HPV integration in head and neck cancer: downstream splicing events and expression ratios linked with poor outcomes.

PURPOSE: HPV integration (HPVint) is associated with carcinogenesis and tumor progression in HPV-associated cancers, including head and neck squamous cell carcinomas (HNSCC). While its impact on human DNA has been well characterized, its relationship with clinical outcomes remains unconfirmed. EXPERIMENTAL DESIGN: We analyzed HPVint events from 261 HPV-associated HNSCC bulk and single-cell RNA-seq samples from five cohorts, including 62 from a new University of Michigan cohort, and DNA HPVint events from 102 HPV(+) HNSCC participants in two of the cohorts. We investigated the consequences of HPVint both with respect to human and HPV gene expression and clinical outcomes (recurrence and overall survival). RESULTS: By leveraging this large meta-cohort of HNSCC, we first reveal an oncogenic gene network based on the recurrent HPV integration locations in the human genome and gene expression alterations, highlighting key recurrent and overexpressed genes including NR4A2, CD274, CCER1 and genes from the CAMK and KLF families. We then stratify HPVint-positive participants by risk using HPV RNA features, specifically spliced HPV-human fusion transcripts (E1* integration) and HPV gene expression ratios (HGER), showing that subsets of participants have worse clinical outcomes based on these two candidate biomarkers. CONCLUSIONS: By focusing on RNA instead of DNA, we expand our understanding of the carcinogenic mechanisms of HPVint, in part addressing the conflicting findings of whether HPVint is associated with aggressive phenotypes and worse clinical consequences and provide potential biomarkers to advance precision oncology in HPV-associated HNSCC. Newly identified genes with recurrent integration events may serve as candidates for targeted therapy.