Longitudinal changes in circulating HPV16 DNA during chemoradiotherapy and their association with tumor burden in oropharyngeal cancer

Circulating HPV DNA has been investigated as a biomarker in HPV-positive oropharyngeal cancer, but its longitudinal behavior during chemoradiotherapy remains incompletely characterized. We evaluated dynamic changes in plasma HPV16 ctDNA and their association with tumor burden and clinical outcomes. In this prospective study, patients with p16-positive oropharyngeal squamous cell carcinoma underwent serial plasma HPV16 ctDNA measurements using droplet digital PCR before, during, and after definitive chemoradiotherapy. ctDNA levels were analyzed in relation to tumor volume, treatment response, and recurrence. Total 61 patients with p16-positive oropharyngeal cancer were enrolled. Among 42 patients with HPV16-positive tumors, baseline HPV16 ctDNA levels were at or above the predefined limit of detection (LoD) in 26 patients (61.9%). Three distinct clearance patterns were observed: continuous decline (Type I, n = 16), transient increase followed by decline (Type II, n = 12), and persistently below-LoD levels (Type III, n = 14). Maximum ctDNA levels were significantly correlated with nodal tumor volume (Type I: r = 0.57, p = 0.02; Type II: r = 0.61, p = 0.03), but not with primary tumor volume. All patients achieved complete response. During follow-up, ctDNA elevation preceded radiologic recurrence in two patients. Notably, some patients with persistently undetectable ctDNA subsequently developed distant metastases, suggesting that absence of detectable ctDNA does not necessarily indicate favorable disease biology. Longitudinal assessment of circulating HPV16 ctDNA during chemoradiotherapy reflects tumor burden, particularly nodal disease, and may provide clinically relevant information for treatment monitoring.