Clinical validation of an HPV whole-genome sequencing assay for MRD detection in patients with HPV+ head and neck cancer treated with surgery.

Surgery is a standard treatment for early-stage human papillomavirus-associated head and neck cancer (HPV+HNC), yet adjuvant therapy decisions rely on clinicopathologic features with limited prognostic accuracy. Circulating tumor HPV DNA is a promising biomarker, but current assays lack the sensitivity needed for detecting minimal residual disease after surgery. This study evaluated the prognostic value of an HPV whole-genome sequencing assay, HPV-DeepSeek, and compared this with existing methods and clinical standards. One hundred three patients with stage I to IV HPV+HNC treated with surgery were prospectively enrolled (NCT06730412). Blood samples were collected before surgery, postoperatively, and during surveillance and were analyzed using HPV-DeepSeek and droplet digital polymerase chain reaction (PCR) assays. After a median follow-up of 27 months, patients with circulating tumor HPV DNA detected after surgery had worse 2-year disease-free and overall survival than those without detection. Detection after completion of all treatment was also associated with poorer outcomes. Minimal residual disease status was a stronger predictor of recurrence than standard clinicopathologic criteria. Molecular recurrence was identified up to 17.5 months earlier than clinical recurrence and nearly twice as early as with droplet digital PCR. These findings demonstrate that sensitive HPV whole-genome sequencing enables accurate detection of minimal residual disease, early identification of recurrence, and improved postoperative risk stratification, supporting its potential as a tool for guiding personalized adjuvant therapy in HPV+HNC.