Frequent mutation of the PI 3 K pathway in head and neck cancer defines predictive biomarkers

Vivian Wai Yan Lui, Matthew L. Hedberg, Hua Li, Bhavana S. Vangara, Kelsey Pendleton, Yan Zeng, Yiling Lu, Qiuhong Zhang, Yu Du, Breean Gilbert, Maria Freilino, Sam Sauerwein, Noah Peyser, Dong Xiao, Brenda Diergaarde, Lin Wang, Simion Chiosea, Raja Seethala, Jonas T. Johnson, Seungwon Kim, Umamaheswar Duvvuri, Robert L. Ferris, Marjorie Romkes, Tomoko Nukui, Patrick Kwok-Shing Ng, Levi A. Garraway, Peter S. Hammerman, Gordon B. Mills, Jennifer R. Grandis


Sentence Statement of Significance:
Treatment options for HNSCC are limited by an incomplete understanding of the targetable mutations that "drive" tumor growth.Here, we define a subgroup of HNSCC harboring activating mutations of genes in the PI3K pathway where targeting the pathway demonstrates antitumor efficacy.These results suggest that PI3K pathway mutation assessment may be used to guide HNSCC therapy.

Introduction
Head and neck squamous cell carcinoma (HNSCC) is a frequently lethal cancer with few effective therapeutic options.Recent genomic findings in head and neck cancer revealed a wide spectrum of unexpected genetic aberrations (1,2).This genomic heterogeneity of HNSCC tumors underscores an obstacle to the identification of effective molecular targeting agents likely to benefit the majority of HNSCC patients.To date, there is a translational gap between genomics and treatment selection for HNSCC patients.TP53 mutation is the single most common mutational event.Yet the loss of function of this tumor suppressor gene has remained challenging to exploit therapeutically.Mitogenic pathways are crucial for cancer development and progression.In other malignancies, mutations of growth pathway genes have been shown to result in pathway activation, enhanced tumor growth, and increased sensitivity to agents targeting the mutated pathway.However, the potential of genomics-based therapy selection has not been widely investigated in HNSCC.
We and others recently reported genomic mutational profiles of over 100 HNSCC tumors (1,2).
Here, we analyzed an additional 45 HNSCC tumors by whole exome sequencing using the Illumina platform.In an effort to identify mutationally altered, targetable mitogenic pathways in HNSCC, we combined all currently available mutational data (from whole-exome sequencing) of 151 HNSCC primary tumors and evaluated the mutational events of genes in three major mitogenic pathways that have been previously implicated in HNSCC pathophysiology, namely the MAPK(3), JAK/STAT(4), and the PI3K pathways (3).These key mitogenic pathways are targetable in human cancers with a variety of agents currently in various stages of clinical development. Research.
on July 4, 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nearly one third of HNSCC tumors harbor PI3K pathway mutations
To date, whole-exome sequencing data of 106 HNSCC tumors are available.Here, we reported the whole-exome sequencing data of additional 45 HNSCC tumors collected at the University of Pittsburgh (Supplementary Table 1).Our results, similar to previous reports, showed a high degree of inter-tumor mutation heterogeneity, further confirming the complexity of HNSCC biology and the associated challenges in defining subsets of patients likely to respond to specific targeted therapies.With the aim of identifying mutationally altered, targetable mitogenic pathways in HNSCC, we assessed the mutational events of genes comprising three major mitogenic and targetable pathways in HNSCC; the JAK/STAT, MAPK and PI3K pathways.
A detailed analysis of the PI3K pathway mutational events showed that 19 of the 151 tumors (12.6%) harbor a PIK3CA mutation (Figure 1B).This mutation rate is similar to that detected in prior reports of HNSCC tumors (7.4% and 10.8% rate (6,7)).Further, we found PIK3CG and PTEN mutations in 4.0% (6/151) of HNSCC tumors, while PIK3R1 (also known as p85), PIK3R5 and PIK3AP1 were mutated in 2.7% tumors (4/151).Other components of the PI3K pathway were mutated in <2% cases (Figure 1B).Major downstream effectors of the PI3K pathway, including PDK1, AKT1 were not mutated, while AKT2 and mTOR were only mutated in 1.3% (2 mutations) of HNSCC tumors.Although PIK3CA gene amplification data is not available for the previously sequenced tumors, in the newly added cohort, PIK3CA was amplified in 24.4% (11/45) of the cases.
Previous reports noted loss of heterozygosity (LOH) of the tumor suppressor PTEN in HNSCC, by PCR-based microsatellite analysis primarily using D10S215 and/ D10S541 probes in relatively small HNSCC cohorts (e.g. 17 and 39 tumors, respectively) (8,9).Although comprehensive analysis of PTEN copy number was not available in the published genomic HNSCC studies (1,2), PTEN gene copy number change was analyzed using a highly sensitive Affymetrix Genome-Wide Human SNP Array 6.0 platform in our 45 newly sequenced HNSCC tumors.Our results showed that PTEN gene copy loss (≥1 copy loss) was only found in 8.16% of cases (4/45), indicating that PTEN loss is not likely to be the primary mediator of PI3K pathway alteration in HNSCC (unlike other cancers such as glioblastoma, where PTEN loss can be as high as 20-60%) (10,11).However, all 4 tumors with PTEN gene copy loss expressed relatively low levels of PTEN protein when compared to HNSCC tumors without PTEN gene copy alteration (P<0.001,Supplementary Figure 1).

PI3K pathway-mutated HNSCC tumors demonstrate an increased rate of cancer gene mutations
To determine if HNSCC tumors harboring mutations in PI3K pathway genes contained a higher number of mutations in other cancer-associated genes, we compared the mutation rates of PI3K pathway-mutated tumors to non-PI3K pathway-mutated tumors.We found that tumors harboring PI3K pathway mutations have higher rates of mutation than non-PI3K-mutated HNSCC tumors.
Further, cancer gene filtering analysis showed that these PI3K pathway-mutated HNSCC tumors harbored twice as many cancer gene mutations than those without PI3K pathway mutations (Figure 1D, 7.2±0.8vs 3.6±0.3,P<0.0001: defined by the Cancer Gene Census, COSMIC Database) (12).These results suggest that PI3K pathway mutations in HNSCC may facilitate the expansion or selection of tumor cells that are already genetically unstable and thus harbor more genomic aberrations including aberrations in known cancer genes.This contention is supported by further analysis demonstrating that DNA damage/repair genes (based on the DNA damage gene list in the cBio portal database (13), which includes ATM, ATR, CHEK1/2, BRCA1/2, FANCF, MLH1, MSH2, MDC1, PARP1 and RAD51) were found to be mutated at a significantly higher frequency in the PI3K-mutated tumors (average mutation rate of 37.0%; 17 mutations in 46 tumors) compared to tumors without PI3K pathway mutations (average mutation rate of 15.2%; 16 mutations in 105 tumors) (P=0.033).The association between PI3K pathway mutations and genomic instability is observed in HNSCC derived from all anatomic sites in our cohort (e.g.oral cavity, pharynx and larynx, data not shown).Mutation rates in laryngeal tumors (186.3±27.1,n=32, data not shown) are significantly higher than the rates of mutation in tumors from the other anatomical locations (78.6±8.3,n=116, P=0.0005, data not shown).Additionally, the prevalence of PI3K pathway mutations is higher in laryngeal tumors (53.1%±9.0%,n=32, data not shown) compared with tumors from the other anatomical locations (25.0%±4.0%,n=116, P=0.0005, data not shown).
Co-mutation analysis showed that tumors with PI3K pathway mutation(s) are also associated with mutations of several known tumor suppressor genes including ARID1A, MLL and MLL3 (P<0.05;Supplementary Table 3), which contribute to chromatin remodeling and transcriptional regulation in cancers (14)(15)(16)(17).Intriguingly, ARID1A has been shown to influence signaling through the PI3K pathway, suggesting that ARID1A may regulate the PI3K pathway and expand the number of tumors susceptible to targeting the PI3K pathway (18).Of note, PI3K pathwaymutated tumors are not associated with TP53 (P=1.0) or NOTCH1 mutations (P=0.34,data not shown).
Interestingly, we also identified 3 tumors where PIK3CA or PIK3R1 was the only known mutated cancer gene [HN_00361, HN_63027 and HN41PT with the respective PI3K-mutations of PIK3R1 (453_454insN), PIK3CA(E542K) and PIK3CA(H1047L)].Strikingly, all three tumors were associated with infection by the human papillomavirus (HPV).Although the number of HPV-positive HNSCC tumors in this cohort is relatively small (15 cases, see Supplementary Table 4), 5 of these tumors harbored PI3K pathway mutations (33.3) suggesting that a subset of HPV-positive HNSCC tumors (20%; 3/15 cases) may be driven by PI3K-pathway mutation(s) alone, without an associated increase in underlying genomic instability.

Only advanced stage HNSCC tumors harbor multiple PI3K-pathway mutations
In HNSCC cancers containing PI3K pathway mutations, 21.7% (10/46) harbored mutations in more than one PI3K pathway member genes, indicating that genetic alterations at multiple levels in the PI3K pathway are relatively common in HNSCC (Table 1).In contrast, HNSCC tumors rarely, if ever, harbored multiple mutations in the MAPK pathway (0 tumors), or the JAK/STAT pathway (only one contained both JAK3 and STAT1 mutations; HN_63080) (Figure 1C).Strikingly, all of these HNSCC tumors (100%; 10/10 cases) with concurrent PI3K mutational events were advanced (Stage IV) (Table 1).None of these tumors was associated with HPV infection.These findings suggest that concerted PI3K pathway aberrations may contribute to HNSCC progression.This finding appears to be unique to HNSCC.Examination of recently published tumor datasets including breast, colon and lung SCC showed that only 1/25 breast tumors, 1/27 colon carcinoma, and 0/31 lung SCC tumors harbored multiple PI3K pathway mutations were stage IV (data not shown; cBio portal (13)).While all 10 tumors with multiple PI3K pathway mutations were advanced (Stage IV), there is no significant association between advanced disease and individual PI3K pathway mutations (data not shown).

Additionally, mutation rates do not vary significantly between Stage IV and earlier Stage (I-III)
disease (data not shown).In the absence of models assessing the specific contribution of each mutation to cell growth or survival, it is not possible to determine the precise effect of individual mutations in tumors that harbor more than one mutation of genes in the PI3K pathway.

PIK3CA canonical and novel mutations increase survival and pathway activation in HNSCC tumors
PIK3CA is a critical gene in the PI3K signaling pathway.In HNSCC tumors, the most common sites of PIK3CA mutations included H1047R/L (8 mutations total), E545K/G (4 mutations) and E542K (3 mutations) (Figure 2A).All of which represent previously reported canonical ("hotspot") mutation sites.This HNSCC-PIK3CA mutation pattern (~90% of mutations found in the helical/kinase domains), is similar to that observed in cervical, breast and lung SCC cancers, but is distinct from other tumors such as endometrial cancer, lung adenocarcinoma, glioblastoma multiforme, and prostate carcinoma (Supplementary Table 5).In addition, we detected 4 previously unreported, novel PIK3CA mutations (R115L, G363A, C971R, R975S).To determine the functional consequences of these mutations we stably expressed, by retroviral infection, each of the novel mutations, and a hotspot mutation (H1047R), in a representative HNSCC cell line that is WT for all PI3K pathway components.Overexpression of WT PIK3CA (mimicking PIK3CA gene amplification), and expression of all the engineered PIK3CA mutants individually, resulted in enhanced growth compared to infection with EGFP control.Furthermore, the canonical hotspot mutation showed significantly enhanced growth compared to overexpression of WT PIK3CA (p = 0.0001).The novel mutations were found to confer moderate growth advantage compared to simulated WT amplification (R115L; p = 0.1174, G363A; p = 0.9637, C971R; p = 0.6503, R975S; p = 0.0958).Immunoblotting of cell lysates revealed that enhanced HNSCC growth conferred by introduction of the novel mutations was associated with increased PI3K pathway activation as reflected by elevated expression of phosphorylated AKT (Figures 2B & C).In the absence of complete functional characterization of these novel mutations, these findings should be considered supportive but not definitive evidence of oncogenic function.

HNSCC patient tumorgrafts with PIK3CA mutations are exquisitely sensitive to BEZ-235
Reports in other cancers suggest that tumors with PI3K pathway activation may be more sensitive to PI3K pathway inhibitors (19).To determine the predictive value of PIK3CA mutational status in HNSCC, we examined the sensitivity of HNSCC cell lines that did and did not harbor intrinsic activating driver PIK3CA(H1047R) hotspot mutations to PI3K pathway inhibitors.As shown in Figure 3A 3E).Another HNSCC patient-derived tumorgraft model (HPVnegative) harboring a PIK3CA mutation (E110K) was also found to be sensitive to BEZ-235 treatment (Supplementary Figure 2).In contrast, patient tumorgrafts with WT PIK3CA and low baseline p-AKT levels were not sensitive to the growth inhibitory effects of BEZ-235 (Figure 3F and Supplementary Figure 3).agent in HNSCC) compared with cetuximab alone (Supplemental Figure 4), suggesting that targeting PI3K in the setting of PIK3CA mutant tumors can enhance treatment responses to cetuximab.

Discussion:
The increase in targeted agents for cancer treatment results in an unprecedented opportunity for personalized cancer medicine.Selection of therapies based on mutation status of molecular targets has transformed clinical management and survival of several human malignancies.The EGFR monoclonal antibody cetuximab is the only targeted therapy that is FDA-approved to date for HNSCC treatment, yet there are no biomarkers that can be assessed in the primary tumor to predict clinical responses to this agent.The recent elucidation of HNSCC genomics offers an opportunity to identify genetic subgroups of HNSCC tumors to guide treatment decisions.
In this report, we employed a bioinformatic approach to identifying mutationally altered, targetable mitogenic pathways in HNSCC.Analyses of all currently available HNSCC wholeexome sequencing data (a total of 151 primary HNSCC tumors) revealed several key findings with important implications for HNSCC pathobiology and treatment.The PI3K pathway is the most frequently mutated oncogenic pathway in HNSCC, with the relative number of PI3Kmutated tumors compared to RAS/MAPK and JAK/STAT-mutated tumors being approximately 3-fold greater.Similar ratios of PI3K pathway mutations (relative to RAS/MAPK or JAK/STAT) are seen in squamous cell carcinoma of the lung and in cervical cancer; both of which share common risk factors with HNSCC, including tobacco and HPV infection, respectively.In contrast, the RAS/MAPK pathway is more frequently mutated than the PI3K pathway in colon and thyroid cancers, and both the PI3K and RAS/MAPK pathways are mutated at comparable rates in lung adenocarcinomas (13).The percentage of HNSCC tumors harboring multiple mutations in the PI3K pathway is similar to that observed in breast cancers (4.9%, 25/507 tumors) and glioblastomas (9.1%, 25/276 tumors) , higher than in thyroid cancer (0.3%, 1/323 tumors) and much lower than most other cancers, including uterine carcinoma (65.7%, 163/248 tumors), melanoma (24.9%, 63/253 tumors), and interestingly lung squamous cell carcinoma (17.4%, 31/178 tumors), which otherwise shares common risk factors and similar relative rates of pathway mutations with HNSCC.(13) Using novel patient-derived tumorgraft models with an oncogenic PIK3CA(E542K) mutation we demonstrated that these tumors are exquisitely sensitivity to a PI3K pathway inhibitor (BEZ-235).Similar results were demonstrated in another HNSCC patient-derived tumorgraft model with a PIK3CA (E110K) mutation, previously reported in breast cancer (21).In contrast, treatment of human-derived heterotopic tumorgrafts with wildtype PIK3CA and low basal expression levels of phospho-AKT, with a PI3K pathway inhibitor, was ineffective.These findings suggest that: 1) PI3K-pathway inhibitors can be effective for treating HNSCC tumors with PI3K mutations; and 2) mutation-guided treatment responses can be evaluated/monitored using patient-derived HNSCC tumorgraft models in vivo.In fact, early-phase clinical trial results showed that patients with solid tumors harboring a PIK3CA hotspot mutation (H1047R) were found to be responsive to PI3K pathway inhibitors (22).However, the effects of other PIK3CA mutations on mediating drug sensitivity in HNSCC preclinical models or clinical trials has not been previously reported.Findings from our study implicate that PIK3CA(E542K) mutation, as well as other non-hotspot mutations (such as E110K) may also identify an HNSCC subgroup potentially responsive to PI3K-pathway inhibitors.In particular, our results using HNSCC patient-derived tumorgrafts suggest that HNSCC tumors with activating PIK3CA mutations may be more sensitive to a dual PI3K/mTOR inhibitor (such as BEZ-235) compared to tumors with wildtype PIK3CA (Figures 3E and Supplementary Figure 3), as indicated by significant inhibition of p-S6 expression in the PIK3CA mutated, but not in the wildtype tumorgrafts.In fact, a recent report of five HNSCC cases found that mTOR-based targeted therapy may be more effective in HNSCC tumors harboring PIK3CA mutation and/or PTEN loss (23).PI3K pathway-mutated HNSCC tumors were found to have a higher rate of non-synonymous mutations, including an increased number of defined cancer gene mutations, compared to tumors without PI3K pathway mutations.This observation implies that the PI3K pathwaymutated HNSCC tumors have "oncogenic" advantage even with genomic instability, and/or that PI3K-mutated HNSCC tumors intrinsically harbor a "mutator" phenotype rendering them more prone to mutation.The oncogenic advantage of PI3K pathway-mutated tumors can be partly explained by PIK3CA "driver" mutations' growth-promoting activity(24) (Figure 2C), while the "mutator" phenotype of these tumors is supported by the our finding that PI3K pathway-mutated tumors are associated with ARID1A and MLL3 mutations, which are important tumor suppressor genes (15,16,25).It is possible that both the "oncogenic/growth" advantage and "mutator" phenotype associated with PI3K pathway-mutated HNSCC tumors are necessary for HNSCC progression; especially since PI3K pathway mutations in these tumors are not associated with TP53 mutation, a previously recognized tumor suppressor alteration that contributes to HNSCC carcinogenesis.Although the relationship of PI3K pathway mutations and TP53 mutation has not been carefully examined in most cancers, a recent study in bladder cancer showed that PIK3CA mutations were significantly more common in TP53 WT tumors (26).Hence, PI3K pathway mutations may mediate tumor progression in the absence of TP53 genetic alteration.
Our finding that all 10 HNSCC tumors with concurrent mutations of multiple PI3K-pathway genes were advanced stage cancers (Stage IV) suggests the potential involvement of concurrent alterations of multiple nodes of the PI3K pathway in HNSCC progression.This agrees with the recent report that in addition to PIK3CA mutation, other pathway components such as PIK3R1 and PIK3R2, when mutated, can also serve to drive cell growth/survival (27).
Although the effects of multiple PI3K pathway mutations on cancer cell growth or progression has not been previously investigated, our results support the possibility that genetic alterations at multiple nodes in this oncogenic pathway, a common feature of many solid tumors, may identify a subgroup of cancer patients most likely to respond to PI3K pathway inhibitors.These cumulative findings identify the PI3K pathway as the most frequently mutated mitogenic pathway in HNSCC tumors.Prospective identification of patients whose tumors harbor these mutations is likely to identify a subgroup of individuals who may benefit from treatment with PI3K pathway inhibitors.

Materials and Methods
Additional methods are detailed as Supplementary Information.

Cancer Gene Census Comparison and Co-mutation Analysis
A mutation comparison program was written in Visual Basic for Microsoft Excel to compare the existence of HNSCC mutations vs. a reference list of mutations of interest (in this case, cancer genes).The program allows side-by-side comparison between multiple groups (2 or more) to find out common mutational events, as well as the number of common events in multiple groups.A cancer gene list was generated in each subgroup of tumors by comparing the Cancer Gene Census list (COSMIC Database) with non-synoynomous mutation gene list of each tumor subgroup (the PI3K-mutated tumors, tumors without PI3K-mutation, PIK3CA-mutated tumors, PIK3CA WT tumors) using this comparison program.This analysis allows us to find out the number of cancer genes mutated in each subgroup.

Mutation Validation by Sanger Sequencing
Sanger sequencing was performed on patient tumors that were grafted for tumorgraft studies.
2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103 , HNSCC cell lines containing endogenous PIK3CA(H1047R) mutations (CAL-33 and Detroit 562)(20) demonstrated increased sensitivity to PI3K pathway inhibition by the mTOR/PI3K inhibitor BEZ-235 compared to representative HNSCC cells with WT PIK3CA [SCC-9 and PE/CA-PJ34(clone C12)].Next, mice bearing CAL-33 xenografts were found to be sensitive to BEZ-235 treatment in vivo when compared to vehicle control (Figure 3B).Due to the lack of HPV-HNSCC cell line models that contain PIK3CA mutations, we developed an HPV-positive PIK3CA-mutated HNSCC patient tumorgraft model (E542K) (Figure 3C) to determine the sensitivity of HPV-positive PIK3CA-mutated HNSCC tumors to PI3K pathway targeting.As shown in Figure 3D, BEZ-235 treatment (at 25mg/kg/day by oral gavage) significantly inhibited the growth of HPV-positive PIK3CA-mutated patient tumorgraft in vivo (P<0.0001).Inhibition of tumor growth was accompanied by decreased PI3K signaling as evident by down regulation of p-AKT(S473) (P=0.0124), and p-S6(S235/236) (P<0.0001) in the BEZ-235-treated tumors (Figure These results indicate that activating mutations of PI3K pathway have the potential to serve as biomarkers for treatment selection in HNSCC.Xenografts developed from a HNSCC cell line harboring a PIK3CA mutation (H1047R) were more sensitive to the combination of BEZ-235 plus cetuximab (the only FDA-approved molecular targeting Research.on July 4, 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103

About 25 -
50mg of tumor tissues (pathologically confirmed HNSCC with >70% tumor cell contents) were used for extraction of DNA by QIAamp DNA Mini Kit (Qiagen, Inc, Valencia, CA).Sequencing primers for HNSCC-associated PIK3CA hotspot mutations were synthesized (Sigma-Aldrich, St. Louis, MO) and used for Sanger sequencing.The primer sequences for E542 site mutation are: 5'-cacgagatcctctctctaaaatcactgagcaggag-3' (forward) and 5'ctcctgctcagtgattttagagagaggatctcgtg-3' (reverse).Sanger sequencing was performed at the Genomics and Proteomics Core Laboratories at the University of Pittsburgh.HNSCC Tumorgraft Model and Drug Treatment BEZ-235 was obtained as a kind gift from Novartis, USA.HPV-positive HNSCC patient tumorgrafts were derived under the auspices of an IRB-approved protocol, with PIK3CA WT or PIK3CA(E542K) mutation were implanted into the flanks of NOD SCIDγ mice and treatment was started when tumors became palpable.BEZ-235 (25mg/kg) or vehicle control was given daily by oral gavage.Tumor volumes were measured every two days.Research.on July 4, 2017.© 2013 American Association for Cancer cancerdiscovery.aacrjournals.orgDownloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103

Figure 1 .
Figure 1.Mutations in oncogenic signaling pathways in HNSCC.(A) Mutation rates of the major mitogenic pathways (the PI3K pathway, the MAPK pathway and the JAK/STAT pathway) in 151 HNSCC patient tumors determined by whole exome sequencing.Components of each pathway examined are displayed underneath each pie chart.(B) Bar graph detailing the number of mutations (dark bars) of each particular component of the PI3K pathway as well as the number of HNSCC tumors harboring these mutations (grey bars).(C) PI3K pathway-mutated HNSCC tumors have higher of non-synonymous mutations and (D) cancer gene mutations when compared to HNSCC tumors without any PI3K pathway mutations.Bar graph representing the average number of non-synonymous mutations per tumor (C) and the average number of cancer gene mutations (D) in 151 HNSCC tumors.Statistical significance was calculated by Fisher's Exact test, P<0.0001 (N=151).(E) Graphical representation of the number of HNSCC tumors with mutation of multiple components of the PI3K, MAPK and the JAK/STAT pathways, respectively.

Figure 2 .
Figure 2. PIK3CA mutations in HNSCC tumors.(A) Schematic of all PIK3CA mutations found in 151 HNSCC tumors by whole exome sequencing.The amino acid (a.a.) positions of each domain is shown in grey below each domain.The number of mutational events at each site is indicated by a filled triangle (▲) in the graph above.Blue triangles indicate mutations found in HPV/HNSCC tumors.Keys: ABD: p85 binding domain; RBD: Ras binding domain; C2 Superfamily; Helical: PIK domain; Kinase: Kinase domain of PIK3CA.(B) Effects of PIK3CA mutations on PI3K signaling in HNSCC cells.WT PIK3CA, hotspot mutant H1047R, and novel mutants: R115L, G363A, C971R, and R975S were stably expressed in an HNSCC cell line harboring no endogenous mutations in the PI3K pathway, PE/CA-PJ34 (clone C12)

Table 1 : HNSCC Tumors with Multiple Mutations in a Single Mitogenic Pathway. Table 1: Describes all tumors in our cohort harboring mutations in more than one gene in a defined mitogenic pathway by mutation type and pathological stage.
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on April 25, 2013; DOI: 10.1158/2159-8290.CD-13-0103