A seven-gene 5-hydroxymethylcytosine signature in circulating cell-free DNA for prognostic stratification of nasopharyngeal carcinoma

Abstract

Background: Circulating cell-free DNA (cfDNA) 5-hydroxymethylcytosine (5hmC) is a promising epigenetic biomarker in cancer. Its prognostic role in nasopharyngeal carcinoma (NPC), however, remains unclear.

Methods: Genome-wide 5hmC profiling was conducted using 5hmC-Seal sequencing on cfDNA from 174 newly diagnosed NPC patients. Patients were randomly assigned to training (n = 105) and test (n = 69) sets. Differential analysis was performed by survival outcome, EBV status, and tumor stage. The primary endpoint was overall survival (OS); the secondary endpoint was event-free survival (EFS). A prognostic score based on a seven-gene 5hmC signature was developed using LASSO-Cox regression in the training set and validated in the test cohort. A nomogram integrating the 5hmC score, tumor stage, and EBV status was constructed. Model performance was assessed via Kaplan-Meier survival analysis, time-dependent ROC curves, calibration plots, and decision curve analysis (DCA).

Results: Marked 5hmC differences were detected between survivors and non-survivors. The 5hmC score stratified OS with AUCs of 0.83 (3-year) and 0.87 (5-year) in the training set and 0.78 (3-year) and 0.80 (5-year) in the test set, and remained predictive across EBV and stage subgroups. The integrated nomogram demonstrated robust calibration and offered the greatest net clinical benefit in DCA, with a 5-year C-index of 0.796.

Conclusions: cfDNA 5hmC profiling enables noninvasive prognostic risk stratification in NPC. The proposed model may support personalized treatment planning and long-term management.

Keywords: 5-hydroxymethylcytosine; Epigenetic biomarker; Nasopharyngeal carcinoma; Prognosis; cfDNA.