Surg Oncol Clin N Am

14 (2005) 69–84

Beyond lymph node staging: molecular

predictors of outcome in breast cancer
David G. Sheldon, MD*
Section of Surgical Oncology, Geisinger Health System, 100 North Academy Avenue,
MC 21–70, Danville, PA 17822-2170, USA

How do the characteristics of a breast tumor define the outcome for the
disease? Clinicians have a wide array of prognostic and predictive factors at
their disposal that provide useful information concerning outcomes in breast
cancer. Prognostic factors are patient or tumor characteristics that are used
to estimate outcomes for the disease, independent of treatment variability.
They can be used to select appropriate patients for therapy. Well-recognized
prognostic factors in breast cancer include tumor size, grade, and the
presence or absence of lymph node metastases. These variables have been
incorporated into a widely used, validated scheme—the Nottingham
Prognostic Index [1,2].
Predictive factors are clinical, pathologic, or biologic features of the
tumor that are used to estimate a response to a treatment, such as systemic
chemotherapy or endocrine therapy [3,4]. There has been an increased
emphasis on identifying and validating predictive factors for outcomes in
breast cancer as more sophisticated molecular techniques define tumor
characteristics. Predictive factors, based on an individual molecular analysis
of the tumor, can be used to tailor therapy. We are on the threshold of
routinely implementing targeted therapies with ‘‘designer’’ regimens based
on the genetic signatures of an individual tumor.
Recent guidelines for adjuvant therapy of breast cancer result in treatment
of greater than more than 90% of patients, despite benefit in less than 10% of
all patients who are exposed to the hazards of chemotherapy [5–8]. The
reason for casting such a broad net is that we are unable to predict accurately
which tumors will have a bad outcome. Most node-negative breast cancers
are cured with surgery and local radiation alone, yet 30% to 40% [9,10] of

* Geisinger Health System, Department of General Surgery, Surgical Oncology and

Endocrinology, 100 North Academy Avenue, Danville, PA 17822-2170.
E-mail address: dgsheldon@geisinger.edu

1055-3207/05/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.soc.2004.07.007
70 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

these patients relapse and ultimately may die of disseminated disease. Why?
Overall, up to 40% of patients who are node-positive will be alive at 10 years,
despite having tumor characteristics that are similar to the other 60% [6].
New insights into the molecular biology of breast cancer may clarify this
problem and tell us how a particular tumor will behave in the future; how it
will respond to cytotoxic, endocrine, or biologic therapy; and whether the
tumor has metastasized—all from a core needle biopsy that is performed in
the surgeon’s office.
This article briefly reviews the exciting, yet unproven, reports of alternate
methods of predicting outcomes in breast cancer and highlights new
molecular methods of diagnosing, classifying, and treating this disease. The
author begins by reviewing well-known factors in breast cancer with which
all alternate methods of staging and predicting outcomes must be compared
prospectively.

Histologic factors of the tumor

Tumor size and grade
Tumor size long has been viewed as a primary indicator of prognosis in
breast cancer. It is clear from screening mammography trials that if tumors
are detected earlier (ie, at a smaller size), survival is improved [11–13].
Multiple randomized, controlled studies have confirmed that tumor size
alone is a primary indicator of outcome in breast cancer. Using Surveillance,
Epidemiology and End-Results data, Carter et al [14] demonstrated that
tumor size correlated with recurrence-free and overall survival. The risk of
harboring axillary lymph node metastases from breast cancer also is
a function of the size of the tumor; the rate of node positivity and the rate of
systemic metastases increase linearly with increasing tumor size [15,16].
Patients who have small tumors have a better outcome than those who have
large tumors [14,17].
For any given size of tumor, a higher nuclear or pathologic grade leads to
a worse outcome [18]. Tumor grade is prognostic for recurrence-free and
overall survival. Higher-grade tumors also are more responsive to cytotoxic
chemotherapy [19]. Pathologists commonly report a histologic grade of
1 (best), 2, or 3 (worst), although the subjective nature of the assessment
makes observations that are based solely on tumor grade difficult [19,20].

Angiolymphatic invasion
Vascular or angioinvasion, as identified by careful histologic examination
of the primary tumor, is correlated highly with local, regional, and systemic
metastases and is a marker for recurrence of disease and overall survival
[21–23].
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 71

Lymphovascular invasion (LVI) is difficult to discern from angioinvasion

on histologic examinations, but is associated with a worse outcome. A review
of the University of Southern California/Van Nuys database indicated that
LVI is an independent marker for risk of death from breast cancer [24]. It
was suggested that LVI can be a surrogate for nodal metastasis; it is a strong
negative prognostic factor and is an indication for aggressive chemotherapy
in the absence of nodal metastasis [25].
Tumor angiogenesis, a necessary component of the metastatic phenotype,
can be assayed by immunohistochemistry. High levels of angiogenesis are
correlated independently with poor survival and metastasis [26].

Axillary nodal metastases

In 2004, the most relevant prognostic factor in breast cancer remains
axillary nodal status. The gold standard for staging of breast cancer is the
histologic examination of the axillary lymph nodes because it is the most
sensitive indicator of prognosis. Axillary nodal staging is performed most
reliably by a level I and II axillary nodal dissection, when greater than 10
lymph nodes are examined [27,28]. Clinical staging of axillary lymph nodes
by physical examination is, by itself, highly inaccurate with false-negative
rates of 30% to 40% [29].
For any given size of a primary breast tumor, axillary nodal metastases
imply a significantly poorer prognosis for the patient as compared with
patients who have similar tumor size but no nodal metastases [14]. Nodal
metastases reduce the mean 10-year overall survival by approximately half
[30,31]. The presence of nodal metastases, the total numbers of nodes, and
the level from which they were harvested are important [17,32].
Despite the fact that nodal status is the most sensitive factor for the
estimation of risk, it is not a good indicator of outcome. Nodal status
cannot predict reliably which of the approximately 30% of patients who are
node-negative will relapse and which of the approximately 40% of patients
who are node-positive will survive for 10 years without evidence of disease
[33,34].
Perhaps the most controversial report of axillary nodal status as it relates
to outcome is the National Surgical Adjuvant Breast and Bowel Project
(NSABP) study, B-04 [35,36]. The node-negative portion of this large trial
randomized women who had clinically-staged early breast cancer to (1)
modified radical mastectomy (MRM); (2) total mastectomy with axillary
radiation (TM/XRT); or (3) total mastectomy alone (TM). Patients did not
receive adjuvant systemic therapy. At 10 and 25 years of follow-up, the
recurrence rates and axillary failure rates in the arms who received MRM
and TM/XRT were similar. As expected, the axillary failure rate in the arm
that underwent TM was much higher than the rate in the two arms that
included axillary treatments (24% versus 4%); however, this rate was only
half of the nodal positivity rate of the arm that underwent MRM which
72 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

indicated that positive axillary lymph nodes always do not become clinically
apparent. Another important finding was that nodal relapse in the arm that
received no axillary treatment (ie, TM) did not diminish survival at 25 years
of follow-up. This raises an interesting biologic question—how can axillary
lymph node metastases be the most sensitive indicator of future systemic
disease recurrence and death from breast cancer when half never become
clinically apparent and delayed treatment does not affect survival?
Although lymph node status is a strong predictor of outcome, axillary
dissection is not without morbidity. The risk of permanent lymphedema is
4% to 36%, depending on the extent of surgery and whether nodal basin
irradiation is used postoperatively [37]. With the advent of sentinel node
biopsy as the standard of care for axillary nodal staging in high-volume
centers [38], the morbidity rate is reduced significantly, but is not zero. With
accurate nodal staging alone, it is not possible to predict reliably which
patients will experience eventual systemic relapse, and thus, die of their
disease.

Molecular predictors and prognostic indicators in breast cancer

Steroid receptors
Decades of quality data validate the use of steroid receptors as a predictor
of outcome in primary breast cancer [4]. Estrogen receptors and progesterone
receptors are detected on tumors by ligand-binding assays, or more recently,
by immunohistochemistry (IHC). Estrogen receptor–positive (ERþ) tumors
have a better prognosis compared with estrogen receptor–negative (ER
)
tumors. The presence of estrogen receptors on a tumor and intact cellular
machinery that is regulated by estrogen is an independent prognostic factor
for survival in breast cancer [39]. Estrogen receptors on a breast tumor also
predict response to systemic endocrine therapy (tamoxifen), and thus,
a better outcome. A large meta-analysis of more than 30,000 treated women
provided level I evidence of a treatment effect for tamoxifen in ERþ tumors.
The proportional recurrence reduction at 1, 2, and 5 years was 21%, 29%,
and 47%, respectively [40]. The corresponding reduction in mortality was
12%, 17%, and 26%, respectively.

erbB-2 (Her2/neu)
The proto-oncogene, erbB-2 (also known as Her2/neu), is located on
chromosome 17 and encodes a 185-kd transmembrane protein, p185, a
homolog of the epidermal growth factor receptor (EGFR). EGFR is a classic
transmembrane tyrosine kinase–type receptor that initiates signaling path-
ways upon ligand binding. It normally is present in small quantities on breast
epithelium. The literature is inconsistent with regards to the predictive
abilities of Her2 in breast cancer, because of methodologic differences in
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 73

assaying for gene abnormalities and the expression of the corresponding

protein receptor. A distinction must be made between gene amplification and
protein overexpression. Identification of gene amplification seems to be a
more sensitive indicator of poor prognosis as compared with protein
overexpression and is associated with newer, more sophisticated techniques
in molecular diagnosis [41–43]. An amplification of the erbB-2/Her2 gene
results in the overexpression of erbB-2/Her2 receptor protein in approxi-
mately one third of invasive breast carcinomas and is associated with poor
outcome [44–46]. ErbB-2 receptor protein overexpression in breast cancer
was associated with lymph node metastases, higher nuclear grade, younger
age at diagnosis, resistance to endocrine therapy, and a poorer prognosis [47–
49]. Similarly, the overexpression of EGFR is a predictor of early recurrence
and death [50,51]. erbB-2/Her2 overexpression was the basis for multiple
trials of combined cytotoxic chemotherapy and targeted biologic therapy
with a monoclonal antibody (trastuzumab) against the receptor [52,53]. The
finding that Her2 status is concordant between primary tumor and the
systemic metastasis supports the biologic basis for this treatment [54]. Long-
term data as to the efficacy of trastuzumab are forthcoming and are derived
mostly from trials in the metastatic setting.

p53
The TP53 gene also is on chromosome 17 and encodes for the p53 protein.
It is the most commonly mutated gene in human cancer [55]. The p53 protein
has many functions that relate to cell cycle arrest and induction of apoptosis
in response to cellular stress. Mutation or inactivation of p53 is an almost
universal requirement for human cancers to proliferate and metastasize. It
is no surprise that mutant TP53 gene products are associated with a poor
prognosis in breast cancer [56]. Mutations in the TP53 gene were
demonstrated in node-negative and node-positive tumors. This finding
independently implied reduced disease-free and overall survival and im-
proved response to regional radiotherapy [57]. Conflicting data concerning
outcomes arise when archival tissue blocks are immunohistochemically
stained for abnormal protein accumulation as compared with newer
techniques that use genome TP53 mutation screening on fresh tissue; the
latter is much more sensitive. A recent meta-analysis of 11 studies with more
than 2300 patients demonstrated that the relative risk of death from breast
cancer is doubled for tumors with somatic TP53 mutations as compared with
those without such mutations [58]. When p53 mutations are noted in
conjunction with Her2/neu gene amplification, outcomes are significantly
worse than for tumors that contain only one or neither of these abnor-
malities. In a large (n = 580), prospective study of Her2/neu amplification in
frozen breast tumor specimens, a Toronto group noted a significant
reduction in disease-free survival in patients whose tumors had the Her2/
neu gene amplified. In a subset of these tumors (n = 108), genome
74 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

abnormalities in TP53 exons 4–10 were noted; this group independently had
worse disease-free and overall survival. This group of tumors also was more
likely to be ER
, high-grade, and to harbor LVI [59].

Cyclins and cyclin-dependent kinase inhibitors

Cyclins are a family of nuclear proteins that accelerate the nuclear mitotic
cycle from G1 to S phase by inactivating the retinoblastoma protein, Rb.
High levels of cyclin-dependent kinases (CDK) and cyclins are seen in breast
cancer cell lines and were associated with poor outcomes in retrospective
clinical series [60]. A recent study from the University of Houston M.D.
Anderson Cancer Center showed convincingly that deregulated expression of
cyclin E is associated with poor survival in breast cancer. These results are
conflicting with older series [61]; the explanation is that IHC detection is
unreliable for the more active, truncated isoforms of cyclin E, whereas
Western blot analysis (used in the Houston series) is much more sensitive.
Western blot–detected cyclin E upregulation was associated with a hazard
ratio for death of 33 as compared with 2.9 for IHC-detected cyclin E on the
same specimens. No patient who had normal cyclin E levels and stage I
cancer (n = 102) died during the follow-up period, whereas all of the patients
who had stage I disease (n = 12) and overexpressed cyclin E died of breast
cancer within 48 months. This is a provocative finding, despite the fact that
no information concerning adjuvant systemic therapy was given in the
report. Similar results were seen in patients who had stage II or III disease.
Another nuclear protein group, the cyclin-dependent kinase inhibitors
(CDKI), play a role in cell cycle regulation. p27kip is a CDKI that is
a negative regulator of cyclin-dependent kinases and antagonizes the activity
of cyclin E. Decreased levels of p27, as demonstrated by IHC, imply increased
mitotic activity and worse outcome [60]. A small study that used IHC to assay
p27 activity confirmed that a decreased level of this protein in breast cancer is
associated independently with shorter overall survival in patients who are
node-positive [62,63]. Results of ongoing research into the CDK/CDKI
system are awaited eagerly to validate cyclin expression as a predictive factor
in breast cancer.

Urokinase-type plasminogen activator system

Extracellular matrix degradation by proteases has been evaluated
extensively and is believed to be a necessary step in the malignant progression
of tumors. Serine proteases, such as urokinase-type plasminogen activator
(uPA), are necessary enzymes for angiogenesis, invasion, and metastasis.
There is considerable evidence that increased levels of uPA and its in-
activator, plasminogen activator inhibitor–1 (PAI-1), and decreased levels of
the inactivator, plasminogen activator inhibitor–2, are correlated with
systemic relapse and death from breast cancer [64]. A large, pooled clinical
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 75

analysis confirmed that tumor immunoassays that are positive for uPA or
PAI-1 are associated independently with a poor outcome for patients who
are node-negative or node-positive [65]. Further prospective study of the
uPA system as it relates to metastasis is warranted; this may represent an
attractive target for antimetastatic therapy.

Other markers of risk in breast tumors

A multitude of other molecular predictors have been studied in breast
cancer. A comprehensive review of them all is not the intent of this article.
Cathespin D, Ki-67, bcl-2, E-Cadherin, nuclear S-phase fraction, and ploidy
status are some of the many molecular markers that have been examined in
high-risk breast cancer [66]. None has been studied in a prospective fashion
in a clinical trial to demonstrate efficacy. One notable study recruited
patients from a neoadjuvant protocol, examined some of these biomarkers
pre- and posttreatment, and noted that a clinical response to cytotoxic
chemotherapy in conjunction with favorable biomarker findings may be
a surrogate for predicting survival in breast cancer [47]. None has gained
prominence in daily oncology practice.

Disseminated tumor cells

Prospective studies of blood and bone marrow samples in unselected
patients who had breast cancer revealed the presence of disseminated tumor
cells (DTCs) in 20% to 40% of cases [67–69]. These cells necessarily are not
metastases per se—rather, they are solitary, nonproliferating tumor cells that
have malignant potential [70,71]. Fluorescent in situ hybridization analysis
revealed that DTCs have malignant genotypes [72] but may not be responsive
to cytotoxic chemotherapy, given their dormancy [73]. DTCs in bone
marrow are strongly predictive of eventual disease relapse and do not
correlate with the presence or absence of lymph node micrometastases
[74,75]. Klein et al [76] studied breast tumor DNA from DTCs in 386
unselected patients; they noted that the DNA aberrations in the disseminated
cells were distinct from the DNA aberrations of the lymph node metastases
and the primary tumor [76]. DTCs do not express ERs consistently when
compared with the primary tumor and lymph node metastases [77]. These
findings are consistent with the theory that breast cancer cells are shed
peripherally early in the course of disease and later acquire the genetic
changes that are characteristic of metastases [78–81].
The presence of DTCs in the marrow is associated strongly with bony
metastases; however, patients who have DTCs can experience long tumor-
free intervals or may not ever relapse [75,82,83]. Diel et al [84] reported that
DTCs, that were detected prospectively with tumor-associated glycoprotein
(TAG-12), were present in 43% of patients who had primary breast cancer.
At 36 months of follow-up, DTCs were superior to lymph node status as
a predictor of eventual metastasis and disease-free and overall survival. This
76 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

study has been criticized for its short follow-up interval and because TAG-12
may be expressed in marrow myeloid precursors and could have confounded
the results. An often-cited report by Braun, [67] however, confirmed the
negative prognostic impact of DTCs in a well-controlled, prospective study
of bone marrow aspirates in benign and malignant disease of the breast that
used IHC-detected cytokeratins. The false positive rate was only 1% (2 of
191 control specimens) as determined by analysis of marrow specimens from
patients who had benign breast disease. Multivariate analysis confirmed that
DTC is associated independently with early systemic recurrence and death
from breast cancer. This study provided strong evidence that DTCs are
independently predictive of outcome, compared with other tumor factors
and lymph node status, and contradicted earlier findings [82,83]. More recent
reports confirmed the findings of Braun and Diel et al and concluded that
bone marrow aspiration for detection of DTC is complementary to
traditional prognostic indicators in breast cancer [85].
DTCs can overexpress Her-2/neu and other tumor markers and this
finding is a poor prognostic indicator [86]. Labeling with IHC revealed that
approximately 70% of DTCs express Her2/neu, 65% express epithelial cell
adhesion marker, 77% express uPA, and up to 100% express extracellular
matrix metalloproteinase inducer [68]. These molecular markers of
phenotypic malignancy represent interesting targets for novel biologic
therapies after systemic therapy with minimal residual microscopic disease.

Tumor genome analysis: microarrays

In DNA microarray analysis, mRNA from snap frozen fresh tissue is
extracted and complimentary double-stranded DNA is created. Reverse
transcription is undertaken and the resultant amplified cRNA is labeled with
fluorescent dye and hybridized to a panel of up to 25,000 oligonucleotides;
each one represents a single gene on a chip that is similar to a glass
microscope slide. Qualitative and quantitative assessment of the amount of
fluorescence enables computer-aided statistical programs to discern patterns
of gene expression—either up-regulated or down-regulated. The presence or
absence of tumor mRNA for each of the 25,000 genes is indicated on the
chip and this is interpreted as evidence of gene expression, but not
necessarily protein product translation or functional gene products. This
extremely powerful tool generates vast amounts of data that can
revolutionize the diagnosis and management of many diseases, most notably
cancer.
Although long-term prospective data are lacking, initial findings from
microarray analysis of tumor tissue has yielded extremely provocative
information. Expression profiling demonstrates that breast tumors are
genetically heterogenous and that they could be subclassified by the
differential expression of known genes [87,88]. Analysis of these patterns
can predict ER status, response to cytotoxic chemotherapy, and potentially
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 77

tailor such therapy, based on specific profiles [89–93]. Based on their genetic
classification of breast tumors, Hedenfalk et al [94] were able to identify
hereditary versus sporadic breast cancer and correctly classify BRCA1- and
BRCA2-mutated tumors. This information can be obtained simply from an
in vivo fine needle aspiration biopsy of tumor tissue [95].
Some of the most exciting data from using DNA expression profiles
showed that the ultimate outcome of the disease may be predicted from the
genetic make-up of the tumor at the time of harvesting. In a small,
prospective study of 55 patients who had breast cancer, Ahr et al [96]
classified all patients, regardless of nodal status, into two groups according
to a clustered, 41-gene analysis. The analysis successfully identified patients
who had synchronous metastatic disease and early relapse; this finding was
independent of nodal status. Similarly, the group that was headed by Van’t
Veer [97] demonstrated in a pilot study that gene expression profiling could
predict clinical outcome in lymph node–negative breast cancer. They
established a 70-gene profile that could predict outcome reliably in terms of
systemic relapse (odds ratio of 15 for development of metastases). In
a follow-up study of 295 consecutive patients (61 patients were included in
the original report), Van de Vijver et al [98] demonstrated the usefulness of
the same 70-gene profile in predicting the outcome of early (stage I or II)
breast cancer. Using the profile, they retrospectively assigned each patient to
a ‘‘good prognosis’’ (n = 115) or ‘‘bad prognosis’’ (n = 180). The profile
was strongly predictive of ultimate outcome; the 5-year freedom from
distant metastasis rate (DFS) was 61% in the group that had a poor
prognosis and was 95% in the group that had a good prognosis. At 10
years, the DFS rates were 50% and 85%, respectively. What is most
remarkable in this report is that the patients who had lymph node
metastases were distributed evenly in the two groups. Most patients who
were lymph node–negative (141/151) did not receive adjuvant therapy,
whereas most patients who were node-positive (120/144) did receive
adjuvant systemic chemotherapy or endocrine therapy. This exciting study
has been criticized for its retrospective nature, patient selection bias, and the
inclusion of pilot data in the manuscript; however, it is a preview of how
microarray data may be used in daily oncology practice. The finding that
the expression profile of a primary tumor is identical to its distant metastasis
that appears later validates the theory that outcomes can be determined at
the initial presentation of the tumor [99]. What cannot be determined by the
Van de Vijver et al [98] study is the fate of patients who do not receive
adjuvant systemic therapy but are lymph node–positive and have a good
prognosis. Gene-expression profiling, if validated in ongoing trials, has the
potential to answer the question of when to treat aggressively the lymph
node–negative patient who ultimately will relapse and when not to treat the
lymph node–positive patient who will have a good outcome, regardless of
therapy. This represents the ‘‘holy grail’’ for clinicians who treat cancer
patients.
78 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

Perspective
Powerful, exciting, thought-provoking, expensive—all of these words
describe the new tools that are available to the physician collaborator who
treats cancer. The sequence of the human genome was reported in 2001
[100,101]; genome research is revealing a plethora of translational appli-
cations for molecular diagnostic techniques. The new discipline of ‘‘discovery
science’’ turns out information concerning the molecular biology of
neoplasia almost daily. What should be done with all of the data? First,
comparisons of the most promising of the molecular prognostic variables (eg,
DTCs, cyclin E, or gene-expression profiles) need to be examined in
a prospective manner with a critical light and be compared with accepted
standards. To say that long-term data are lacking is almost superfluous.
Advancements in cancer treatments are based on observations from
prospective clinical trials. Data from DTCs and molecular profiling can be
obtained in almost real-time and may accelerate the pace of discovery if, for
instance, the eradication of viable DTCs in the marrow [102] or the
normalization of gene-expression profile with therapy, is validated as an
outcome equivalent. The surgeon must play an integral role in this process.
The paradigm of neoadjuvant therapy for breast carcinoma that provides
linear data points by interrogating the tumor genome pretherapy,
posttherapy, and postexcision is well-suited for the multi-disciplinary clinic.
Soon, we should have prospective gene-expression data on individual breast
tumors and their associated in situ carcinoma, lymph node metastases, and
DTCs. The differences in expression profiles of each of these tissues should
shed much light on the molecular biology of breast cancer.
The corollary to this paradigm is the necessity of a well-run team of
clinicians and scientists with an infrastructure that is designed to handle large
amounts of information that are derived from tissues as soon as it is
procured. Many of these new techniques require high-quality RNA that is
obtained from immediately frozen tissue and an accessible tissue bank.
Recently, a technique to obtain gene expression data from paraffin-
embedded archival tissue blocks using reverse transcriptase polymerase
chain reaction was reported in abstract form and in the lay press, before any
formal peer-reviewed process [103]. Private industry has reserved the rights to
the 21-gene profile that is specific to the report and has marketed the test
commercially to clinicians. This should be cause for alarm among academic
oncologists; the data that were obtained from the test have not been
examined critically before commercial application. If validated, however, this
process could have a tremendous impact on routine examinations of breast
cancer specimens.
Can molecular profiles predict who does not need systemic chemother-
apy? This should be a component of future study design. If and when tumor
genetic profiles confidently predict who does and who does not need
aggressive treatment we will have arrived as clinicians. When the current
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 79

standard of care calls for treatment of more than 90% patients to the benefit
of less than 10%, much work needs to be done.

References
[1] Galea MH, Blamey RW, Elston CE, Ellis IO. The Nottingham Prognostic Index in primary
breast cancer. Breast Cancer Res Treat 1992;22:207–19.
[2] Balslev I, Axelsson CK, Zedeler K, Rasmussen BB, Carstensen B, Mouridsen HT. The
Nottingham Prognostic Index applied to 9,149 patients from the studies of the Danish
Breast Cancer Cooperative Group (DBCG). Breast Cancer Res Treat 1994;32:281–90.
[3] Harris JR, Lippman ME, Veronesi U, Willett W. Breast cancer (3). N Engl J Med 1992;327:
473–80.
[4] Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast
cancer by immunohistochemical analysis. Mod Pathol 1998;11:155–68.
[5] Goldhirsch A, Glick JH, Gelber RD, Coates AS, Senn HJ. Meeting highlights:
International Consensus Panel on the Treatment of Primary Breast Cancer. Seventh
International Conference on Adjuvant Therapy of Primary Breast Cancer. J Clin Oncol
2001;19:3817–27.
[6] Polychemotherapy for early breast cancer: an overview of the randomized trials. Early
Breast Cancer Trialists’ Collaborative Group. Lancet 1998;352:930–42.
[7] Cole BF, Gelber RD, Gelber S, Coates AS, Goldhirsch A. Polychemotherapy for early
breast cancer: an overview of the randomized clinical trials with quality-adjusted survival
analysis. Lancet 2001;358:277–86.
[8] Eifel P, Axelson JA, Costa J, et al. National Institutes of Health Consensus Development
Conference Statement: adjuvant therapy for breast cancer, November 1–3, 2000. J Natl
Cancer Inst 2001;93:979–89.
[9] Fisher B, Redmond C, Dimitrov NV, et al. A randomized clinical trial evaluating sequential
methotrexate and fluorouracil in the treatment of patients with node-negative breast cancer
who have estrogen-receptor-negative tumors. N Engl J Med 1989;320:473–8.
[10] Mansour EG, Gray R, Shatila AH, et al. Survival advantage of adjuvant chemotherapy in
high-risk node-negative breast cancer: ten-year analysis–an intergroup study. J Clin Oncol
1998;16:3486–92.
[11] Wald N, Frost C, Cuckle H. Breast cancer screening: the current position. BMJ 1991;302:
845–6.
[12] Fletcher SW, Elmore JG. Clinical practice. Mammographic screening for breast cancer.
N Engl J Med 2003;348:1672–80.
[13] Tabar L, Smith RA, Vitak B, et al. Mammographic screening: a key factor in the control of
breast cancer. Cancer J 2003;9:15–27.
[14] Carter CL, Allen C, Henson DE. Relation of tumor size, lymph node status, and survival
in 24,740 breast cancer cases. Cancer 1989;63:181–7.
[15] Silverstein MJ, Gierson ED, Waisman JR, Colburn WJ, Gamagami P. Predicting axillary
node positivity in patients with invasive carcinoma of the breast by using a combination of
T category and palpability. J Am Coll Surg 1995;180:700–4.
[16] Koscielny S, Tubiana M, Le MG, et al. Breast cancer: relationship between the size of the
primary tumour and the probability of metastatic dissemination. Br J Cancer 1984;49:
709–15.
[17] Fisher ER, Sass R, Fisher B. Pathologic findings from the National Surgical Adjuvant
Project for Breast Cancers (protocol no. 4). X. Discriminants for tenth year treatment
failure. Cancer 1984;53:712–23.
[18] Henson DE, Ries L, Freedman LS, Carriaga M. Relationship among outcome, stage of
disease, and histologic grade for 22,616 cases of breast cancer. The basis for a prognostic
index. Cancer 1991;68:2142–9.
80 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

[19] Neville AM, Bettelheim R, Gelber RD, et al. Factors predicting treatment responsiveness
and prognosis in node-negative breast cancer. The International (Ludwig) Breast Cancer
Study Group. J Clin Oncol 1992;10:696–705.
[20] Le Doussal V, Tubiana-Hulin M, Friedman S, Hacene K, Spyratos F, Brunet M.
Prognostic value of histologic grade nuclear components of Scarff-Bloom-Richardson
(SBR). An improved score modification based on a multivariate analysis of 1262 invasive
ductal breast carcinomas. Cancer 1989;64:1914–21.
[21] Pinder SE, Ellis IO, Galea M, O’Rouke S, Blamey RW, Elston CW. Pathological
prognostic factors in breast cancer. III. Vascular invasion: relationship with recurrence and
survival in a large study with long-term follow-up. Histopathology 1994;24:41–7.
[22] Davis BW, Gelber R, Goldhirsch A, et al. Prognostic significance of peritumoral vessel
invasion in clinical trials of adjuvant therapy for breast cancer with axillary lymph node
metastasis. Hum Pathol 1985;16:1212–8.
[23] Bettelheim R, Penman HG, Thornton-Jones H, Neville AM. Prognostic significance of
peritumoral vascular invasion in breast cancer. Br J Cancer 1984;50:771–7.
[24] Woo CS, Silberman H, Nakamura SK, et al. Lymph node status combined with
lymphovascular invasion creates a more powerful tool for predicting outcome in patients
with invasive breast cancer. Am J Surg 2002;184:337–40.
[25] Lauria R, Perrone F, Carlomagno C, et al. The prognostic value of lymphatic and blood
vessel invasion in operable breast cancer. Cancer 1995;76:1772–8.
[26] Viens P, Jacquemier J, Bardou VJ, et al. Association of angiogenesis and poor prognosis in
node-positive patients receiving anthracycline-based adjuvant chemotherapy. Breast
Cancer Res Treat 1999;54:205–12.
[27] Axelsson CK, Mouridsen HT, Zedeler K. Axillary dissection of level I and II lymph nodes is
important in breast cancer classification. The Danish Breast Cancer Cooperative Group
(DBCG). Eur J Cancer 1992;28A:1415–8.
[28] Mathiesen O, Carl J, Bonderup O, Panduro J. Axillary sampling and the risk of erroneous
staging of breast cancer. An analysis of 960 consecutive patients. Acta Oncol 1990;29:
721–5.
[29] Sacre RA. Clinical evaluation of axillary lymph nodes compared to surgical and
pathological findings. Eur J Surg Oncol 1986;12:169–73.
[30] Ferguson DJ, Meier P, Karrison T, Dawson PJ, Straus FH, Lowenstein FE. Staging of
breast cancer and survival rates. An assessment based on 50 years of experience with radical
mastectomy. JAMA 1982;248:1337–41.
[31] Veronesi U, Cascinelli N, Mariani L, et al. Twenty-year follow-up of a randomized study
comparing breast-conserving surgery with radical mastectomy for early breast cancer.
N Engl J Med 2002;347:1227–32.
[32] Veronesi U, Luini A, Galimberti V, Marchini S, Sacchini V, Rilke F. Extent of metastatic
axillary involvement in 1446 cases of breast cancer. Eur J Surg Oncol 1990;16:127–33.
[33] Overgaard M, Hansen PS, Overgaard J, et al. Postoperative radiotherapy in high-risk
premenopausal women with breast cancer who receive adjuvant chemotherapy. Danish
Breast Cancer Cooperative Group 82b Trial. N Engl J Med 1997;337:949–55.
[34] Ragaz J, Jackson SM, Le N, et al. Adjuvant radiotherapy and chemotherapy in node-
positive premenopausal women with breast cancer. N Engl J Med 1997;337:956–62.
[35] Fisher B, Redmond C, Fisher ER, et al. Ten-year results of a randomized clinical trial
comparing radical mastectomy and total mastectomy with or without radiation. N Engl J
Med 1985;312:674–81.
[36] Fisher B, Jeong JH, Anderson S, Bryant J, Fisher ER, Wolmark N. Twenty-five-year
follow-up of a randomized trial comparing radical mastectomy, total mastectomy, and
total mastectomy followed by irradiation. N Engl J Med 2002;347:567–75.
[37] Larson D, Weinstein M, Goldberg I, et al. Edema of the arm as a function of the extent of
axillary surgery in patients with stage I–II carcinoma of the breast treated with primary
radiotherapy. Int J Radiat Oncol Biol Phys 1986;12:1575–82.
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 81

[38] Edge SB, Niland JC, Bookman MA, et al. Emergence of sentinel node biopsy in breast
cancer as standard-of-care in academic comprehensive cancer centers. J Natl Cancer Inst
2003;95:1514–21.
[39] Thompson AM, Elton RA, Hawkins RA, Chetty U, Steel CM. PS2 mRNA expression
adds prognostic information to node status for 6-year survival in breast cancer. Br J Cancer
1998;77:492–6.
[40] Early Breast Cancer Trialists’ Collaborative Group. Systemic treatment of early breast
cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000
recurrences and 24,000 deaths among 75,000 women. Lancet 1992;339:1–15.
[41] Andrulis IL, Bull SB, Blackstein ME, et al. neu/erbB-2 amplification identifies a poor-
prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study
Group. J Clin Oncol 1998;16:1340–9.
[42] Riou G, Mathieu MC, Barrois M, et al. c-erbB-2 (HER-2/neu) gene amplification is a better
indicator of poor prognosis than protein over-expression in operable breast-cancer
patients. Int J Cancer 2001;95:266–70.
[43] Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, Bernstein L. Evaluation of HER-2/neu
gene amplification and overexpression: comparison of frequently used assay methods in
a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 2002;20:
3095–105.
[44] Ciocca DR, Fujimura FK, Tandon AK, et al. Correlation of HER-2/neu amplification with
expression and with other prognostic factors in 1103 breast cancers. J Natl Cancer Inst
1992;84:1279–82.
[45] Borg A, Tandon AK, Sigurdsson H, et al. HER-2/neu amplification predicts poor survival
in node-positive breast cancer. Cancer Res 1990;50:4332–7.
[46] Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, MCGuire WL. Human breast
cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
Science 1987;235:177–82.
[47] Chang J, Powles TJ, Allred DC, et al. Biologic markers as predictors of clinical outcome
from systemic therapy for primary operable breast cancer. J Clin Oncol 1999;17:3058–63.
[48] Agrup M, Stal O, Olsen K, Wingren S. C-erbB-2 overexpression and survival in early onset
breast cancer. Breast Cancer Res Treat 2000;63:23–9.
[49] Hartmann LC, Ingle JN, Wold LE, et al. Prognostic value of c-erbB2 overexpression in
axillary lymph node positive breast cancer. Results from a randomized adjuvant treatment
protocol. Cancer 1994;74:2956–63.
[50] Sainsbury JR, Farndon JR, Needham GK, Malcolm AJ, Harris AL. Epidermal-growth-
factor receptor status as predictor of early recurrence of and death from breast cancer.
Lancet 1987;1:1398–402.
[51] Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in
human breast and ovarian cancer. Science 1989;244:707–12.
[52] Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal
antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J
Med 2001;344:783–92.
[53] Vogel C, Cobleigh MA, Tripathy D, et al. First-line, single-agent Herceptin(R)
(trastuzumab) in metastatic breast cancer. a preliminary report. Eur J Cancer 2001;
37(Suppl 1):25–9.
[54] Gancberg D, Di Leo A, Cardoso F, et al. Comparison of HER-2 status between primary
breast cancer and corresponding distant metastatic sites. Ann Oncol 2002;13:1036–43.
[55] Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor
suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:
4855–78.
[56] Overgaard J, Yilmaz M, Guldberg P, Hansen LL, Alsner J. TP53 mutation is an
independent prognostic marker for poor outcome in both node-negative and node-positive
breast cancer. Acta Oncol 2000;39:327–33.
82 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

[57] Jansson T, Inganas M, Sjogren S, et al. p53 Status predicts survival in breast cancer patients
treated with or without postoperative radiotherapy: a novel hypothesis based on clinical
findings. J Clin Oncol 1995;13:2745–51.
[58] Pharoah PD, Day NE, Caldas C. Somatic mutations in the p53 gene and prognosis in breast
cancer: a meta-analysis. Br J Cancer 1999;80:1968–73.
[59] Bull SB, Ozcelik H, Pinnaduwage D, et al. The combination of p53 mutation and neu/erbB-
2 amplification is associated with poor survival in node-negative breast cancer. J Clin Oncol
2004;22:86–96.
[60] Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and
cyclin E, alone and in combination, correlate with survival in young breast cancer patients.
Nat Med 1997;3:222–5.
[61] Donnellan R, Kleinschmidt I, Chetty R. Cyclin E immunoexpression in breast ductal
carcinoma: pathologic correlations and prognostic implications. Hum Pathol 2001;32:
89–94.
[62] Fredersdorf S, Burns J, Milne AM, et al. High level expression of p27(kip1) and cyclin
D1 in some human breast cancer cells: inverse correlation between the expression of
p27(kip1) and degree of malignancy in human breast and colorectal cancers. Proc Natl
Acad Sci USA 1997;94:6380–5.
[63] Tsuchiya A, Zhang GJ, Kanno M. Prognostic impact of cyclin-dependent kinase inhibitor
p27kip1 in node-positive breast cancer. J Surg Oncol 1999;70:230–4.
[64] Foekens JA, Peters HA, Look MP, et al. The urokinase system of plasminogen activation
and prognosis in 2780 breast cancer patients. Cancer Res 2000;60:636–43.
[65] Look MP, van Putten WL, Duffy MJ, et al. Pooled analysis of prognostic impact of
urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer
patients. J Natl Cancer Inst 2002;94:116–28.
[66] Beenken SW, Grizzle WE, Crowe DR, et al. Molecular biomarkers for breast cancer
prognosis: coexpression of c-erbB-2 and p53. Ann Surg 2001;233:630–8.
[67] Braun S, Pantel K, Muller P, et al. Cytokeratin-positive cells in the bone marrow and
survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:
525–33.
[68] Pantel K, Muller V, Auer M, Nusser N, Harbeck N, Braun S. Detection and clinical
implications of early systemic tumor cell dissemination in breast cancer. Clin Cancer Res
2003;9:6326–34.
[69] Pierga JY, Bonneton C, Vincent-Salomon A, et al. Clinical significance of immunocyto-
chemical detection of tumor cells using digital microscopy in peripheral blood and bone
marrow of breast cancer patients. Clin Cancer Res 2004;10:1392–400.
[70] Mansi JL, Berger U, McDonnell T, et al. The fate of bone marrow micrometastases in
patients with primary breast cancer. J Clin Oncol 1989;7:445–9.
[71] Solakoglu O, Maierhofer C, Lahr G, et al. Heterogeneous proliferative potential of occult
metastatic cells in bone marrow of patients with solid epithelial tumors. Proc Natl Acad Sci
USA 2002;99:2246–51.
[72] Muller P, Weckermann D, Riethmuller G, Schlimok G. Detection of genetic alterations in
micrometastatic cells in bone marrow of cancer patients by fluorescence in situ
hybridization. Cancer Genet Cytogenet 1996;88:8–16.
[73] Braun S, Kentenich C, Janni W, et al. Lack of effect of adjuvant chemotherapy on the
elimination of single dormant tumor cells in bone marrow of high-risk breast cancer
patients. J Clin Oncol 2000;18:80–6.
[74] Braun S, Cevatli BS, Assemi C, et al. Comparative analysis of micrometastasis to the bone
marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant
therapy. J Clin Oncol 2001;19:1468–75.
[75] Gebauer G, Fehm T, Merkle E, Beck EP, Lang N, Jager W. Epithelial cells in bone marrow
of breast cancer patients at time of primary surgery: clinical outcome during long-term
follow-up. J Clin Oncol 2001;19:3669–74.
 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84 83

[76] Klein CA, Schmidt-Kittler O, Schardt JA, Pantel K, Speicher MR, Riethmuller G.
Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of
single cells. Proc Natl Acad Sci USA 1999;96:4494–9.
[77] Ditsch N, Mayer B, Rolle M, Untch M, Schildberg FW, Funke I. Estrogen receptor
expression profile of disseminated epithelial tumor cells in bone marrow of breast cancer
patients. Recent Results Cancer Res 2003;162:141–7.
[78] Fisher B. Laboratory and clinical research in breast cancer–a personal adventure: the David
A. Karnofsky memorial lecture. Cancer Res 1980;40:3863–74.
[79] Klein CA, Blankenstein TJ, Schmidt-Kittler O, et al. Genetic heterogeneity of single
disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683–9.
[80] Schmidt-Kittler O, Ragg T, Daskalakis A, et al. From latent disseminated cells to overt
metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci USA
2003;100:7737–42.
[81] Klein CA. Gene expression signatures, cancer cell evolution and metastatic progression.
Cell Cycle 2004;3:29–31.
[82] Mansi JL, Easton D, Berger U, et al. Bone marrow micrometastases in primary breast
cancer: prognostic significance after 6 years’ follow-up. Eur J Cancer 1991;27:1552–5.
[83] Mansi JL, Gogas H, Bliss JM, Gazet JC, Berger U, Coombes RC. Outcome of primary-
breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999;
354:197–202.
[84] Diel IJ, Kaufmann M, Costa SD, et al. Micrometastatic breast cancer cells in bone marrow
at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst
1996;88:1652–8.
[85] Wiedswang G, Borgen E, Karesen R, et al. Detection of isolated tumor cells in bone marrow
is an independent prognostic factor in breast cancer. J Clin Oncol 2003;21:3469–78.
[86] Braun S, Schlimok G, Heumos I, et al. ErbB2 overexpression on occult metastatic cells in
bone marrow predicts poor clinical outcome of stage I–III breast cancer patients. Cancer
Res 2001;61:1890–5.
[87] Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature
2000;406:747–52.
[88] Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in
independent gene expression data sets. Proc Natl Acad Sci USA 2003;100:8418–23.
[89] Staunton JE, Slonim DK, Coller HA, et al. Chemosensitivity prediction by transcriptional
profiling. Proc Natl Acad Sci USA 2001;98:10787–92.
[90] Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas
distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001;98:
10869–74.
[91] Gruvberger S, Ringner M, Chen Y, et al. Estrogen receptor status in breast cancer is
associated with remarkably distinct gene expression patterns. Cancer Res 2001;61:
5979–84.
[92] Chang JC, Wooten EC, Tsimelzon A, et al. Gene expression profiling for the prediction of
therapeutic response to docetaxel in patients with breast cancer. Lancet 2003;362:362–9.
[93] Buchholz TA, Stivers DN, Stec J, et al. Global gene expression changes during neoadjuvant
chemotherapy for human breast cancer. Cancer J 2002;8:461–8.
[94] Hedenfalk I, Duggan D, Chen Y, et al. Gene-expression profiles in hereditary breast cancer.
N Engl J Med 2001;344:539–48.
[95] Symmans WF, Ayers M, Clark EA, et al. Total RNA yield and microarray gene expression
profiles from fine-needle aspiration biopsy and core-needle biopsy samples of breast
carcinoma. Cancer 2003;97:2960–71.
[96] Ahr A, Karn T, Solbach C, et al. Identification of high risk breast-cancer patients by gene
expression profiling. Lancet 2002;359:131–2.
[97] van’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical
outcome of breast cancer. Nature 2002;415:530–6.
84 D.G. Sheldon / Surg Oncol Clin N Am 14 (2005) 69–84

[98] van de Vijver MJ, He YD, van’t Veer LJ, et al. A gene-expression signature as a predictor
of survival in breast cancer. N Engl J Med 2002;347:1999–2009.
[99] Weigelt B, Glas AM, Wessels LF, Witteveen AT, Peterse JL, van’t Veer LJ. Gene
expression profiles of primary breast tumors maintained in distant metastases. Proc Natl
Acad Sci USA 2003;100:15901–5.
[100] Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human
genome. Nature 2001;409:860–921.
[101] Venter JC, Adams MD, Myers EW, et al. The sequence of the human genome. Science 2001;
291:1304–51.
[102] Janni W, Hepp F, Rjosk D, et al. The fate and prognostic value of occult metastatic cells
in the bone marrow of patients with breast carcinoma between primary treatment and
recurrence. Cancer 2001;92:46–53.
[103] Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. Multi-gene RT-PCR assay for
predicting recurrence in node negative breast cancer patients—NSABP studies B-20 and
B-14. Oncology Times 2004;26(Suppl):2–3.