Next-Generation Sequencing of HPV ctDNA Detects Copy Number Dynamics and Integration as Prognostic Biomarkers in Metastatic Anal Cancer

Traditional circulating HPV DNA tests have had limited scope, so we examined the clinical usefulness of a newly developed next-generation sequencing (NGS)–based HPV ctDNA platform for people with locally advanced or metastatic squamous cell carcinoma of the anal canal (mSCCA). Plasma samples from patients with mSCCA were processed to extract ctDNA, which was analyzed using a 1.4 Mb hybrid-capture panel designed to cover the complete genomic sequences of all 193 known HPV types. A specialized analytic workflow identified HPV genotype, copy number (CN), and integration breakpoints. Seventy-seven plasma specimens collected from 28 individuals with HPV-associated SCCA were reviewed retrospectively. HPV ctDNA was measurable in 26 of these patients (93%), including those with rare viral subtypes. Median HPV CN values were significantly higher in individuals with metastatic disease compared with those with locally recurrent or unresectable tumors (p = 0.043). Fluctuations in HPV CN tracked closely with imaging-based treatment response (p = 0.027). Viral integration was observed in 23 patients (82%), while the remainder showed patterns consistent with episomal HPV. Patients with more extensive integration (mean ≥1 event across submitted samples) experienced notably poorer overall survival once immunotherapy was initiated (13.6 months compared with 36.0 months; p = 0.003). This HPV-focused NGS ctDNA approach demonstrated that changes in HPV CN parallel therapeutic outcomes, and that HPV integration detectable in blood is associated with inferior prognosis.