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Integration of viral DNA into the host genome has been implicated in human papillomavirus (HPV) associated tumorigenesis, and studies in HPV associated head and neck squamous cell carcinoma (HNSCC) attempting to classify viral integration status have reported associations among HPV status, viral load, and overall survival. However, few genomic studies have correlated HPV structure with commonly occurring somatic mutations in HNSCC. Here, we conducted hybrid-capture DNA sequencing using a panel of over 800 cancer related genomic targets, as well as probes specific for the full HPV16 and HPV18 genomes, in a novel cohort of 525 HNSCC cases. Paired reads were aligned to a human reference containing complete viral genomes, including all high-risk HPV types, which allowed for robust identification of HPV(+) tumors and specific HPV type. Variant calling and filtering, followed by pathway analysis of significantly mutated genes, defined the catalog of somatic driver alterations in HNSCC. Viral genome structure, integration status, and viral load were determined via initial copy number analysis of HPV-specific reads, followed by identification of viral/human breakpoints and indels with variant calling algorithms. Manual review was required in some cases due to variability in read coverage and alignment to HPV genome. We identified 252 HPV(+) tumors, the majority of which were HPV16 (n=228, 90%). Analysis of HPV16(+) tumors revealed five classes with variable viral loads: pure episome (EPI, n=54), episomal rearranged (EPI-R, n=51), episomal chimeric (EPI-C, n=22), integrated (INT, n=92), and integrated deleted (INT-A, n=12). Integrated tumors had at least one integration event (INT) and a few exhibited recurrent deletions in the viral genome (INT-A). Episomal samples had either complete HPV genomes (EPI), circular genomes with one or more indels (EPI-R), or circular genomes with HPV and human DNA (EPI-C). Interestingly, PIK3CA, the most frequently mutated gene in HPV(+) HNSCC, was mutated at a higher frequency in all integrated samples compared to all episomal types (44% vs 23%, p=0.005). We also noticed more deleterious mutations in innate immunity genes (HLA-A/B, B2M, TRAF3, BIRC3) in the episomal samples (16% vs 8%), which may allow cells with intact HPV genomes to evade anti-viral immune responses. Additionally, mutations in oxidative stress response genes that lead to the constitutive activation of NRF2 (NFE2L2, CUL3, KEAP1) were more frequent in integrated samples (11% vs 4%), which could confer radiotherapy resistance and a worse prognosis. Overall, using DNA sequencing and integrative genomic analysis in the largest cohort of HPV(+) HNSCC to date, we developed a novel classification method based on viral genome structure which associates with host somatic alterations that may contribute to variable outcomes in HNSCCs. Citation Format: Jeremiah Ray Holt, Xiaobei Zhao, Hyo Young Choi, Paul Little, Angela L. Mazul, Benjamin Wahle, Vonn Walter, Jose P. Zevallos, David Neil Hayes. Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6062.

Purpose: Previous studies implicated viral oncoproteins E6 and E7, of which targets include but are not limited to tumor suppressors p53 and pRb, in human papillomavirus (HPV) associated tumorigenesis. However, HPV-associated tumors must accumulate additional genomic alterations as HPV oncoproteins alone are insufficient for tumor formation in vivo. Experimental Design: Targeted sequencing was conducted with a panel of over 800 cancer related genomic regions and genes in a novel cohort of 525 head and neck squamous cell carcinoma (HNSCC) patients, including 252 HPV(+) and 273 HPV(-) tumors. Variant calling and filtering, followed by analysis with computational tools such as MutSig for somatic mutations and DiNAMIC.Duo for recurrent copy number alterations (CNAs), defined the catalog of driver alterations that are specific to HPV(+) and HPV(-) tumors. Pathway analysis was then used to elucidate the functional role of somatic alterations in the context of previously described HNSCC perturbations and HPV pathophysiology. Results: Integrative genomic analysis in one of the largest HPV(+) HNSCC cohorts to date revealed differential patterns of somatic mutations and CNAs in E6/E7 targets between HPV(+) and HPV(-) tumors. Specifically, EP300, an activator of p53 and target of E6, and retinoblastoma (RB) family members RB1 (pRb) and RBL2 (p130), which are known targets of E7 mediated degradation, were selected as significantly mutated by MutSig in HPV(+) samples only (EP300 & RB1 P Conclusions: Previous studies suggest HPV-associated tumors lack significant levels of somatic alterations in canonical tumor suppressor genes that are known targets of viral oncoproteins. Here, we show additional selective pressure exists for somatic alteration of E6/E7 target genes in at least a subset of HPV(+) HNSCCs, which further deregulate cell cycle and apoptosis pathways in these tumors. Citation Format: Jeremiah Holt, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Paul Little, Benjamin Wahle, Angela Mazul, Jose P. Zevallos, David Neil Hayes. Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 49.