Abstract: HPV positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) comprises a progressively rising epidemic that will be a public health problem for several decades both because of the persistently low adoption of the vaccine in the United States and the delay from HPV infection to the development of malignancy. For this disease, treatment paradigms have been successful for some patients but are both highly morbid and limited by the need to identify patients at increased risk of treatment failure. We believe that developing an improved understanding of interactions between HPV and the host tumor genome will help resolve this problem, but until now the technical approaches needed to study viral-host interactions remain limited. Recently, we developed a novel targeted capture sequencing (TCS)-based HPV integration caller and assembler. Through analysis of a pilot cohort of tumors and models profiled with matched TCS, long read genome sequencing and transcriptome sequencing, we show our approach has a higher sensitivity and specificity than two other published approaches. Unexpectedly, this analysis uncovered additional preliminary data suggesting that multiple unique structural mechanisms drive HPV integration, and that the structural mechanism of HPV integration may have utility in predicting the impact on chromatin accessibility and transcription of integration adjacent genes. Further, by using our technology to evaluate multiple spatial locations within individual HPV+ tumors, we also develop pilot data suggesting that HPV integration structures are spatially heterogeneous, and that clonal outgrowth of specific “driver” integrations may be a key feature of tumor progression. Now, by leveraging our new technology and biospecimen resources, we propose to test the hypothesis that the structure of an HPV integration event determines the impact on local chromatin accessibility as well as adjacent transcript expression and structure, such that key structural features can be used to identify functionally pivotal integration events as “drivers” of HPV+ OPSCC. We propose the following aims: 1) To determine the accuracy of using local sequence features from short read technologies (TCS and ATAC) as a surrogate for the structural mechanism of HPV integration in recurrent HPV+ OPSCC, 2) To define structural features of HPV integration events critical for regulating the expression, and structure of adjacent host and viral- host fusion transcripts in recurrent HPV+ OPSCC, 3) To identify functionally pivotal driver integration structures through analysis of clonally enriched integrations in HPV+ OPSCCs with poor outcome. Our primary goal is to improve the overall survival of patients with HPV+ OPSCC, while reducing treatment-related morbidity. Our immediate objective is to advance the basic knowledge of HPV+ OPSCC tumor genetics, such that in the future HPV integration structures may be used to help understand treatment outcomes and/or to guide treatme...