Blood plasma and oral rinse liquid profiling for human papillomavirus in head and neck cancer – Unmasking false-positive p16 tissue cases and tracking disease dynamics

Abstract Background Human papillomavirus (HPV) is a major etiological factor in a subset of head and neck squamous cell carcinomas (HNSCC), particularly in the oropharynx, and reliable detection is critical for prognosis and treatment decisions. Methods We applied droplet digital PCR (ddPCR) assays targeting HPV16 oncogenes (E6, E7) and the non-oncogenic gene E2 to tumor tissue, blood plasma, and oral rinse samples from 58 HNSCC patients. Results were compared with p16 INK4a immunohistochemistry (IHC). A cohort of 44 non-cancer controls was included. Longitudinal monitoring was performed in 33 patients with serial liquid biopsies. Results ddPCR identified 41% of patients as HPV16-positive in tumor tissue, with higher specificity than p16 INK4a IHC, which yielded 10 likely false positives. Baseline liquid biopsy results were highly concordant with tissue findings, and all 44 controls tested negative. Both blood plasma and oral rinse performed reliably, though oral rinse may be particularly well suited for HPV-related HNSCC due to direct mucosal shedding. In longitudinal analyses, all initially HPV-positive patients showed HPV clearance after surgery. In two cases, loss of clearance (i.e. re-detection of HPV DNA in plasma and oral rinse) preceded clinically confirmed progression, with lead times of 87 and 122 days. Conclusion ddPCR-based HPV testing provides high diagnostic specificity compared with p16 INK4a IHC and allows minimally invasive monitoring. Plasma and oral rinse represent complementary liquid biopsy sources, with oral rinse offering particular advantages for mucosal tumors. Longitudinal testing revealed early molecular recurrence, supporting its potential as a prognostic tool. While promising, the use of liquid biopsy–based HPV detection for screening at-risk but undiagnosed populations remains exploratory and requires further evaluation in larger cohorts.