HPV16 Genomic Status Stratifies Risk of Disease Recurrence in Oropharyngeal Squamous Cell Carcinoma

Abstract Purpose: A subset of patients with human papillomavirus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) develops disease recurrence despite generally favorable outcomes. Reliable biomarkers for risk stratification remain lacking. We investigated whether comprehensive viral genomic profiling refines prognostic classification. Experimental Design: Tumor samples from 100 patients with p16-positive and HPV DNA–positive OPSCC enrolled in 2 independent cohorts were analyzed using targeted viral capture sequencing. Viral genotype, sublineage, single-nucleotide variation, virus–host integration sites, viral structural variants (SV), clonality, and viral copy number were characterized. Associations with cumulative incidence of progression, progression-free survival (PFS), and overall survival were assessed at 60 months. Results: HPV16 was detected in 94 tumors, 83 of which belonged to the HPV16_A1 sublineage. Within this sublineage, 20 tumors harbored strictly episomal viral genomes, whereas 63 exhibited integration and/or viral SV. No progression events occurred in the strictly episomal HPV16_A1 subgroup, whereas 12 occurred among HPV16_A1 tumors with an integration and/or SV. At 60 months, no progression events were observed in strictly episomal HPV16_A1 tumors compared with a 23.1% cumulative incidence of progression in HPV16_A1 tumors with an integration and/or SV. Previously reported high-risk single-nucleotide variants and a novel upstream regulatory region variant were associated with worse PFS. Integration sites were enriched in open chromatin and promoter regions and clustered within viral E2 and E4 genes. Conclusions: Within HPV16_A1 tumors, viral genomic architecture defines clinically distinct subgroups. Strictly episomal (wild-type) genomes identify a low-risk population, whereas integration and SV characterize higher-risk disease.