Spatiotemporal Heterogeneity of HPV Integration Architectures Drive Oncogenic Features of Oropharyngeal Cancer

Viral integration is common in human papillomavirus-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC), but its structural and clinical significance remains unclear. We integrated targeted capture sequencing of 197 HPV+ OPSCCs with long-read nanopore sequencing of 17 tumors and models to characterize HPV-host rearrangements. We developed a novel quantitative informatics framework that classifies integration structures as Type1 (simple, single/low-copy) or Type2 (clustered, high-copy, rearranged). HPV integration occurred in 83% of tumors, with recurrent hotspots including TP63, MYC, CD274, TRAF2, and KMT2C. Type2 events were enriched for HPV E2 disruption, focal human amplifications, preferential transcription, and noncanonical HPV-host RNA fusion isoforms. Type2 tumors showed increased HPV oncogene expression and activation of MYC, NF-{kappa}B/TNF, PI3K-AKT-mTOR, Hippo/YAP-TAZ, and epithelial stemness programs. Multi-region and longitudinal analyses revealed intratumoral heterogeneity and clonal selection of Type2 architectures during progression, supporting integration structure as a potential marker of aggressive behavior and risk stratification.