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

Summary 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-κB/TNFα, 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.