BACKGROUND
the 5-year survival rate for primary locally advanced head and neck squamous cell carcinoma  is approximately 50 %  <cit> , however recurrent disease carries a dismal prognosis of  <dig>  months with first line chemotherapy  <cit> .
hnscc recurs ~30 % of the time, most often within the first 1– <dig> years of definitive treatment.
pathways associated with progression have been identified using array-based gene expression analysis; however these studies are limited by the lack of rigor using older analysis techniques and normalization techniques, and heterogeneously treated patients.
identification of specific pathways linked to progression after radiation has the promise of informing targeted strategies to improve the prognosis of head and neck cancer.
the cancer genome atlas  is a joint effort of the national cancer institute and the national human genome research institute and has revolutionized the ability of investigators to ask prognostic questions about tumor biology previously limited by suboptimal sample sizes, non-standard sample normalization, and outdated techniques.
hnscc lends itself to this study, as there are diverse epidemiologic risk factors , anatomic subsites , and issues with heterogeneity even within subpopulations of tumor cells within a given patient.
to this end, tcga has generated whole exome sequencing, snp array, dna methylation, rna-seq, and mirna-seq data for a large collaborative cohort of hnscc patients.
their results have recently been published highlighting distinct subgroups within newly diagnosed hnscc patients e.g.
different mutational profiles between hpv-driven and tobacco-related tumors  <cit> .
tcga annotated data on these patients provides an unprecedented opportunity to determine which molecular pathways are most associated with disease progression and survival, in order to gain insight into potentially targetable biology.
we sought to determine what molecular alterations were unique among hnscc progressors in tcga to help inform future patient stratification and adjuvant treatment.
RESULTS
patient demographics
we annotated  <dig> patients in tcga with a median follow up time of  <dig>  days and determined that 29 % of them had progressed .
the mean age was 62 years old, 74 % of patients were male, 90 % where white, and 77 % of patients smoked.
primary sites included the oral cavity , oropharynx , and larynx .
unfortunately, 71 % of patients did not have known hpv status .
stage distribution included 41 % stage i-iii and 57 % stage iv, with 17 % having close or positive margins and 20 % with gross or microscopic nodal extracapsular extension.
at the time of last follow up,  <dig>  % of patients were deceased with a median of 456 days to death.
radiation treatment was part of initial treatment in 70 % of patients and was used as a single modality in 26 % of patients.
chemotherapy was used in 43 % of patients but only in 2 % as a single modality.
among the progressor cohort, primary sites similarly included oral cavity , oropharynx , and larynx .
stage distribution was  <dig>  % stage i-iii and 62 % stage iv, with  <dig>  % having close or positive margins and 36 % with gross or microscopic nodal extracapsular extension.
radiation treatment was used in 88 % of these patients.
median follow up was 411 days and 62 % of patients were deceased at a median of  <dig>  days to death.
the overall demographics reflect what we would expect clinically, and we used this sample set to make comparisons between progressors and nonprogressors.table  <dig> demographics for tcga hnscc patients analyzed in this study
race a
site b
nodal extracapsular spread c
curated therapy: therapy d
a
w white, b black, a asian, ai american indian
b
oc oral cavity, opx oropharynx, l larynx
c
ge gross extension, me microscopic extension, ne no extranodal extension
d
c chemotherapy only, r radiation only, cr chemotherapy & radiation, crtm chemotherapy, radiation & targeted molecular therapy, crtmv chemotherapy, radiation, targeted molecular therapy & vaccine
gray shaded rows indicate significant differences between progressors and nonprogressors
differentially expressed genes
for the primary comparison of progressors versus nonprogressors,  <dig> differentially expressed candidate genes were identified .
to provide biological context, we examined if curated pathway models were enriched for any of the genes in the candidate gene list.
there were a striking number of integrin-related pathways  that were significantly enriched for several of the differentially expressed candidate genes .
in addition, the biocarta fibrinolysis pathway, il- <dig> anti-inflammatory signaling pathway , as well as the kegg complement and coagulation cascades were among other pathways identified as significantly enriched .
we then were interested in determining whether these pathways were reflected in progressors assigned to radiation treatment, as this is the primary adjuvant treatment modality in hnscc.fig.
<dig> examination of differentially expressed  and mutated genes in tcga hnscc subjects in the context of a stylized pathway representation of the reactome integrin cell surface interactions pathway, which was significantly enriched for the putative de candidate genes .
note that three of these genes are also ctcf binding sites: vtn, fgg, and fgb
fig.
<dig> the il- <dig> anti-inflammatory signaling pathway is enriched for putative differentially expressed genes  figure is a stylized representation of the biocarta pathway.
note the following synonyms: jak: jak <dig>  ho-1: hmox <dig>  il-1: il1a.
also note that stat <dig> is also a ctcf binding site
radiation treatment assignment
we examined differential expression in progressors and nonprogressors assigned to radiation treatment to determine the extent of overlap in enriched pathways between this subset and the entire group.
there were  <dig> progressors assigned radiation treatment and  <dig> nonprogressors.
from the  <dig> differentially expressed genes , the overlap of differentially expressed genes from the  <dig> list with the  <dig> list was  <dig>  %., with respect to shared pathways, we identified that the kegg complement and coagulation cascades pathway was also enriched in the progressors assigned to radiation treatment .
other pathways enriched in progressors assigned to radiation included mapk signaling , cell adhesion , fgfr ligand binding and activation, gpcr ligand binding, pi-3 k cascades, and cell-cell junction organization  .
of note, in this cohort as well, interactions with the extracellular environment appeared to be important for progression.
while this was an interesting finding, given our modest sample size after stratification by treatment assignment, we carried out the rest of our analyses with the entire cohort.
we next moved to look at somatic mutations in the entire cohort, comparing progressors and nonprogressors.
somatic mutations
we examined the somatic mutations in progressors and nonprogressors to examine both shared and “unique” mutations at both the variant and gene level .
we note that “unique” is defined as a mutation only being seen in one group and could reflect sampling issues.
when we examined the ratio of variant to gene level mutation for the unique mutations, the ratio was higher for progressors than for nonprogressors .
this is in contrast to the larger range of mutations seen in nonprogressors as well a higher median number of mutations .
this suggests that even though there are more mutations seen in the nonprogressors , there appears to be higher variability relative to the number of genes mutated in progressors compared to nonprogressors.
when pathways were examined for enrichment of somatic mutations , pre-notch transcription and translation , as well as ecm receptor interaction  were among those identified , again highlighting the significance of microenvironment interactions.table  <dig> pathways enriched for genes with increased frequency of mutations in the progressor cohort  compared to nonprogressors
bold indicates genes found mutated only in the progressor cohort
fdr false discovery rate
*same genes are also enriched in pre-notch expression and processing
**same genes are also enriched in ion channel transport
among the somatic mutations, the distribution for only the truncating mutations was examined .
again a larger ratio of variants to gene level mutations was seen in progressors compared to nonprogressors .
we then examined which genes had a significant increase in truncating mutations in progressors compared to nonprogressors.
we filtered the list of genes examined to those genes significant by mutsig annotation .
for the gene ccctc-binding factor , zinc finger chromatin-binding factor and transcriptional regulator, there was a significant occurrence of truncating mutations  in progressors, as well as an increased overall frequency of mutations in the progressors compared to the nonprogressors .
all of the truncating mutations were seen in progresssors .
ctcf was ranked 21st of all mutations in the hnscc complete tcga cohort based on mutsig2cv analysis .
we then examined the differentially expressed candidate genes for those with ctcf binding sites .
when only these genes were utilized in the pathway enrichment analysis, integrin-related pathways were again identified from both pid and reactome .
after curating the differential expression and mutational analysis for progressors and nonprogressors, we chose to focus on integrin and il- <dig> pathways, given these pathways were clearly enriched in the entire progressor cohort.fig.
<dig> lollipop plot highlighting mutations on a linear protein and its domains to examine somatic mutations within the ctcf gene in tcga hnscc annotated progressors and nonprogressors.
green indicates missense mutations and red indicates a truncating mutation.
note  <dig> silent mutations  are not shown.
plot was generated by the mutationmapper visualization tool
a
a
bold indicates genes found mutated only in the progressor cohort
fdr false discovery rate
asame genes also enriched in urokinase-type plasminogen activator  and upar-mediated signaling ; beta <dig> integrin cell surface interactions ; integrin cell surface interactions
integrin signaling deregulation in hnscc progressors
as mentioned above, the reactome integrin cell surface interaction pathway was significantly enriched for the putative differentially expressed candidate genes .
in addition, the frequency of uniquely mutated genes in progressors ranged from  <dig>  to  <dig>  %, with  <dig>  % of the progressors having at least one gene mutated in the pathway .
for the progressors,  <dig>  % had overexpression of at least one gene in this pathway representation .
when evaluating combined mutation and overexpression,  <dig>  % of the progressors had at least one gene aberrant in this pathway.
in addition to this specific reactome pathway, four other reactome pathways relating to integrin signaling were deregulated as well as the pid beta <dig>  beta <dig>  and beta <dig> integrin cell surface interactions pathways .
as previously noted, the radiation treatment assignment cohort also exhibited gene expression aberrancies in extracellular matrix interactions indicating similar biology in this subgroup .
this supports the concept that microenvironmental interactions involving integrins are essential for hnscc progression.
il- <dig> signaling alterations in hnscc progressors
notably, the il- <dig> anti-inflammatory signaling pathway was significantly enriched for putative differentially expressed genes among hnscc progressors .
in addition, hnscc progressors also harbored unique mutations in several members of the pathway including il- <dig>  stats including stat5a, and blvrb .
specifically,  <dig>  % of the progressors had at least one gene overexpressed in this pathway  and  <dig>  % of the progressors had at least one gene mutated.
this was intriguing given the early promise of immune-based therapies in hnscc.
in addition, multiple clotting pathways were found to be differentially expressed  including the fibrinolysis pathway , the intrinsic prothrombin activation pathway , genes involved in platelet aggregation , and complement and coagulation cascades .
these pathways are linked to inflammation  <cit>  as well, and could also point to the importance of this microenvironmental alteration in hnscc progression.
again, similar gene expression deregulation was seen in these pathways for the radiation treatment assignment cohort.
gene signature predicting survival
given the clear unique molecular alterations in hnscc progressors, we were able to generate an 11-gene signature predicting survival based on those genes both differentially expressed and mutated only in progressors.
overall survival using kaplan-maier estimates for those patients harboring the gene signature  was significantly diminished .
median survival for those with alterations in the gene signature was  <dig>  months compared to  <dig>  months for those without the alterations .
of note several of these genes are involved in interactions with the extracellular environment including vtn  and krtap19- <dig> .fig.
<dig> overall survival kaplan-maier estimates for gene signature .
censored patients are indicated by tick marks.
signature is based on those genes differentially expressed and also mutated only in progressors.
red indicates cases with alterations in those genes  median survival in months for those with alterations in the gene signature is  <dig>  months compared to  <dig>  months for those without the alterations
cox modeling of molecular and clinical data
given the prognostic ability of the gene signature above, we were interested in whether we could model survival based on these molecular changes and historically important clinical factors.
we examined key clinical features  as well as the combined mutation and expression gene signature using a multivariate cox proportional hazards modeling approach.
while the overall model was significant , the key factors were interestingly nodal extracapsular spread and alcohol use .
CONCLUSIONS
mining the tcga database provides unprecedented opportunities to unravel unique feature of tumorigenesis in hnscc.
recent published analysis of primary hnscc molecular alterations in patient data from tcga reiterated known hnscc drivers and uncovered distinct molecular alterations between hpv and tobacco-driven tumors  <cit> .
tumor heterogeneity regardless of the pathways involved was linked to reduced overall survival in a companion study of this population, highlighting the genomically unstable nature of this cancer  <cit> .
the heterogeneity of hnscc, in part, has limited improved therapeutic targeting of this disease.
in fact, cetuximab, targeting egfr overexpression, is the only targeted agent used in the treatment of hnscc.
however single agent response rates are low, and in combination with standard chemotherapy for progressive disease, overall survival remains less than one year  <cit> .
many groups are harnessing the power of the tcga data to characterize molecular changes that might predict survival, exemplified by a recent study suggesting an 11-gene signature was able to predict nodal extracapsular spread and also overall survival  <cit> .
our study was designed to identify the genomic differences between progressors and nonprogressors at both the dna and rna level in order to highlight important pathways associated with progression.
interestingly, we uncovered a significant increase in deleterious mutations of ctcf, which is a master chromatin regulator associated with genomic instability and cancer progression  <cit> .
deregulation of this gene could be a contributor to the genomic instability and heterogeneity in hnscc although further mechanistic studies would be required for evaluation.
interestingly, progressors displayed differentially expressed genes harboring ctcf binding sites that participated integrin-related pathways.
this indicates that at least one potential downstream effect of ctcf deregulation could be aberrant microenvironmental interactions involving integrins facilitating hnscc progression.
we identifed integrin and il- <dig> signaling as unique prognostic pathways for hnscc progression.
microenvironmental interaction aberrancies were confirmed by both mutational and expression analysis, and was revealed in progressor cohorts irrespective of their radiation treatment assignment.
this implicates tumor microenvironment interactions in the driving biology of tumor progression for all hnscc patients including those that require radiation as part of their treatment.
intriguingly, both of these pathways have potential promise for guiding targeting therapies.
recently, targeting both egfr overexpression and integrin b <dig> signaling was shown to radiosensitize hnscc cells, building on previous literature demonstrating integrin aberrations in hnscc  <cit> .
further, cilengitide, an αvβ <dig> and αvβ <dig> integrin inhibitor, has been tested in the recurrent/metastatic hnscc setting in combination with cytotoxic chemotherapy, however there was no improvement in progression free survival with addition of cilengitide  <cit> .
this should be reevaluated with the improved biomarkers identified in this study or in the definitive rather than metastatic setting.
alterations in il- <dig> signaling uncovered in this study suggests to an interesting therapeutic angle.
inflammation is a hallmark of hnscc progression based on both animal and human studies  <cit> .
il- <dig> signaling plays a key role in regulation of cancer-associated inflammation including regulation of cd <dig> t cells  <cit> .
as targeting of cd <dig> cells with pd <dig>  pathway inhibitors has shown significant promise in multiple similar tumor types, it has emerged as a attractive targetable pathway in hnscc  <cit> .
potentially, deregulation of the il- <dig> pathway could be used as a biomarker to stratify patients more likely to respond to this therapy.
finally, we identified an 11-gene signature to predict for progression.
in addition to alcohol use and nodal extracapsular spread, a known poor prognostic pathologic factor utilized by other groups  <cit> , this pathway was very powerful in stratifying patients with poor prognosis.
our novel gene signature could be used to identify patients that could benefit from intensified therapy .
the limitations of our study include that we were unable to stratify by hpv status given incomplete clinical annotation within the tcga dataset, we did not have access to recurrent tumor tissue, and we did not stratify by stage.
nevertheless we were able to uncover significant aberrant pathways that, after further mechanistic validation, have potential to open new avenues for therapeutic treatment of recurrent hnscc.

 the 5-year survival rate for primary locally advanced head and neck squamous cell carcinoma is approximately 50 %, however recurrent disease carries a dismal prognosis of  months with first line chemotherapy. hnscc recurs ~30 % of the time, most often within the first 1– years of definitive treatment. pathways associated with progression have been identified using array-based gene expression analysis; however these studies are limited by the lack of rigor using older analysis techniques and normalization techniques, and heterogeneously treated patients. identification of specific pathways linked to progression after radiation has the promise of informing targeted strategies to improve the prognosis of head and neck cancer. the cancer genome atlas is a joint effort of the national cancer institute and the national human genome research institute and has revolutionized the ability of investigators to ask prognostic questions about tumor biology previously limited by suboptimal sample sizes, non-standard sample normalization, and outdated techniques. hnscc lends itself to this study, as there are diverse epidemiologic risk factors, anatomic subsites, and issues with heterogeneity even within subpopulations of tumor cells within a given patient. to this end, tcga has generated whole exome sequencing, snp array, dna methylation, rna-seq, and mirna-seq data for a large collaborative cohort of hnscc patients. their results have recently been published highlighting distinct subgroups within newly diagnosed hnscc patients e.g. different mutational profiles between hpv-driven and tobacco-related tumors. tcga annotated data on these patients provides an unprecedented opportunity to determine which molecular pathways are most associated with disease progression and survival, in order to gain insight into potentially targetable biology. we sought to determine what molecular alterations were unique among hnscc progressors in tcga to help inform future patient stratification and adjuvant treatment.  patient demographics we annotated patients in tcga with a median follow up time of  days and determined that 29 % of them had progressed. the mean age was 62 years old, 74 % of patients were male, 90 % where white, and 77 % of patients smoked. primary sites included the oral cavity, oropharynx, and larynx. unfortunately, 71 % of patients did not have known hpv status. stage distribution included 41 % stage i-iii and 57 % stage iv, with 17 % having close or positive margins and 20 % with gross or microscopic nodal extracapsular extension. at the time of last follow up,  % of patients were deceased with a median of 456 days to death. radiation treatment was part of initial treatment in 70 % of patients and was used as a single modality in 26 % of patients. chemotherapy was used in 43 % of patients but only in 2 % as a single modality. among the progressor cohort, primary sites similarly included oral cavity, oropharynx, and larynx. stage distribution was  % stage i-iii and 62 % stage iv, with  % having close or positive margins and 36 % with gross or microscopic nodal extracapsular extension. radiation treatment was used in 88 % of these patients. median follow up was 411 days and 62 % of patients were deceased at a median of  days to death. the overall demographics reflect what we would expect clinically, and we used this sample set to make comparisons between progressors and nonprogressors.table demographics for tcga hnscc patients analyzed in this study race a site b nodal extracapsular spread c curated therapy: therapy d a w white, b black, a asian, ai american indian b oc oral cavity, opx oropharynx, l larynx c ge gross extension, me microscopic extension, ne no extranodal extension d c chemotherapy only, r radiation only, cr chemotherapy & radiation, crtm chemotherapy, radiation & targeted molecular therapy, crtmv chemotherapy, radiation, targeted molecular therapy & vaccine gray shaded rows indicate significant differences between progressors and nonprogressors differentially expressed genes for the primary comparison of progressors versus nonprogressors, differentially expressed candidate genes were identified. to provide biological context, we examined if curated pathway models were enriched for any of the genes in the candidate gene list. there were a striking number of integrin-related pathways that were significantly enriched for several of the differentially expressed candidate genes. in addition, the biocarta fibrinolysis pathway, il- anti-inflammatory signaling pathway, as well as the kegg complement and coagulation cascades were among other pathways identified as significantly enriched. we then were interested in determining whether these pathways were reflected in progressors assigned to radiation treatment, as this is the primary adjuvant treatment modality in hnscc.fig. examination of differentially expressed and mutated genes in tcga hnscc subjects in the context of a stylized pathway representation of the reactome integrin cell surface interactions pathway, which was significantly enriched for the putative de candidate genes. note that three of these genes are also ctcf binding sites: vtn, fgg, and fgb fig. the il- anti-inflammatory signaling pathway is enriched for putative differentially expressed genes figure is a stylized representation of the biocarta pathway. note the following synonyms: jak: jak ho-1: hmox il-1: il1a. also note that stat is also a ctcf binding site radiation treatment assignment we examined differential expression in progressors and nonprogressors assigned to radiation treatment to determine the extent of overlap in enriched pathways between this subset and the entire group. there were progressors assigned radiation treatment and nonprogressors. from the differentially expressed genes, the overlap of differentially expressed genes from the list with the list was  %., with respect to shared pathways, we identified that the kegg complement and coagulation cascades pathway was also enriched in the progressors assigned to radiation treatment. other pathways enriched in progressors assigned to radiation included mapk signaling, cell adhesion, fgfr ligand binding and activation, gpcr ligand binding, pi-3 k cascades, and cell-cell junction organization. of note, in this cohort as well, interactions with the extracellular environment appeared to be important for progression. while this was an interesting finding, given our modest sample size after stratification by treatment assignment, we carried out the rest of our analyses with the entire cohort. we next moved to look at somatic mutations in the entire cohort, comparing progressors and nonprogressors. somatic mutations we examined the somatic mutations in progressors and nonprogressors to examine both shared and “unique” mutations at both the variant and gene level. we note that “unique” is defined as a mutation only being seen in one group and could reflect sampling issues. when we examined the ratio of variant to gene level mutation for the unique mutations, the ratio was higher for progressors than for nonprogressors. this is in contrast to the larger range of mutations seen in nonprogressors as well a higher median number of mutations. this suggests that even though there are more mutations seen in the nonprogressors, there appears to be higher variability relative to the number of genes mutated in progressors compared to nonprogressors. when pathways were examined for enrichment of somatic mutations, pre-notch transcription and translation, as well as ecm receptor interaction were among those identified, again highlighting the significance of microenvironment interactions.table pathways enriched for genes with increased frequency of mutations in the progressor cohort compared to nonprogressors bold indicates genes found mutated only in the progressor cohort fdr false discovery rate *same genes are also enriched in pre-notch expression and processing **same genes are also enriched in ion channel transport among the somatic mutations, the distribution for only the truncating mutations was examined. again a larger ratio of variants to gene level mutations was seen in progressors compared to nonprogressors. we then examined which genes had a significant increase in truncating mutations in progressors compared to nonprogressors. we filtered the list of genes examined to those genes significant by mutsig annotation. for the gene ccctc-binding factor, zinc finger chromatin-binding factor and transcriptional regulator, there was a significant occurrence of truncating mutations in progressors, as well as an increased overall frequency of mutations in the progressors compared to the nonprogressors. all of the truncating mutations were seen in progresssors. ctcf was ranked 21st of all mutations in the hnscc complete tcga cohort based on mutsig2cv analysis. we then examined the differentially expressed candidate genes for those with ctcf binding sites. when only these genes were utilized in the pathway enrichment analysis, integrin-related pathways were again identified from both pid and reactome. after curating the differential expression and mutational analysis for progressors and nonprogressors, we chose to focus on integrin and il- pathways, given these pathways were clearly enriched in the entire progressor cohort.fig. lollipop plot highlighting mutations on a linear protein and its domains to examine somatic mutations within the ctcf gene in tcga hnscc annotated progressors and nonprogressors. green indicates missense mutations and red indicates a truncating mutation. note silent mutations are not shown. plot was generated by the mutationmapper visualization tool a a bold indicates genes found mutated only in the progressor cohort fdr false discovery rate asame genes also enriched in urokinase-type plasminogen activator and upar-mediated signaling; beta integrin cell surface interactions; integrin cell surface interactions integrin signaling deregulation in hnscc progressors as mentioned above, the reactome integrin cell surface interaction pathway was significantly enriched for the putative differentially expressed candidate genes. in addition, the frequency of uniquely mutated genes in progressors ranged from to  %, with  % of the progressors having at least one gene mutated in the pathway. for the progressors,  % had overexpression of at least one gene in this pathway representation. when evaluating combined mutation and overexpression,  % of the progressors had at least one gene aberrant in this pathway. in addition to this specific reactome pathway, four other reactome pathways relating to integrin signaling were deregulated as well as the pid beta beta and beta integrin cell surface interactions pathways. as previously noted, the radiation treatment assignment cohort also exhibited gene expression aberrancies in extracellular matrix interactions indicating similar biology in this subgroup. this supports the concept that microenvironmental interactions involving integrins are essential for hnscc progression. il- signaling alterations in hnscc progressors notably, the il- anti-inflammatory signaling pathway was significantly enriched for putative differentially expressed genes among hnscc progressors. in addition, hnscc progressors also harbored unique mutations in several members of the pathway including il- stats including stat5a, and blvrb. specifically,  % of the progressors had at least one gene overexpressed in this pathway and  % of the progressors had at least one gene mutated. this was intriguing given the early promise of immune-based therapies in hnscc. in addition, multiple clotting pathways were found to be differentially expressed including the fibrinolysis pathway, the intrinsic prothrombin activation pathway, genes involved in platelet aggregation, and complement and coagulation cascades. these pathways are linked to inflammation as well, and could also point to the importance of this microenvironmental alteration in hnscc progression. again, similar gene expression deregulation was seen in these pathways for the radiation treatment assignment cohort. gene signature predicting survival given the clear unique molecular alterations in hnscc progressors, we were able to generate an 11-gene signature predicting survival based on those genes both differentially expressed and mutated only in progressors. overall survival using kaplan-maier estimates for those patients harboring the gene signature was significantly diminished. median survival for those with alterations in the gene signature was  months compared to  months for those without the alterations. of note several of these genes are involved in interactions with the extracellular environment including vtn and krtap19-.fig. overall survival kaplan-maier estimates for gene signature. censored patients are indicated by tick marks. signature is based on those genes differentially expressed and also mutated only in progressors. red indicates cases with alterations in those genes median survival in months for those with alterations in the gene signature is  months compared to  months for those without the alterations cox modeling of molecular and clinical data given the prognostic ability of the gene signature above, we were interested in whether we could model survival based on these molecular changes and historically important clinical factors. we examined key clinical features as well as the combined mutation and expression gene signature using a multivariate cox proportional hazards modeling approach. while the overall model was significant, the key factors were interestingly nodal extracapsular spread and alcohol use.  mining the tcga database provides unprecedented opportunities to unravel unique feature of tumorigenesis in hnscc. recent published analysis of primary hnscc molecular alterations in patient data from tcga reiterated known hnscc drivers and uncovered distinct molecular alterations between hpv and tobacco-driven tumors. tumor heterogeneity regardless of the pathways involved was linked to reduced overall survival in a companion study of this population, highlighting the genomically unstable nature of this cancer. the heterogeneity of hnscc, in part, has limited improved therapeutic targeting of this disease. in fact, cetuximab, targeting egfr overexpression, is the only targeted agent used in the treatment of hnscc. however single agent response rates are low, and in combination with standard chemotherapy for progressive disease, overall survival remains less than one year. many groups are harnessing the power of the tcga data to characterize molecular changes that might predict survival, exemplified by a recent study suggesting an 11-gene signature was able to predict nodal extracapsular spread and also overall survival. our study was designed to identify the genomic differences between progressors and nonprogressors at both the dna and rna level in order to highlight important pathways associated with progression. interestingly, we uncovered a significant increase in deleterious mutations of ctcf, which is a master chromatin regulator associated with genomic instability and cancer progression. deregulation of this gene could be a contributor to the genomic instability and heterogeneity in hnscc although further mechanistic studies would be required for evaluation. interestingly, progressors displayed differentially expressed genes harboring ctcf binding sites that participated integrin-related pathways. this indicates that at least one potential downstream effect of ctcf deregulation could be aberrant microenvironmental interactions involving integrins facilitating hnscc progression. we identifed integrin and il- signaling as unique prognostic pathways for hnscc progression. microenvironmental interaction aberrancies were confirmed by both mutational and expression analysis, and was revealed in progressor cohorts irrespective of their radiation treatment assignment. this implicates tumor microenvironment interactions in the driving biology of tumor progression for all hnscc patients including those that require radiation as part of their treatment. intriguingly, both of these pathways have potential promise for guiding targeting therapies. recently, targeting both egfr overexpression and integrin b signaling was shown to radiosensitize hnscc cells, building on previous literature demonstrating integrin aberrations in hnscc. further, cilengitide, an αvβ and αvβ integrin inhibitor, has been tested in the recurrent/metastatic hnscc setting in combination with cytotoxic chemotherapy, however there was no improvement in progression free survival with addition of cilengitide. this should be reevaluated with the improved biomarkers identified in this study or in the definitive rather than metastatic setting. alterations in il- signaling uncovered in this study suggests to an interesting therapeutic angle. inflammation is a hallmark of hnscc progression based on both animal and human studies. il- signaling plays a key role in regulation of cancer-associated inflammation including regulation of cd t cells. as targeting of cd cells with pd pathway inhibitors has shown significant promise in multiple similar tumor types, it has emerged as a attractive targetable pathway in hnscc. potentially, deregulation of the il- pathway could be used as a biomarker to stratify patients more likely to respond to this therapy. finally, we identified an 11-gene signature to predict for progression. in addition to alcohol use and nodal extracapsular spread, a known poor prognostic pathologic factor utilized by other groups, this pathway was very powerful in stratifying patients with poor prognosis. our novel gene signature could be used to identify patients that could benefit from intensified therapy. the limitations of our study include that we were unable to stratify by hpv status given incomplete clinical annotation within the tcga dataset, we did not have access to recurrent tumor tissue, and we did not stratify by stage. nevertheless we were able to uncover significant aberrant pathways that, after further mechanistic validation, have potential to open new avenues for therapeutic treatment of recurrent hnscc. 
mutational analysis highlighted a potential role for ctcf, a crucial regulator of long-range chromatin interactions, in head and neck cancer progression. head and neck cancer is morbid with a poor prognosis that has not significantly improved in the past several decades. cox modeling of both key clinical features and the signature was significant with the greatest prognostic improvement seen in the model based on nodal extracapsular spread and alcohol use alone. we identified head and neck cancer patients in the cancer genome atlas with sufficient clinical annotation regarding therapeutic treatment and disease progression to identify progressors and non-progressors. an 11-gene signature derived from a combination of unique mutations and differential expression was identified. in addition, we identified an 11-gene signature with implications for patient prognostication. a number of genes were uniquely mutated in the progressor cohort including increased frequency of truncating mutations in ctcf. the purpose of this study was to identify biological pathways underlying progressive head and neck cancer to inform prognostic and adjuvant strategies. molecular analyses of head and neck cancer tumors that progressed despite treatment, identified il- and integrin pathways to be strongly associated with cancer progression. hnsccctcfhead and neck canceril-10integrinrna-seqtcgamutationsignaturenih/nci5p30ca06533)mcweeney shannon issue-copyright-statement© the author 2016 this signature was associated with decreased overall survival.
 head and neck cancer is morbid with a poor prognosis that has not significantly improved in the past several decades. the purpose of this study was to identify biological pathways underlying progressive head and neck cancer to inform prognostic and adjuvant strategies. we identified head and neck cancer patients in the cancer genome atlas with sufficient clinical annotation regarding therapeutic treatment and disease progression to identify progressors and non-progressors. we compared primary tumor gene expression and mutational status between these two groups.  genes were differentially expressed between progressors and nonprogressors. pathway analyses revealed deregulation of multiple pathways related to integrin signaling as well as il- signaling. a number of genes were uniquely mutated in the progressor cohort including increased frequency of truncating mutations in ctcf. an 11-gene signature derived from a combination of unique mutations and differential expression was identified. this signature was associated with decreased overall survival. cox modeling of both key clinical features and the signature was significant with the greatest prognostic improvement seen in the model based on nodal extracapsular spread and alcohol use alone.  molecular analyses of head and neck cancer tumors that progressed despite treatment, identified il- and integrin pathways to be strongly associated with cancer progression. in addition, we identified an 11-gene signature with implications for patient prognostication. mutational analysis highlighted a potential role for ctcf, a crucial regulator of long-range chromatin interactions, in head and neck cancer progression. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hnsccctcfhead and neck canceril-10integrinrna-seqtcgamutationsignaturenih/nci5p30ca06533)mcweeney shannon issue-copyright-statement© the author 2016 