Defining genome-wide cfDNA methylation landscapes in pediatric and adolescent and young adult (AYA) bone and soft-tissue sarcomas

Background: Pediatric and AYA sarcomas are rare, biologically heterogeneous tumors with limited noninvasive biomarkers to guide diagnosis, personalized treatment, and disease monitoring. Standard-of-care evaluation relies on invasive biopsies that carry procedural risks and imaging modalities with limited sensitivity for detecting minimal residual disease and distinguishing tumor types. Cell-free DNA (cfDNA) methylation profiling offers a minimally invasive alternative capable of capturing tumor-derived epigenomic alterations and has consistently shown strong clinical utility in adult malignancies. However, genome-wide cfDNA methylation patterns across diverse pediatric and AYA sarcoma subtypes remain poorly defined. Methods: Plasma was collected from 93 pediatric and AYA patients (pediatric <18 years; AYA 15-39 years) representing 27 Ewing sarcoma, 17 rhabdomyosarcoma, 11 liposarcoma, 10 chordoma, 8 synovial sarcoma, 7 chondrosarcoma, 6 osteosarcoma, and 6 fibrosarcoma cases, plus 19 matched healthy donors. cfDNA was isolated from 1 mL plasma, quantified by Qubit, and assessed by Bioanalyzer. Libraries were prepared using the NEBNext EM-seq v2 workflow with PCR cycles scaled to cfDNA input amount. Genome-wide sequencing (15× target depth) was followed by alignment, deduplication, and methylation calling. MultiQC was used to assess depth, duplication rate, conversion efficiency, and CpG coverage. Global methylation summaries were computed, and methylKit was used to generate per-CpG and 1-kb tiling matrices and initiate differential methylation analyses. Results: Ewing sarcoma patients had significantly higher plasma cfDNA concentrations than age- and sex-matched healthy donors (24.4 vs 4.6 ng/mL, p < 0.0001), whereas cfDNA levels for other sarcoma subtypes overlapped with the healthy cohort. Low or undetectable cfDNA concentrations by Qubit were far more common among non-Ewing sarcoma samples (23 of 66) than Ewing sarcoma (1 of 27). All samples achieved sufficient CpG coverage for genome-wide methylation modeling. Global methylation summaries revealed no significant differences in mean beta-values across sarcoma subtypes or versus healthy donors. An independent analysis of the Ewing subset identified 299 significant differentially methylated regions (DMRs), supporting the detectability of disease-specific methylation changes. Conclusions: These results demonstrate the feasibility of high-quality cfDNA methylome profiling across a large and diverse pediatric and AYA sarcoma cohort, including in plasma samples with cfDNA concentrations below the limit of detection. While global methylation levels appear broadly similar across subtypes, proof-of-concept DMR analysis in Ewing sarcoma suggests that subtype-specific epigenomic signatures could enable future noninvasive diagnostic tools for pediatric and AYA sarcomas.