Methylation-Based ctDNA for Post-Treatment Surveillance of Non-Viral Head and Neck Cancer

Importance: Recurrence rates in head and neck squamous cell carcinoma (HNSCC) remain high, yet current post-treatment surveillance relies primarily on clinical examination and imaging with known limitations. Circulating tumor DNA (ctDNA) has shown promise for recurrence detection in HPV-positive HNSCC, but less is known about its role in non-virally associated disease. Objective: To evaluate the diagnostic performance of a methylation-based ctDNA assay for detecting recurrence during post-treatment surveillance of non-virally associated HNSCC and to assess its prognostic association with survival outcomes. Design, Setting, and Participants: This retrospective single-institution cohort study included patients with non-metastatic non-virally associated HNSCC who underwent definitive therapy and had at least one Guardant360 ctDNA test during post-treatment surveillance between January 2024 and October 2025. Guardant360 identifies tumor-specific DNA methylation patterns via next-generation sequencing of plasma cell-free DNA. A positive result was defined by a methylation signal of 0.05% or greater. Main Outcomes and Measures: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic curve (AUC). Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: Fifty patients were included; most had larynx (38%) or oral cavity (36%) primaries, AJCC stage IVA-IVB disease (72%), T3-T4 tumors (69%), and N2-N3 nodal classification (48%). Treatment was chemoradiation (54%) or surgery (46%). At a median follow-up of 18.0 months, 13 patients recurred, and 12 had positive ctDNA results. Sensitivity, specificity, PPV, and NPV were 76.9%, 94.6%, 83.3%, and 92.1%, respectively (AUC, .858; 95% CI, .733-.982). Positive ctDNA was associated with worse PFS (1-year: 25.0% vs 96.4%; P < .001) and OS (1-year: 75.0% vs 100%; P < .001). Among 10 patients with recurrence and positive ctDNA, ctDNA preceded clinical detection in 6 (60%) by a median of 131 days. An exploratory analysis identified TP53, SETD2, and ROBO2 mutations as associated with recurrence. Conclusions and Relevance: A methylation-based ctDNA assay demonstrated high sensitivity and specificity for detecting disease recurrence in non-virally associated HNSCC. Positive post-treatment ctDNA was associated with inferior survival and preceded clinical detection in most cases, supporting ctDNA integration into surveillance strategies, though prospective validation is warranted.