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Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

ABSTRACT: High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes.Integration of transcriptionally active hrHPV into cellular intergenic regions associates with tumor behavior by altering gene expression. Mol Cancer Res; 14(10); 941-52. ©2016 AACR.

SUBMITTER: Walline HM

PROVIDER: S-EPMC5065754 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

Walline Heather M HM Komarck Christine M CM McHugh Jonathan B JB Bellile Emily L EL Brenner J Chad JC Prince Mark E ME McKean Erin L EL Chepeha Douglas B DB Wolf Gregory T GT Worden Francis P FP Bradford Carol R CR Carey Thomas E TE

Molecular cancer research : MCR 20160715 10

High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those ...[more]

PMID: 27422711

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Project description:IntroductionMost patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy.MethodsPatients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors.ResultsTen patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples.ConclusionChemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Project description:The global rise of HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) has generated considerable interest underlying its etiology and management. Despite an overall decline in head and neck malignancies, the incidence in OPSCC has by contrast sharply risen, with HPV(+) subtypes now comprising 80% of all OPSCC.1,2 Relative to HPV(-) OPSCC, HPV(+) patients are more responsive to chemoradiation and harbor a 52% risk-of-death reduction.3 Despite this distinct outcome, treatment regimens remain the same for all OPSCC subtypes (smoking-driven and virus-driven), rather than an adaptive approach to what most consider distinct diseases. The short-term effects (e.g., mucositis, odynophagia) and long-term toxicities (e.g., xerostomia, dysphagia, ototoxicity) from treatment substantially affect quality of life, and rival the impact of the cancer itself. Recently published as well as ongoing trials are actively examining deintensification approaches2,4-9, with the goal of diminishing treatment sequelae for HPV(+) subtypes. While deintensification may decrease chemoradiation-related toxicities, it nonetheless may also undertreat a meaningful percentage of HPV(+) patients who may then recur. In examining national trials, 19% of HPV(+) patients had disease progression after therapy, with a two-year survival rate of 60%.10 Current methods to ascertain HPV status involve p16 staining. However, on comparison with gold standard E6/E7 expression by qPCR, p16 harbored a 15% false positivity rate11, suggesting limited utility as a sole biomarker for deintensification. Improved molecular stratification would greatly enhance the clinician’s ability to precisely tailor treatment while minimizing the risk of jeopardizing outcomes. One approach encompasses in-depth proteomic profiling of HPV(+) OPSCC to reveal distinct protein expression profiles and delineate clinically relevant upstream pathways. In turn, these proteomic differences may distinguish higher-risk disease (cases predisposed to recurrence that may benefit from treatment intensification) from lower-risk phenotypes (cases whose treatment response is sufficiently robust to warrant deintensification). Here, two HPV(+) OPSCC cohorts stratified by treatment response are compared via a hybrid data dependent acquisition/data independent acquisition (DDA/DIA) approach via mass spectrometry. We focused on detection of low-abundance proteins to highlight proteomic signatures that can be potentially exploited for treatment stratification.

Project description:IntroductionIn North America and in most European countries, Human Papillomavirus (HPV) is responsible for over 70% of oropharyngeal squamous cell carcinomas. The burden of OPSCC, in high-income countries, has been steadily increasing over the past 20 years. As a result, in the USA and in the UK, the burden of HPV-related oropharyngeal squamous cell carcinoma in men has now surpassed that of cervical cancer in women. However, the oncogenic impact of high-risk HPV integration in oropharyngeal squamous cell carcinomas hasn't been extensively studied. The present study aimed to explore the patterns of HPV integration in oropharyngeal squamous cell carcinomas and to assess the feasibility and reliability of long-read sequencing technology in detecting viral integration events in oropharyngeal head and neck cancers.MethodsA cohort of eight HPV-positive OPSCC pre-treatment patient tumors (four males and four females), were selected. All patients received a p16INK4A positive OPSCC diagnosis and were treated at the McGill University Health Centre, a quaternary center in Montreal. A minimum of 20mg of tumor tissue was used for DNA extraction. Extracted DNA was subjected to Nanopore long-read sequencing to detect and analyze for the presence of high-risk HPV sequences. PCR and Sanger sequencing experiments were performed to confirm Nanopore long-read sequencing readings.ResultsNanopore long-read sequencing showed that seven out of eight patient samples displayed either integrated or episomal high-risk HPV sequences. Out of these seven samples, four displayed verifiable integration events upon bioinformatic analysis. Integration confirmation experiments were designed for all four samples using PCR-based methods. Sanger sequencing was also performed. Four distinct HPV integration patterns were identified: concatemer chromosomal integration in a single chromosome, bi-chromosomal concatemer integration, single chromosome complete integration and bi-chromosomal complete integration. HPV concatemer integration also proved more common than full HPV integration events.Conclusion and relevanceLong-read sequencing technologies can be effectively used to assess HPV integration patterns in OPSCC tumors. Clinically, more research should be conducted on the prognostication value of high-risk HPV integration in OPSCC tumors using long-read sequencing technology.

Project description:BACKGROUND:A subset of patients with human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV-OSCC) experience poor clinical outcomes. The authors of this report explored prognostic risk factors for overall survival (OS) and recurrence-free survival (RFS). METHODS:Patients with incident HPV-OSCC who received treatment at the Johns Hopkins Hospital between 1997 and 2008 and who had tissue available for HPV testing as well as demographic and clinicopathologic information (N?=?176) were included. Tissue was tested for HPV by in situ hybridization (ISH) and/or p16 immunohistochemistry. Demographic and clinicopathologic information was extracted from medical records. RESULTS:In total, 157 of 176 patients (90%) with OSCC had HPV-associated disease (HPV-OSCC). In the patients with HPV-OSCC, the 3-year and 5-year OS rates were 93% (95% confidence interval [CI], 88%-98%) and 89% (95% CI, 81%-97%), respectively. Shorter survival was observed among older patients (hazard ratio [HR], 2.33 per 10-year increase; 95% CI, 1.05-5.16 per 10-year increase; P?=?.038), patients with advanced clinical T classification (HR, 5.78; 95% CI, 1.60-20.8; P?= .007), and patients who were currently using tobacco (HR, 4.38; 95% CI, 1.07-18.0; P?= .04). Disease recurrence was associated with advanced clinical T-classification (HR, 8.32; 95% CI, 3.06-23; P?<?.0001), current/former alcohol use (HR, 13; 95% CI, 1.33-120; P?= .03), and unmarried status (HR, 3.28; 95% CI, 1.20-9.00; P?= .02). Patients who remained recurrence free for 5 years had an 8.6% chance of recurrence by 10 years (1-sided 95% CI upper bound, 19%; P?= .088). CONCLUSIONS:In this study, prognostic risk factors were identified for patients with HPV-OSCC. The observed recurrence rates between 5 years and 10 years after definitive therapy need to be validated in additional studies to determine whether extended cancer surveillance is warranted in this cancer population. Cancer 2013;119:3462-3471.. © 2013 American Cancer Society.

Project description:BackgroundOropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+).MethodsPatients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(-) data, survival analysis for HPV(-) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1.ResultsThe patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(-) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(-) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(-) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(-) HNSCC.ConclusionsHigh CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(-) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(-) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states.

Dataset Information

Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

Publications

Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

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