Methylation-Based ctDNA for Post-Treatment Surveillance of Non-Viral Head and Neck Cancer
- 1Department of Radiation Oncology, Montefiore Einstein Comprehensive Cancer Center
Abstract
ImportanceRecurrence 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.
ObjectiveTo 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 ParticipantsThis 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 MeasuresSensitivity, 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.
ResultsFifty 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 RelevanceA 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.
Key PointsO_ST_ABSQuestionC_ST_ABSCan a methylation-based circulating tumor DNA assay detect recurrence during post-treatment surveillance of non-virally associated head and neck squamous cell carcinoma?
FindingsIn this cohort study of 50 patients, the ctDNA assay had a sensitivity of 76.9% and specificity of 94.6% for detecting recurrence (AUC of 0.858). Positive ctDNA preceded clinical detection in 60% of recurrences by a median of 131 days and was associated with worse progression-free and overall survival.
MeaningMethylation-based ctDNA may be a useful tool for surveillance of recurrence in non-virally associated HNSCC.
Related
- Dynamic ctDNA-MRD in breast cancer: subtype-specific implications for perioperative decision-making, postoperative surveillance, and MRD-guided therapy.
- Urinary dual-site proenkephalin (PENK) methylation testing for detection of high-grade or invasive recurrence during surveillance of non-muscle-invasive bladder cancer
- Urine DNA methylation as a potential diagnostic and surveillance tool in non-muscle invasive bladder cancer in Thailand: A pilot study
- Post-Treatment Surveillance to Detect Recurrent Disease in Esophagogastric Cancer: A Review of Current Practice and Future Perspectives
- From Radical Treatment to Structured Active Surveillance: A Narrative Review of Low-Risk Localized Prostate Cancer
BibTeX
@misc{tomer2026methylationbased,
title = {{Methylation-Based ctDNA for Post-Treatment Surveillance of Non-Viral Head and Neck Cancer}},
author = {D. Tomer and P. Brodin and N. Malik and S. Mayat and C. Velten and B.-H. Rhieu and E. Castellucci and B. Schiff and V. Mehta and R. Smith and S. Kalnicki and M. Garg and R. Kabarriti},
howpublished = {medRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.17.26363361},
url = {https://www.medrxiv.org/content/10.64898/2026.09.17.26363361v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Methylation-Based ctDNA for Post-Treatment Surveillance of Non-Viral Head and Neck Cancer
AU - D. Tomer
AU - P. Brodin
AU - N. Malik
AU - S. Mayat
AU - C. Velten
AU - B.-H. Rhieu
AU - E. Castellucci
AU - B. Schiff
AU - V. Mehta
AU - R. Smith
AU - S. Kalnicki
AU - M. Garg
AU - R. Kabarriti
PY - 2026
DA - 2026/09/17
PB - medRxiv
DO - 10.64898/2026.09.17.26363361
UR - https://www.medrxiv.org/content/10.64898/2026.09.17.26363361v1
AB - ImportanceRecurrence 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. ObjectiveTo 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 ParticipantsThis 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 MeasuresSensitivity, 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. ResultsFifty 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 RelevanceA 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. Key PointsO_ST_ABSQuestionC_ST_ABSCan a methylation-based circulating tumor DNA assay detect recurrence during post-treatment surveillance of non-virally associated head and neck squamous cell carcinoma? FindingsIn this cohort study of 50 patients, the ctDNA assay had a sensitivity of 76.9% and specificity of 94.6% for detecting recurrence (AUC of 0.858). Positive ctDNA preceded clinical detection in 60% of recurrences by a median of 131 days and was associated with worse progression-free and overall survival. MeaningMethylation-based ctDNA may be a useful tool for surveillance of recurrence in non-virally associated HNSCC.
ER -