Abstract 49: Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets
Authors: Holt Jeremiah , Jo Heejoon , Zhao Xiaobei , Choi Hyo Young , Walter Vonn , Little Paul , Wahle Benjamin , Mazul Angela , Zevallos Jose P. , Hayes David Neil 
Published: June 2022

Abstract
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&lt;0.001, RBL2 P=0.01), while RBL1 (p107) was significant for both HPV(+) and HPV(-) samples (P&lt;0.001, P=0.02 respectively). Higher fractions of mutations in HPV(+) vs. HPV(-) tumors were observed for EP300 (13% vs 6%, Fisher’s exact test P=0.006), RB1 (7% vs 3%, P=0.07), RBL2 (3% vs 1%, P=0.09), and FOXM1 (4% vs 1%, P=0.02), another cell cycle pathway target of E7, but not for RBL1 (4% vs. 3%, P=1). Lower copy numbers were detected in HPV(+) tumors compared to HPV(-) for chromosome regions containing RB1 (P=0.03), RBL1 (P=0.08), RBL2 (P&lt;0.001), and for another inhibitory target of E6, FADD (P&lt;0.001), which is an activator of the proapoptotic protein caspase-8. Additionally, significant copy number gains were detected in HPV(+) vs. HPV(-) tumors within the genomic region containing E2F1 (P&lt;0.001), the transcription factor whose inhibition by RB family proteins is abrogated in the presence of E7, thus leading to abnormal cell cycle progression and proliferation.
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.