Abstract LB252: HPV integration events are heterogenous, clonally selected and associated with spatially distinct transcriptomic profiles in aggressive HPV positive oropharyngeal squamous cell carcinoma
Authors: Gu Wenjin , Bhangale Apurva , Brummel Collin V. , Gensterblum-Miller Elizabeth , Spector Matthew E. , Mills Ryan E. , Brenner Chad 
Published: April 2023

Abstract
HPV positive Oropharyngeal Squamous Cell Carcinoma (HPV positive OPSCC) is rising in incidence and, for patients that recur, survival is extremely poor. Recent technological advances have led to a better understanding of patterns of HPV integration in primary tumors but have not yet tested the evolution of these events in deadly forms of the disease. Therefore, we performed multi-region sequencing analysis on a cohort of 34 recurrent and metastatic patients with HPV positive OPSCC (29% survival rate) plus one recurrent HPV positive model pair, UPCI-90 and UPCI-152. We then used our recently developed capture-based sequencing HPV integration caller and assembler pipeline, SearcHPV, to identify patterns of viral integration into the host genome and Oxford Nanopore long-read whole genome sequencing to validate the integration events in selected cases. In total, 1591 HPV integrations were called and assembled. Unexpectedly, our analysis revealed that HPV integration events are heterogenous as multiple independent integration events were observed in different spatial regions of the same tumor. Further, when comparing independent and anatomically distinct regions of recurrence from the same patient, independent integration events were also observed. Heterogeneity of integration structures was found to include variation of local HPV copy number, distribution of the specific HPV integration sites within the structures as well as distinct characteristics of rearrangement breakpoints (human-HPV, HPV-HPV, human-human). Given this observation, we then evaluated the hypothesis that HPV integration events could be clonally selected by comparing the integration profiles from a cohort of matched primary and recurrent tumors. In this cohort, 64 integration events (11 exact same; 53 clustered in the same genes) were maintained throughout tumor progression to recurrence, while other integration events were lost, supporting a model of clonal evolution with driver and passenger integrations. We then explored this further using one recurrent HPV positive model pair, UPCI-90 and UPCI-152, using long-read whole genome sequencing which validated the heterogeneity and clonal selection of HPV integration events. Finally, to test if differences in HPV integration content in spatially distinct regions could drive spatially distinct phenotypic differences, we performed multi-region RNA-seq analysis on 8 HPV16 positive OPSCCs, which demonstrated spatial differences in HPV16 E7 expression, that were validated with RNAscope assays, as well as statistically significant spatial enrichment of independent oncogenic gene sets. Collectively, we report evidence showing that HPV integration events are heterogenous, clonally selected during progression and associated with distinct transcriptomic profiles. Based on this data, we now propose a new working model of disease pathogenesis in which heterogeneity and clonal evolution of HPV integration events serve as a critical driver of the disease process.
Citation Format: Wenjin Gu, Apurva Bhangale, Collin V. Brummel, Elizabeth Gensterblum-Miller, Matthew E. Spector, Ryan E. Mills, Chad Brenner. HPV integration events are heterogenous, clonally selected and associated with spatially distinct transcriptomic profiles in aggressive HPV positive oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB252.