath tumors, 535 Small round cell tumors, 538, 540, 541
Sheep, 419 SMM. See Stabilized matrix method (SMM)
Shingles, 350 Smoking, 410
SHM. See Somatic hypermutation (SHM) SNP. See Single nucleotide polymorphism (SNP)
SHP, 36 Sodium dodecyl sulfate (SDS), 455
SHP-1, 36 Sodium dodecyl sulfate polyacrylamide gel
ShwachmanBodianDiamond syndrome (SBDS) electrophoresis (SDS-PAGE), 455
gene, 352 Soft tissue neoplasm, 422
ShwachmanDiamond syndrome (SDS), 352 Soft tissue sarcomas, 225, 249, 522
Side-by-side analysis, 468 Soft tissue tumor, 497, 522
Side-scattered light (SSC), 472 Solar systems, 430
Siglec-H, 184, 188 Solid cancers, 290
Siglec-H+, 179 Solid malignancies, 220, 344
Index 593
Solid pediatric tumors, 538 Streptavidin, 454, 461

Solid pseudopapillary neoplasm, 511 Streptavidin conjugation, 461
Solid tumors, 9, 10, 13, 93, 99, 104, 122, 186, 220, Stroma, 122, 123, 205
226, 227, 229, 250, 276, 279, 281, 443 Stromal and cancer cells
Soluble allergens, 408 bFGF, 15
Soluble factors, 474 EGF, 15
Soluble proteins, 464 PDGF, 15
Somatic cells, 409 TGF-, 15
Somatic evolution, 297 VEGF, 15
Somatic hypermutation (SHM), 307 Stromal cell-derived factor 1 (SDF-1), 18, 179, 188, 354
Somatic mutations, 356 Stromal cells, 12, 122, 131
Sonic hedgehog (Shh), 212 Stromal compartment, 279
SOPs. See Standard operating procedures (SOPs) Stromal tissue, 277
Sorafenib treatment, 248, 249 Subchronic inammation, 412
SOS proteins, 441 Subepithelial cell dome (SED), 82
Spatial interactions, 432 Subpopulations, 421
Spatial organization, 431 Sunitinib, 249
Spatial scales, 430 Surface antigens, 439, 440
Spatiotemporal regulatory features, 433 Surface marker expression, 462
Spatiotemporal scales, 432 Survival, 186, 357, 472
Spindle cell tumor, 540 Susceptibility
Spleen, 12 loci, 299
Splenomegaly, 148, 355 variants, 300
Splice sites, 301 Sustained oscillations, 440
Splicing variants, 436 Synergistic agents, 226
Spontaneous metastases, 170 Synergistic radiation, 226
Sporadic transmission, 346 Synthetic imidazoquinolines, 181
26S proteasome, 227 Systemic circulation, 166
Squamous cell carcinoma (SCC), 127, 329, 348, 353, Systemic lupus erythematosus (SLE), 153, 185, 356
356, 361, 410, 494, 500, 509, 511, 516, 543 Systemic reaction, 409
Squamous cell carcinoma of head and Systems biology, 430, 432, 434, 440, 441, 443, 444
neck (SCCHN), 226
Squamous lung cancer, 346
Src family kinases, 182, 345 T
Src homology 2 domain-containing gene 1A t(9;22), 312(INITIAL SMALL CASE)
(SH2D1A; OMIM*300490), 355 T10 sarcoma, 167
Src kinase, 153 TAAs. See Tumor-associated antigens (TAAs)
19S RP, 228 TADC. See Tumor-associated dendritic cells (TADC)
SSCC. See Small cell squamous carcinoma (SSCC) TAG-72 (CA72.4), 495
SSO. See Sequence-specic probes (SSO) tagSNPs, 299, 300(INITIAL SMALL CASE)
SSP. See Sequence-specic primers (SSP) TAK1. See TGF--activated kinase 1 (TAK1)
Stabilized matrix method (SMM), 437 TAK-1-binding protein 2/3 (TAK1/TAB2/3)
Stable growth, 421 complex. TAK1, 256
Standard operating procedures (SOPs), 474, 477, 480 TAL1, 352
Staphylococcal infections, 357 T allele, 314, 318, 319, 329, 330
Start codons, 439 TAMC. See Tumor-associated myeloid cells (TAMC)
Starvation, 261 Tamoxifen therapy, 248, 249
STAT, 357 TAM recruitment, 130
STAT3, 53, 80, 81, 100, 357 TAMs. See Tumor-associated macrophages (TAMs)
deciency, 357 TANs. See Tumor-associated neutrophils (TANs)
function, 346 TAP. See Transporter associated with
STAT3 (OMIM*102582), 357 Ag presentation (TAP)
STAT5, 81 TAP-1, 164
Statistical expression patterns, 431 TAP-1 adenovirus vector, 163
Statistical models, 431, 432 TAP-1-negative, 163
Statistics biology, 432 TAP-1-positive, 163
Steatorrhea, 352 TAP-2 gene, 164
Stem cell factor (SCF), 12 Tapasin, 163
Stomach, 67 TApDCs. See Tumor-associated pDCs (TApDCs)
Stop codons, 439 TAP-decient RMA-S cells (H-2 class I negative), 164
594 Index
TAPs. See Peptide transporters (TAPs) TGF-s type II receptor (DNR), 3

Targeted anti-angiogenesis agent, 220 Th. See T-helper (Th) cells
TAS. See Trait-associated SNP (TAS) Th1. See T helper cell type 1 (Th1)
TAs. See Tumor antigens (TAs) Th1/Th2 balance, 408
TAS block, 301 Th2. See T helper cell type 2 (Th2)
Tasmanian devils, 422, 423 Th2-like environment, 188
tBID. See Truncated BID (tBID) Th17 cells, 78, 107, 357, 441
TC-1 (MHC-I-positive), 164 Th17-related cytokines, 356
T-cell(s), 179, 184, 262, 306, 345, 355, 382384, T-helper (Th) cells, 304, 345
386, 437 T helper cell type 1 (Th1), 178, 181, 408, 441
anergy, 6365, 72 bias, 408
CD80/CD86, 380 cell apoptosis, 188
exhaustion, 63, 6568, 72 cytokines, 381, 382
leukemia, 434 IFN-, 408
leukemia-lymphoma, 137, 348 IL-12, 408
lymphoma, 99 T helper cell type 2 (Th2), 188, 408, 409, 414, 415, 441
T-cell immunoglobulin-mucin domain cytokines, 415
protein-4 (TIM-4), 33 lymphocytes, 131
T-cell receptor (TCR), 347, 353, 382384, 386, 473, 477 Therapeutic cancer vaccines, 436
restimulation, 355 Therapeutic vaccines, 436
stimulation, 349 Therapy-mediated apoptosis, 431
T-cell receptor-induced necroptosis, 262 3D structure modeling, 437
T-cell-type lymphomas, 537 Thrombocytopenia, 225, 229, 360
TCR. See T-cell receptor (TCR) Thymic stromal lymphopoietin (TSLP), 13
TCR /+ B220+CD4+CD8+ double-negative T Thymoma tumor cell line (EL4), 130
(DNT) cells, 355 Thyroid, 505
TCR /+ DNT cells, 356 cancer cells, 127
T cytotoxic lymphocytes (CTLs), 162 hormone tests, 457
TDLN. See Tumor-draining lymph node (TDLN) medullary carcinoma, 505
Tec family tyrosine kinases, 347 nodules, 457
T effector memory cell, 383 Thyroid-stimulating hormone (TSH), 457
Telomere shortening, 377 Tie2, 11
TEM. See Tie2-expressing monocytes (TEM) Tie2+, 11
Temozolomide, 227, 248, 250, 251 Tie2-expressing monocytes (TEM), 1113, 15, 16
Temporal scales, 430 Tie2 monocyte, 15
TEMRA (CD45RA+ CCR7), 383 TIL-Bs. See Tumor-inltrating B cells (TIL-Bs)
TEMRA cells. See Terminally differentiated effector TIL prognosis, 280
memory (TEMRA) cells TILs. See Tumor-inltrating lymphocytes (TILs)
Temsirolimus (CCI-779), 249 TIM-3, 63, 6568, 7072
Terminally differentiated effector Time-of-ight mass spectrometry, 485
memory (TEMRA) cells, 386, 473 Time to progression (TTP), 249
Termination codon Tissue homeostasis, 125, 210
TAA, 440 Tissue invasion, 386
TAG, 440 Tissue microenvironment, 166
TGA, 440 Tissue remodeling, 10, 123, 130
Tertiary lymphoid structures (TLS), 542 TL32711, 230
Tertiary lymphoid tissue (TLT), 279 TLR. See Toll-like receptor (TLR)
Testicular carcinoma, 360 TLR1, 380
Testicular tumors, 521 TLR1/TLR2, 380
Tetralogy of Fallot, 361 TLR2, 77, 182, 379, 380
Tetrapeptide, 229 TLR3, 20, 30, 379
TFBSs. See Transcription factor binding sites (TFBSs) TLR4, 32, 77, 182, 379, 380
T-Follicular Cells Help (TFH), 277 TLR7, 180, 181, 185187, 189, 379, 381
TGF. See Transforming growth factor (TGF) TLR7/TLR9, 185
TGF-, 187, 188 TLR8, 346
TGF-. See Transforming growth factor- (TGF-) TLR9, 180, 181, 184187, 189, 346, 381
TGF-1, 421425 TLS. See Tertiary lymphoid structures (TLS)
TGF-1 gene, 331, 333 T-lymphocytes, 128, 146, 152, 280, 345, 348, 383
TGF--activated kinase 1 (TAK1), 256, 260 T lymphocytes helper 17 (Th17), 277
TGF- receptor type 2 (TGF--R2), 18 T lymphopenia, 354
Index 595
TME. See Tumor microenvironment (TME) Transcription factor binding sites (TFBSs), 298, 301
TNF. See Tumor necrosis factor (TNF) Transcription factor nuclear factor (TNF)-B, 412
TNF-. See Tumor necrosis factor-alpha (TNF-) Transcription factors, 301, 313, 357, 379, 386,
TNF-induced necroptosis, 256, 257, 262 432, 436, 461
TNF-induced necrosis, 259 Transforming growth factor (TGF), 256, 412
TNF-induced necrotic cell death, 256 Transforming growth factor- (TGF-), 3, 12, 13, 16,
TNF-induced necrotic death, 258 1820, 30, 38, 63, 101106, 108, 109,
TNF- receptor 1 (TNFR1), 145, 152, 212, 252 187189, 197, 202, 205, 330, 331, 388
TNF-resistant cells, 259 Transitional (urothelial) cell carcinoma, 514
TNF gene, 217 Translational medicine, 443
TNF-induced apoptosis, 256 Transmembrane activator, 346
TNF-induced necroptosis, 257 Transmissible cancers, 424, 425
TNF inhibitors, 145 Transmissible tumors, 426
TNF ligand superfamily member 10 (TNFSF10), 212 Transmission electron microscopy (TEM), 245
TNF-R. See Tumor necrosis factor receptor (TNF-R) Transplantable tumor model systems (GRAFT), 166
TNFR1. See TNF- receptor 1 (TNFR1) Transplant-transmitted cancers, 426
TNFR1 death receptor, 144 Transporter associated with Ag presentation (TAP),
TNFR2 receptor, 144, 252 307, 311, 423
TNF-receptor-associated death domain (TRADD), 252 T regulatory (Treg), 187, 279, 280, 378, 387
TNF-receptor-associated factor 2/5 (TRAF2/5), 252 Trichilemmal carcinoma, 495
TNF-related apoptosis-inducing ligand (TRAIL), 69, Trichostatin A, 169, 290
181, 186, 187, 199, 212, 216, 220, 225 TRIF, 379
TNM-Immune (TNM-I), 543 Trisomy 21, 351
TNM staging, 280 Trophoblasts, 426
Tolerance, 63, 297, 356 Truncated AIF (tAIF), 259
Tolerogenic cells, 131 Truncated BID (tBID), 148, 215, 259
Tolerogenic environments, 77, 184 Tryptophan, 184, 203
Tolerogenic factors, 188 TSA (sarcoma), 51
Tolerogenic responses, 178 TSC1/2, 248
Toll-like receptor (TLR), 6, 32, 33, 54, 80, 100, TSGs. See Tumor suppressor genes (TSGs)
180182, 189, 260, 379381, 386, 387, 485 TSH. See Thyroid-stimulating hormone (TSH)
agonists, 179 TSH-secreting pituitary adenomas, 457
pathway, 276 TSLP. See Thymic stromal lymphopoietin (TSLP)
Topoisomerase I, 213 TT genotype, 330
Topology, 432 t(8;21) translocation, 291
Total body irradiation (TBI), 50 Tuberous sclerosis complex (TSC), 246
Toxicity, 443 TUCAN, 212
Toxic proteins, 408 Tumor, 39, 434
TP53 mutation, 125 Tumoral angiogenesis, 202
TRADD. See Tumor necrosis factor (TNF) receptor Tumor antigens (TAs), 203, 205
1-associated death domain protein (TRADD) Tumor-associated antigens (TAAs), 162, 169, 276,
TRADD-FADD, 256 306, 307, 435, 436
TRAF1, 217 Tumor-associated dendritic cells (TADC), 1113, 18, 20
TRAF2, 145, 217, 256, 257 Tumor-associated macrophages (TAMs), 1115,
TRAF2/5, 252 1720, 64, 98, 103, 108, 123, 130, 131,
TRAF-6-mediated NF-B and 137, 276278, 280, 412
MAP-kinases (MAPKs), 181 Tumor-associated myeloid cells (TAMC), 912,
TRAF family genes, 217 14, 1719
TRAIL. See TNF-related apoptosis-inducing ligand Tumor-associated neutrophils (TANs), 1116, 20, 278
(TRAIL) Tumor-associated pDCs (TApDCs), 186, 187, 189
TRAIL (Apo2 ligand), 220 Tumor-associated proteins, 482
TRAIL-R. See TRAIL receptor (TRAIL-R) Tumor biology, 163, 431, 432
TRAIL-R1, 212, 217, 220, 225, 264 Tumor cell(s), 163, 167, 407, 409, 410
TRAIL-R2, 212, 217, 220, 225 invasion, 133
TRAIL receptor (TRAIL-R), 225, 252 proliferation, 188, 189
TRAIL-resistant cells, 230 Tumor center (CT), 542
TRAIL-sensitive tumors, 225 Tumor-derived PGE2, 188
Trait-associated SNP (TAS), 299301 Tumor-draining lymph node (TDLN), 4851, 54
Transcriptional pathways, 441 Tumor-editing, 123, 128, 196
Transcriptional targets, 442 Tumor elimination, 388
596 Index
Tumor environment, 81, 131, 188 Tumor suppressor genes (TSGs), 165, 248, 286288,
Tumor growth, 126, 162, 187, 281, 408, 415, 442, 445, 451 306, 344, 352, 410
Tumor growth inhibition, 250 Tumor surveillance, 357
Tumorigenesis, 30, 80, 104, 105, 130, 211, 212, 386, 387 Tumor survival, 130
Tumorigenic, 164 Tumor vaccines, 306
capacity, 165 Two-dimensional scatter plots, 480
potential, 357 Type 1 diabetes, 356
Tumorigenicity, 163 Type 2 diabetes, 388
Tumor growth, 166 Type I hypersensitivity, 408
Tumor-immune evasion, 188 Type II inammation, 13
Tumor-immune interactions, 388 Type I interferon, 32, 178, 181183, 185189, 381
Tumor immune microenvironment, 186 Type I interferon production, 187
Tumor-immune system interaction, 434 Type I NKT, 200
Tumor immunity, 313 Typical allergic symptoms, 408
Tumor immunology, 434 Tyrosinase, 306
Tumor-inltrating B cells (TIL-Bs), 52, 53, 55 Tyrosine kinase signaling family, 444
Tumor-inltrating lymphocytes (TILs), 64, 68, 69,
128, 129, 200, 279, 421, 542
Tumor inltrating myeloid cells, 10 U
Tumor invasion, 126, 131 U1 small nuclear ribonucleoprotein (U1snRNP), 213
Tumor like growth, 431 Ubiquitinated proteins, 227, 261
Tumor mass, 186 Ubiquitin C-terminal hydrolases (UCH), 228
Tumor microenvironment (TME), 5, 6, 9, 1113, 17, Ubiquitin-like conjugation systems, 244
18, 29, 30, 32, 33, 39, 61, 64, 67, 72, 77, Ubiquitin-like protein Atg12, 244
7981, 85, 86, 94, 95, 97, 101, 104109, Ubiquitin-like systems, 244
123, 129, 187, 188, 202205, 251, 278, Ubiquitin-proteasome pathway (UPP), 227, 228
383, 388, 432, 541, 542 Ubiquitin-proteasome system (UPS), 243
Tumor milieu, 198 Ubiquitin-protein ligase (E3)-like enzyme, 245
Tumor necrosis factor (TNF), 3, 144, 145, 199, 385 Ubiquitin-specic proteases (USP), 228
Tumor necrosis factor-alpha (TNF-), 15, 16, 33, 38, 110, UCHL5, 228
131, 181, 197, 199, 216, 252, 304, 307, 311, Ulceration, 351
312, 324, 325, 329, 380, 381, 383385, 387 ULK1, 244, 246
antagonists, 145 ULK-Atg13-FIP200, 246
polymorphisms, 326 ULKs. See Unc-51-like kinases (ULKs)
Tumor necrosis factor receptor (TNF-R), 144 Ultraviolet radiation resistance-associated
Tumor necrosis factor (TNF) receptor 1-associated death gene (UVRAG), 244, 245, 261
domain protein (TRADD), 144, 145 Umiquimod-treated cancers, 184
Tumor necrosis factor (TNF) receptor superfamily, UNC-5, 212
212, 344 UNC-5B/DAPK1, 212
13B (TNFRSF13B or TACI; OMIM*604907), 344 UNC-5H2, 212
13C (TNFRSF13C or BAFF-R; OMIM*606269), 344 UNC-5 homolog family receptors, 212
CD19 (OMIM*107265), 344 UNC-5A, 212
CD20 (OMIM*112210), 344 UNC-5B, 212
CD21 (CR2;OMIM*120650), 344345 UNC-5C, 212
CD81 (OMIM*186845), 344 UNC-5D, 212
LRBA (OMIM*606453), 345 Unc-51-like kinases (ULKs), 244, 246
Tumor necrosis factor-related apoptosis-inducing Undifferentiated carcinoma, 496, 499
ligand (TRAIL), 50, 66 Undifferentiated nasopharyngeal carcinoma, 499
Tumor-node-metastasis (TNM), 280 Undifferentiated round-cell neoplasm, 420
Tumor parenchyma, 130, 132 UniProt, 437
Tumor progression, 128, 144, 186, 187, 279, 330, unique long 16 binding proteins (ULBPs), 36
410, 412, 415 Unique polymorphism, 301
Tumor regression, 169, 186189, 421, 425 3-Untranslated region (3-UTR), 285, 287
Tumor shrinking, 451 uPA. See Urokinase plasminogen activator (uPA)
Tumor-specic immune response, 383 Upper aerodigestive tract (UADT) cancer, 329
Tumor stroma, 278 Urokinase plasminogen activator (uPA), 12
Tumor suppressor, 102, 219, 307, 378 Urothelial carcinoma, 514, 515
mechanisms, 198 USP14, 228
protein, 244 Uterine cervical carcinoma, 80
Index 597
Uterine endometrium, 326 W

Uterine tumors, 516 Waldenstrom macroglobulinemia (WM), 288
Uveitis, 356 Warts, hypogammaglobulinemia, infections, and
myelokathexis (WHIM) syndrome, 354
WAS. See WiskottAldrich syndrome (WAS)
V WAS gene (OMIM*300392), 351, 360
Vaccinations, 4, 50, 68, 95, 136, 473, 478, 484 WASP. See WiskottAldrich syndrome protein (WASP)
Vaccines, 5, 34, 97, 105, 169, 434, 437, 472 Western-based tests, 456
Vaccinia virus, 436 Western blotting, 455, 456, 462
Vaporization, 485 WGS. See Whole genome sequencing (WGS)
Variable myelodysplasia, 360 Wheezing, 409
Variable number tandem repeat (VNTR), 297, 316 WHIM syndrome. See Warts, hypogammaglobulinemia,
Varicella zoster (VZ) viruses, 348 infections, and myelokathexis (WHIM)
Vascular abnormalities, 357 syndrome
Vascular endothelial growth factor (VEGF), 12, 13, Whole blood, 472
1517, 20, 37, 98, 101, 104106, 132, 189, Whole genome sequencing (WGS), 311
202, 203, 205, 288, 388, 414 Wildlife species, 424
Vascular endothelial growth factor-A (VEGF-A), 38 Wild-type (WT), 459
Vascularization, 39, 130, 200 Wild vertebrate species, 424
Vascular protein sorting 34 (Vps34), 245 Wilms tumor (WT), 540, 541
Vasculitis, 348, 356 WiskottAldrich syndrome (WAS), 351, 360
VCAM-1, 178 WiskottAldrich syndrome protein (WASP), 348, 360
VEGF. See Vascular endothelial growth Wnt--catenin, 346
factor (VEGF) Wolves, 420, 424
VEGF, 132
VEGF-A. See Vascular endothelial growth
factor-A (VEGF-A) X
VEGF-induced angiogenesis, 250 X-IAP. See X-linked inhibitor of apoptosis protein
VEGF-induced HUVEC cell proliferation, 250 (X-IAP)
VEGFR1, 17 XIAP (OMIM*300079), 355
VEGFR2, 16 XLA. See X-linked agammaglobulinemia (XLA)
Velcade, 228 X-linked agammaglobulinemia (XLA), 345, 346
Venipuncture, 480 X-linked immunodeciency, 350, 360
Vimentin, 498, 522, 541 X-linked inhibitor of apoptosis protein (X-IAP), 148,
VINIII. See Vulvar intraepithelial neoplasia 213, 215, 217, 230, 260, 355
grade III (VINIII) X-linked lymphoproliferative disease (XLP), 346347,
Viral diseases, 186 354, 355
Viral-induced diminished TAP function, 307 X-linked neutropenia (XLN), 360
Viral infections, 5, 183, 184, 186, 188, 345, 347, X-linked recessive, 353
348, 350, 353 X-linked SCN, 351
Virus, 2, 410 X-linked thrombocytopenia (XLT), 360
Virus-infected cells, 260 XLN. See X-linked neutropenia (XLN)
Visceral adiposity, 385 XLP. See X-linked lymphoproliferative disease (XLP)
Visceral metastasis, 166 XLP-like disorder, 355
Vitamin B12, 355 XLT. See X-linked thrombocytopenia (XLT)
Vitiligo, 356 XMEN, 350, 351
V(D)J, 356
VNTR. See Variable number tandem repeat (VNTR)
Voltage-dependent anion channel (VDAC), 259 Y
Vomiting, 409 YM155, 230
Von Hippel-Lindau tumor suppressor, 125 Yo, 456
Von Willebrand factor (vWF), 530, 531
Vorinostat, 250
Vps34. See Vascular protein sorting 34 (Vps34) Z
V-set domain-containing T cell, 5 ZAP70, 349, 353
Vulva, 357 Zinc, 4, 353, 354
Vulvar intraepithelial neoplasia grade III (VINIII), 5 Zinc transporter, 353
vWF. See Von Willebrand factor (vWF) ZnT-1, 353
VZ viruses. See Varicella zoster (VZ) viruses Z-VAD-FMK, 259

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Nima Rezaei

ISBN 978-3-662-44005-6 ISBN 978-3-662-44006-3 (eBook)

Library of Congress Control Number: 2014952677

Springer-Verlag Berlin Heidelberg 2015

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Several empirical observations suggested a long time ago that established

Cancer Immunology: Cancer Immunotherapy for Organ-Specic Tumors,

Nima Rezaei, MD, PhD

I would like to express my gratitude to the technical editor of this book,

Nima Rezaei, MD, PhD

1 Introduction on Cancer Immunology

2 Inammatory and Innate Immune Cells in Cancer

2.3.3 Cancer Cell Dissemination . . . . . . . . . . . . . . . . . . . . . 16

3 Role of Innate Immunity in Cancers and

4 B Cells in Cancer Immunology: For or Against

5 The Role of Exhaustion in Tumor-Induced T Cell

6 Regulatory T Cells and Th17 Cells in Cancer

7 Role of Cytokines in Tumor Immunity and Immune

8 Role of Chemokines and Chemokine Receptors in Cancer . . . 121

9 The CD95/CD95L Signaling Pathway: A Role

10 MHC Class I Molecules and Cancer Progression:

11 Role of Plasmacytoid Dendritic Cells in Cancer . . . . . . . . . . . . 177

12 Cancer Immunoediting: Immunosurveillance,

13 Apoptosis and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

14 Autophagy and Necroptosis in Cancer. . . . . . . . . . . . . . . . . . . . 243

14.5 Necroptosis and Possible Therapeutic

15 Prognostic Value of Innate and Adaptive Immunity

16 Epigenetics and microRNAs in Cancer . . . . . . . . . . . . . . . . . . . 285

17 Immunogenetics of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

17.6.4 HLA Typing and HLA Association Studies:

18 Primary Immunodeciencies and Cancers . . . . . . . . . . . . . . . . 343

18.8 Other Well-Dened Immunodeciencies . . . . . . . . . . . . . . . . 356

19 Immunosenescence, Oxidative Stress, and Cancers . . . . . . . . . 377

20 Nutrition, Immunity, and Cancers . . . . . . . . . . . . . . . . . . . . . . . 395

20.5.4 Nucleotides, Long-Chain Omega-3 Polyunsaturated

21 Allergies and Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

22 Cancer Immunology of Transmissible Cancers . . . . . . . . . . . . 419

23 Envisioning the Application of Systems Biology

24 Principles of Immunological Diagnostic Tests for Cancers . . . 451

25 Flow Cytometry in Cancer Immunotherapy:

26 Immunohistochemistry of Cancers . . . . . . . . . . . . . . . . . . . . . . 491

26.5.4 Small Intestine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

Seyed Hossein Aalaei-Andabili, MD Thoracic and Cardiovascular

Katherine Belov, BSc, PhD Faculty of Veterinary Science,

Hopital Piti Salptrire, Universits, UPMC Univ Paris 06, CR7,

Shailendra K. Gupta, PhD Department of Systems Biology and

Patrick Legembre, PhD Universit Rennes-1, Rennes, France

Christian Ottensmeier, MD, PhD, FRCP Faculty of Medicine,

Susana Romero-Garcia, PhD Departamento de Enfermedades

Xiao-Lian Zhang, PhD State Key Laboratory of Virology,

3-UTR 3-untranslated region

APECED Autoimmune polyendocrinopathy with candidiasis and ecto-

CHL Classic Hodgkin lymphoma

DC-SIGN Dendritic cell-specic ICAM-3 grabbing non-integrin

FasL Fas ligand

HEV High endothelial venules

MAMP Microbe-associated molecular pattern

NAIP Neuronal apoptosis inhibitory protein

PCG Protein coding gene

RHIM RIP homotypic interaction motif

TLR Toll-like receptor

Contents 1.5 Genetic and Environmental