Blood-derived cell-free DNA is a superior biomarker for noninvasive DNA methylation profiling in multiple myeloma

Research during the past decades has revealed a complex, aberrant DNA methylation profile in multiple myeloma (MM), playing a key role in disease progression, prognosis, and therapy response.However, spatial epigenetic heterogeneity may cause underestimation of alterations in the DNA methylation profile when analyzing single bone marrow (BM) aspirates.Liquid biopsies, particularly cell-free DNA (cfDNA), have demonstrated their ability to reflect tumor methylation profiles in solid cancers, but their application in MM remains underexplored.This study evaluates the potential of blood-based liquid biopsies to detect aberrant DNA methylation in MM.Using enzymatic methylation sequencing (EM-Seq), we analyzed 44 matched BM-DNA, cfDNA, circulating tumor cell (CTC), and peripheral blood mononuclear cell (PBMNC) DNA samples from 11 MM patients with advanced disease.Differentially methylated regions (DMRs) were identified by comparing MM samples to healthy control genomic DNA (gDNA), focusing on genome-wide CpG islands.We detected previously described promoter hypermethylation in CDKN2A, CDH1, and RASSF1A in human myeloma cell lines.When comparing circulating biomarkers in patient samples, cfDNA showed a superior performance with 78.2% concordance with BM-DNA while permitting detection of the highest number of DMRs, some of which not detected in BM-DNA.Pathway enrichment analysis highlighted enrichment of transcriptional misregulation, Ras/MAPK signaling, and focal adhesion pathways.Notably, our findings indicate aberrant methylation of extracellular matrix-associated genes.This is the first comprehensive comparative analysis of circulating biomarker-derived methylation profiles in MM.Our findings support cfDNA as a feasible non-invasive biomarker for methylation profiling, paving the way for multi-omics diagnostics in MM clinical practice.