Circulating Tumor DNA Methylation Profiling for Early Diagnosis and Treatment Monitoring in Rhabdomyosarcoma: A Prospective Cohort Study

Background Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents, with limited non-invasive tools available for early detection and real-time therapeutic monitoring. Circulating tumor DNA (ctDNA) methylation profiling via liquid biopsy represents a promising approach, yet its clinical utility in RMS remains inadequately characterized. Methods This prospective observational cohort study enrolled 86 patients aged 0 to 21 years with histopathologically confirmed RMS at a tertiary pediatric oncology center between January 2024 and December 2025. Plasma cfDNA was collected at diagnosis and at serial treatment time points. A targeted bisulfite sequencing panel interrogating five loci (RASSF1A, MYOD1, PAX3, CDKN2A, and CDH13) was applied, and an aggregate ctDNA methylation score was derived. Associations with clinicopathological features, treatment response, and survival outcomes were analyzed. Results ctDNA methylation was detectable in 79 of 86 patients (91.9%) at diagnosis, with an AUC of 0.919 (95% CI 0.872 to 0.967). RASSF1A hypermethylation was identified in 68.6% of patients and was significantly associated with alveolar histology, metastatic disease, and high-risk classification. Molecular response by cycle 2 correlated strongly with imaging-based response (85.4% vs. 42.3%, p < 0.001). RASSF1A hypermethylation was independently prognostic for inferior event-free survival on multivariable analysis (hazard ratio 2.31, 95% CI 1.28 to 4.17, p = 0.006). The 18-month EFS was 44.2% versus 71.8% for RASSF1A-positive and RASSF1A-negative patients, respectively (log-rank p = 0.003). Conclusions Plasma ctDNA methylation profiling demonstrates high sensitivity for RMS detection and provides clinically meaningful prognostic and predictive information. Integration of this liquid biopsy approach into routine oncological care warrants prospective validation in larger multicenter cohorts.