Intrapatient comparative analysis of tumor-informed ctDNA and ctHPV-DNA in patients with HPV-driven OPSCC.

Abstract

6075 Background: The rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) is largely attributable to human papillomavirus associated (HPV+) disease, which accounts for ~70% of OPSCC cases. Circulating tumor (ct)DNA has the potential to enable more accurate treatment response assessment, guide response-adaptive management, and detect minimal residual disease to indicate persistence or recurrence. Both mutation-based tumor-informed ctDNA and ctHPV-DNA testing have demonstrated utility in HPV+ disease, but prospective intrapatient evaluations remain limited. A direct comparison of these approaches is essential to determine redundancy versus complementarity and to guide optimal integration into OPSCC patient management. Methods: In an ongoing prospective study, serial plasma samples were obtained from patients with stages I-IV OPSCC undergoing curative intent treatment. Up to 50 patient specific somatic variants were selected based on tumor whole exome sequencing to develop a personalized tumor-informed next generation sequencing (NGS) ctDNA assay (Haystack MRD) for plasma analysis. In patients with HPV+ disease (determined via ISH, IHC, and/or NGS), plasma was also analyzed using an NGS-based assay interrogating 13 high-risk HPV strains (Haystack HPV). Paired intrapatient samples were analyzed using percent agreement with 95% confidence intervals and Cohen’s kappa; concordance of dynamic changes was assessed using Spearman’s correlation. Results: As of January 2026, ctDNA results were available for 111 serial timepoints from 26 patients. The median number of timepoints per patient was 4 (range 1-9). Seventeen patients (65%) had HPV+, and 9 (35%) had HPV− disease. In HPV+ patients, across 85 longitudinal samples collected during multimodal treatment and post-treatment surveillance, mutation-based ctDNA and ctHPV demonstrated high concordance (91%; 95% CI, 82.5–95.2; κ=0.80). Of 30 ctDNA+ samples, 28 were ctHPV+ (93%; 95% CI, 78.7–98.2%), while 49 of 55 ctDNA- samples were ctHPV- (89%; 95% CI, 78.2–94.9%). Discordance was infrequent (8/85, 9.4%), predominantly ctHPV+/ctDNA- (6/85, 7.1%). All ctHPV+/ctDNA- cases occurred during neoadjuvant treatment monitoring and reflected earlier clearance of ctDNA, with ctHPV clearance lagging by several weeks to months. Two low-level (<100 parts per million) ctDNA+/ctHPV- cases were observed in the adjuvant setting. When both analytes were present, dynamic changes in ctDNA and ctHPV levels were highly concordant (Spearman’s ρ=0.94), although ctHPV was consistently detected at higher absolute levels. Conclusions: In HPV-driven OPSCC, tumor-informed ctDNA and ctHPV show high longitudinal concordance and distinct clearance kinetics, with earlier ctDNA clearance. Ongoing analyses will define how these assays can be optimally integrated into response assessment, treatment adaptation, and surveillance strategies.