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Baltimore, USA
*
Hopkins University School of Medicine, 1650 Orleans Street, CRB-1 Room 344,
Baltimore, MD 21287-0013, USA. Tel: +1 (410) 614-6204, Fax: +1 (410) 502-0677, email: cchung11@jhmi.edu
TheAuthor2015.PublishedbyOxfordUniversityPressonbehalfoftheEuropeanSocietyforMedical
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ABSTRACT
Background: To determine genetic alterations in head and neck squamous cell
carcinoma (HNSCC) using formalin-fixed, paraffin-embedded (FFPE) tumors obtained
through routine clinical practice, selected cancer-related genes were evaluated and
compared with alterations seen in frozen tumors obtained through research studies.
Patients and methods: DNA samples obtained from 252 FFPE HNSCC were analyzed
using next generation sequencing-based (NGS) clinical assay to determine sequence
and copy number variations in 236 cancer-related genes plus 47 introns from 19 genes
frequently rearranged in cancer. Human papillomavirus (HPV) status was determined by
presence of the HPV DNA sequence in all samples and corroborated with high-risk HPV
in situ hybridization (ISH) and p16 immunohistochemical staining (IHC) in a subset of
tumors. Sequencing data from 399 frozen tumors in The Cancer Genome Atlas and
University of Chicago public datasets were analyzed for comparison. Results: Among
252 FFPE HNSCC, 84 (33%) were HPV positive and 168 (67%) were HPV negative by
sequencing. A subset of 40 tumors with HPV ISH and p16 IHC results showed complete
concordance with NGS-derived HPV status. The most common genes with genetic
alterations were PIK3CA and PTEN in HPV-positive tumors and TP53 and CDKN2A/B
in HPV-negative tumors. In the pathway analysis, the PI3K pathway in HPV-positive
tumors and DNA repair-p53 and cell cycle pathways in HPV-negative tumors were
frequently altered. The HPV-positive oropharynx and HPV-positive nasal
cavity/paranasal sinus carcinoma shared similar mutational profiles. Conclusion: The
genetic profile of FFPE HNSCC tumors obtained through routine clinical practice is
Key words: DNA mutation, copy number variation, human papillomavirus, head and
neck squamous cell carcinoma
Key Message: To date, genetic alterations in head and neck squamous cell carcinoma
were determined in frozen tumors through research studies. The genetic profile of
formalin-fixed, paraffin-embedded tumors obtained through routine clinical practice is
comparable to frozen tumors studied in research setting. However, the clinical
significance of these genetic alterations requires further investigation.
INTRODUCTION
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease arising
from the mucosal lining of the upper aerodigestive tract including the nasal cavity,
paranasal sinuses, oral cavity, pharynx, and larynx. Common causes of HNSCC are
tobacco and alcohol use [1]. High-risk human papillomavirus (HPV) is an established
cause of HNSCC arising primarily in the oropharynx [2]. Recent data suggest that a
subset of nasal cavity and paranasal sinus HNSCCs is also associated with HPV [3].
Clinically, patients with HPV-positive HNSCC, especially non-smokers, have
significantly better prognosis compared to patients with HPV-negative HNSCC after
treatments for newly diagnosed as well as recurrent/metastatic diseases [4-7].
To improve understanding of the underlying cancer biology and leverage the findings to
optimize the HNSCC management, there have been concerted efforts to genetically
characterize HNSCC. To date, four large studies have reported the genetic alterations
in HNSCC; three studies evaluated the genetic alterations using whole exome
sequencing (WES) and whole genome sequencing (WGS) [8-10], and one study applied
a targeted approach of evaluating 617 selected cancer-associated genes [11]. However,
these earlier studies were conducted using frozen primary tumors, which are not
common tissue collection or storage method in clinical practice. In this study, we
evaluated genetic alterations of HNSCC using formalin-fixed, paraffin-embedded
(FFPE) tumors obtained through routine clinical practice and compared with publically
available sequencing data generated from frozen tumors. In addition, we have
evaluated carcinomas arising in the sinonasal tract where ~20% of the tumors are highrisk HPV positive [3] and established similarities in their HPV-specific profiles compared
to HPV-positive oropharynx SCC.
Copy number and mutation data from 279 HNSCC samples (TCGA cohort) with known
HPV status were downloaded from cBioPortal (http://www.cbioportal.org/public-portal/);
TCGA copy-number data was GISTIC transformed prior to analysis [10, 13]. The
genomic information of 236 genes contained in the FoundationOne assay was
extracted. Similarly, the genomic information of overlapping 122 genes contained in the
FoundationOne assay was extracted from the University of Chicago dataset (Chicago
cohort) [11]. The genomic features selected from TCGA and Chicago data were
tabulated with the genomic profiles from the FoundationOne assay. Because generearrangement data was not available in the Chicago cohort [11], we considered only
short variants and copy number alterations in 236 cancer-related genes.
Pathway assignments
We mapped the 236 altered genes from the three studies to the set of curated pathways
available from the National Cancer Institute (NCI) Pathway Interaction Database (PID)
[16]. NCI PID contains signaling interactions associated with major biomolecular and
cellular processes. For genes that could be assigned to multiple pathways, we chose
the pathways with the highest fraction of mapped genes from the 236 altered gene set.
Additionally, knowledge of the functional classification of the major signaling molecules
in the pathway was applied. As an example, the ErbB receptor signaling network is a
RESULTS
Patient characteristics and genomic profiles
Genetic alteration data from 252 FFPE HNSCC tumors were available in the FM cohort.
The HPV status of 252 HNSCC patients was determined by NGS; 84 (33%) were HPVpositive and 168 (67%) were HPV-negative. The high-risk HPV ISH and p16 IHC were
available for 40 tumors in the FM-JHU cohort and showed 100% concordance when
compared with HPV tumor status determined by NGS. Twenty-two of 84 (26.2%) HPV-
positive tumors and 37 of 168 (22%) HPV-negative tumors were obtained from distant
metastatic sites. Clinical characteristics are summarized in Table 1.
The most common alteration by mutation and/or copy number variation in HPV-positive
tumors was PIK3CA (30%) in the FM dataset, which is consistent with the TCGA and
Chicago datasets (35% and 56%, respectively; Table 2). The PI3K pathway was also
the most commonly altered pathway seen in HPV-positive tumors including alterations
of PTEN (15%), AKT1 (5%), RICTOR (4%), mTOR (2%), AKT2 (2%), and PIK3R1 (2%)
in addition to PIK3CA (Figure 1A, Table 2, and Supplemental Table 2). The most
common alteration in HPV-negative tumors was TP53 (87%) in the FM dataset, which is
again consistent with TCGA and Chicago cohorts (84% and 80%, respectively; Table 3).
The most commonly altered pathways excluding TP53 are the cell cycle and PI3K
pathways in HPV-negative tumors (Figure 1B, Table 3 and Supplemental Table 3). This
10
suggests that the selected cancer gene analysis using FFPE tumors obtained through
routine clinical practice yield comparable assessment of genomic alterations to frozen
tumors.
DISCUSSION
To realize the goal of personalized medicine, understanding the molecular
underpinnings of HNSCC and the use of clinically applicable biomarker assays are key
factors. Molecular characterization of HNSCC has been extensively published [8-11];
however, the WES or WGS data were generated in research settings using frozen
tumors. While these datasets provide comprehensive evaluation of the molecular
11
In our study, we obtained aggregate genomic profile data from Foundation Medicine,
Inc. for comparison with TCGA and Chicago datasets [10, 11]. Analysis of aggregate
data included an estimated count of gene-specific alterations per patient based on the
alteration frequency in the non-aggregate, patient-specific data (see Materials and
Methods); this approach could be problematic if the distribution of gene-specific
alterations varied significantly among the cohorts (e.g., a gene was, on average, altered
2 times per sample in the aggregate data). However, despite the heterogeneity of the
FM cohort obtained from multiple tumor sites and varying medical practice settings with
non-standardized tissue procurement methods, our data reveal remarkable similarities
12
to the TCGA and Chicago cohorts supporting the feasibility of routine clinical testing to
determine genetic alterations using FFPE tumors.
Corroborating a similar finding from the TCGA Network [10], our multi-cohort study
revealed that HPV-positive and HPV-negative HNSCC have distinct mutation profiles
and most of the discriminating power is in the lack of TP53 mutations in HPV-positive
HNSCCs and in the loss of CDKN2A/B in HPV-negative HNSCC. Indeed, loss of p53
function in HPV-positive HNSCCs through expression of the viral oncoprotein, E6,
would negate selective pressure of gaining TP53 mutations. CDKN2A which encodes
p16 protein is the most widely used surrogate marker of HPV-positive cancers and it is
clearly established that up to 90% of HPV-negative HNSCC tumors lack p16 expression
due to mutations, loss of heterozygosity and/or promoter hypermethylation [10].
We found that PIK3CA was the most frequently mutated oncogene among HPV-positive
patients in this multi-cohort study; a similar finding was recently reported by the TCGA
Network [10]. In addition to confirming the PI3K pathway to be the most commonly
altered pathway and potentially a very promising therapeutic target in HPV-positive
HNSCC, we report the fibroblast growth factor (FGF) pathway may be a relevant
therapeutic target in HPV-positive HNSCC. Novel agents targeting the FGF pathway are
in active development [19-21]. With emerging data showing that there are phenotypical
differences among the TP53 mutations associating with a high or low risk of poor
survival and some mutants displaying oncogenic properties by a gain-of-function [22,
23], relative lack of common TSG mutations in HPV-positive HNSCC may also suggest
13
favorable response to targeted agents such as PI3K or FGFR inhibitors which warrant
clinical evaluation. For HPV-negative patients with mutations predominantly in TSGs,
innovative trial designs to evaluate synthetic lethality through combination regimens are
necessary. Recent advances in understanding of excessive reliance on the G2-M
checkpoint in tumors lacking p53 function have led to evaluation of Wee1 and Chk1
inhibitors, which suggest a significant potential for clinical development in HNSCC [24,
25].
In addition to the oropharynx site within HNSCC, the presence of high-risk HPV is also
found in nasal cavity/paranasal sinus carcinoma suggesting HPV as an important
etiologic agent of carcinomas arising in the sinonasal tract. Unlike in HPV-positive
oropharynx SCC, there is very limited molecular and clinical data for nasal
cavity/paranasal sinus carcinoma, likely due to its rarity with annual incidence of only
0.5 to 1.0 patients per 100,000 [26, 27]. In the absence of the sinonasal tract cancerspecific data, clinicians have a tendency to extrapolate the data from the oropharynx
SCC. Our genomic data suggest that further studies to evaluate the role of HPV and
NFE2L2 (Nrf2) pathway in nasal cavity/paranasal sinus carcinoma are warranted.
Our study expands the current genomic data available surrounding HNSCC; however,
further investigation is required to determine the clinical significance of these genetic
alterations before use in routine clinical decision-making. Our data indicate that
specimens obtained through routine clinical practice exhibit similar genomic profiles
compared to those obtained in an academic setting, and demonstrate the feasibility of
14
ACKNOWLEDGEMENTS
None.
FUNDING
This work is funded, in part, by a NIH grant (NCI CCSG P30 CA006973).
15
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FIGURE LEGENDS
Figure 1: A) Tumor suppressor gene (TSG) and oncogene (OG) classification, and
pathway evaluation of altered genes in human papillomavirus (HPV)-positive head and
neck squamous cell carcinoma (HNSCC) in the Foundation Medicine-Johns Hopkins
University (FM-JHU) cohort. B) TSG and oncogene classification, and pathway
evaluation of altered genes in HPV-negative HNSCC in the FM-JHU cohort. Only
pathways altered in >8 of 40 FM-JHU samples are included in the pie charts and tables.
Figure 2: Genetic alteration profiles of, A) head and neck squamous cell carcinoma
(HNSCC) including oral cavity, oropharynx, larynx and hypopharynx primary sites, and
B) HNSCC including nasal cavity and paranasal sinus primary sites.
18
19
20
Table 1. Clinical characteristics of head and neck squamous cell carcinoma in the
Foundation Medicine cohort
HPV+ (n=84)
56.2 year (30 year, 84 year)
HPV- (n=168)
60.7 year (18 year, 85 year)
65 (77.4%)
19 (22.6%)
120 (71.4%)
48 (28.6%)
6 (7.1%)
8 (9.5%)
1 (1.2%)
10 (11.9%)
10 (11.9%)
0 (0%)
7 (4.2%)
5 (3.0%)
8 (4.8%)
34 (20.2%)
2 (1.2%)
7 (4.2%)
Regional
Lymph Node
9 (10.7%)
17 (10.1%)
Distant
Lung
Bone
Liver
Chest Wall
Skin
Trachea
Adrenal Gland
Pleura
Pleural Fluid
Brain
Parotid Gland
Salivary Gland
12 (14.3%)
2 (2.4%)
2 (2.4%)
0 (0%)
1 (1.2%)
1 (1.2%)
0 (0%)
1 (1.2%)
2 (2.4%)
1 (1.2%)
0 (0%)
0 (0%)
9 (5.4%)
6 (3.6%)
2 (1.2%)
3 (1.8%)
5 (3.0%)
1 (0.6%)
2 (1.2%)
2 (1.2%)
0 (0%)
0 (0%)
2 (1.2%)
1 (0.6%)
Miscellaneous
Head and Neck
Soft Tissue
Ear
Not Provided
8 (9.5%)
6 (7.1%)
0 (0%)
4 (4.8%)
38 (22.6%)
7 (4.2%)
1 (0.6%)
9 (5.4%)
Table 2. Top 30 of 236 most commonly altered genes in HPV-positive head and neck
squamous cell carcinoma
Gene
Name
Alteration
HPV+
FM
(N=84)
HPV+
Chicago
(N=51)
HPV+
TCGA
(N=36)
Pathway
Average
PIK3CA
Mut/Ampl
30%
35%
56%
PI3K-PTEN-AKT-mTOR
37%
SOX2
MLL2
(KMT2D)
RB1
BCL6
EP300
NOTCH1
Ampl
11%
NA
28%
Transcription-Sox2
16%
Mut
13%
20%
17%
NA
16%
Mut/Loss
Ampl
Mut
Mut
7%
1%
10%
6%
24%
18%
12%
18%
6%
25%
14%
11%
Cell Cycle-CCND-RB1
RTK-JAK-STAT
Cell Cycle-CCND-RB1
Development-NOTCH
12%
11%
11%
11%
PTEN
Mut/Loss
15%
8%
3%
PI3K-PTEN-AKT-mTOR
11%
FGFR3
ASXL1
KLHL6
FBXW7
TP53
ATM
BRCA2
BRIP1
(BACH1)
LRP1B
ATRX
KDM6A
BRCA1
BLM
JAK2
NF1
HRAS
MYC
ATR
FGF19
FGF3
FGF4
Mut
Mut
Ampl
Mut/Loss
Mut
Mut
Mut
1%
5%
1%
12%
5%
1%
6%
24%
10%
NA
6%
16%
16%
12%
11%
19%
25%
3%
3%
8%
3%
RTK-GF-FGF
NA
NA
Development-NOTCH
DNA repair
DNA repair
DNA repair
10%
9%
8%
8%
8%
7%
7%
NA
0%
16%
8%
DNA repair
7%
Mut
NA
Mut/Loss
Mut
NA
NA
Mut
Mut
Ampl
Mut
Ampl
Ampl
Ampl
2%
0%
7%
2%
0%
0%
2%
1%
5%
2%
4%
4%
4%
12%
18%
NA
14%
18%
14%
14%
12%
6%
NA
NA
NA
NA
8%
3%
3%
3%
0%
6%
0%
3%
3%
8%
6%
6%
6%
RTK
NA
NA
DNA repair
DNA repair
RTK-JAK-STAT
Ras
Ras
TGFbeta-SMAD
DNA repair
RTK-GF-FGF
RTK-GF-FGF
RTK-GF-FGF
7%
6%
6%
6%
5%
5%
5%
5%
5%
4%
4%
4%
4%
RICTOR
Ampl
4%
NA
6%
PI3K-PTEN-AKT-mTOR
4%
Table 3. Top 30 of 236 most commonly altered genes in HPV-negative head and neck
squamous cell carcinoma
Gene
Name
Alteration
HPVFM
(N=168)
HPVChicago
(N=69)
HPVTCGA
(N=243)
TP53
Mut
87%
80%
84%
CDKN2A/B
Mut/Loss
54%
32%
57%
FGF19
FGF3
FGF4
Ampl
Ampl
Ampl
23%
22%
22%
NA
NA
NA
32%
31%
31%
PIK3CA
Mut/Ampl
16%
29%
34%
CCND1
Ampl
24%
13%
NOTCH1
Mut/Loss
16%
LRP1B
SOX2
MLL2
(KMT2D)
EGFR
KLHL6
BCL6
Mut/Loss
Ampl
Pathway
Average
84%
32%
DNA repair
Cell CycleCDKN2A/B-CDK
RTK-GF-FGF
RTK-GF-FGF
RTK-GF-FGF
PI3K-PTEN-AKTmTOR
Cell Cycle-CCND-RB1
26%
21%
Development-NOTCH
20%
6%
8%
30%
NA
22%
21%
RTK
Transcription-Sox2
18%
16%
Mut
10%
20%
18%
NA
15%
Mut/Ampl
NA
Mut
13%
0%
1%
12%
NA
12%
16%
21%
18%
RTK-GF-EGF
NA
RTK-JAK-STAT
14%
13%
11%
ATR
Mut
2%
NA
16%
DNA repair
10%
NFE2L2
NOTCH2
MYC
FGFR1
ATRX
JAK2
Mut
Mut
Ampl
Mut/Ampl
NA
Ampl
7%
7%
5%
5%
0%
4%
1%
17%
1%
10%
30%
17%
14%
9%
15%
12%
7%
7%
Oxidative stress
Development-NOTCH
TGFbeta-SMAD
RTK-GF-FGF
NA
RTK-JAK-STAT
10%
9%
9%
9%
8%
8%
SMAD4
Mut/Loss
7%
7%
8%
TGFbeta-SMAD
7%
RICTOR
Ampl
5%
NA
9%
ZNF703
BRCA2
Mut/Ampl
Mut
4%
7%
NA
16%
9%
4%
FOXL2
NA
0%
NA
11%
PRKDC
NA
0%
NA
11%
GPR124
KDM6A
APC
NA
Mut
Mut
0%
2%
3%
NA
NA
14%
11%
9%
6%
PI3K-PTEN-AKTmTOR
NA
DNA repair
NA
PI3K-PTEN-AKTmTOR
Cell Adhesion-Integrin
NA
Ras
53%
28%
27%
27%
27%
26%
7%
7%
7%
7%
7%
6%
6%
6%