CfDNA epigenetic profiling for tissue-of-origin mapping, accelerated aging, and cardiotoxicity biomarker discovery in cardio-oncology

Background Early detection of cancer therapy-related cardiotoxicity is critical to individualizing treatment and preserving long-term cardiac health, yet current biomarkers largely capture downstream injury and offer limited insight into underlying biology. Plasma cfDNA methylation profiling provides a non-invasive, integrative readout of tissue-of-origin and epigenetic state, enabling simultaneous assessment of cardiac involvement and mechanistic signals that can power biomarker discovery and risk stratification. Purpose To evaluate whether cfDNA methylation profiling identifies tissue-of-origin shifts, epigenetic ageing signals and methylation biomarkers associated with cardiotoxicity and cardiac dysfunction. Methods Patients receiving anticancer therapy and suspected of cardiotoxicity were enrolled in the Cardio-Oncology outpatient care progran. Plasma cfDNA was analysed from liquid biopsy of cancer patients with proven cardiotoxicity (defined according to 2022 ESC cardio-oncology guidelines) (group Cardio-Onco, n=13) and compared to that of cancer patients without cardiotoxicity (group Onco; n=11) and age- and sex-matched healthy controls (n=12). Targeted cfDNA methylation sequencing was performed. Tissue-of-origin was inferred using UXM (methylation-based) deconvolution. Epigenetic age was estimated using DNA methylation (DNAm) GrimAge and principal component (PC) proxy clocks. Differentially methylated regions (DMRs) were identified and candidate loci prioritised using random forest-based feature selection. Associations between methylation signals and cardiac imaging and laboratory measures were evaluated using multivariable regression adjusting for age, sex and significant deconvolved cell-type proportions. Results CardioOnco samples showed increased estimated contribution of cardiac fibroblast-derived cfDNA compared with Onco and healthy controls. No cohort-level acceleration of epigenetic age was detected using DNAm GrimAge or PC clocks; however, GrimAge-related proxy components linked to TIMP1, B2M and PAI1 were significantly increased in CardioOnco. Feature selection highlighted methylation loci annotated to RHOJ, MAGI1, SPON1, ANKRD2 and PALLD, implicating cell-cell adhesion and communication pathways. Fibroblast-associated DMR signals were associated with reduced left ventricular ejection fraction (LVEF) and right ventricular ejection fraction (RVEF) and with higher troponin T and N-terminal pro-B-type natriuretic peptide (NT-proBNP) (p<0.01). Conclusion Plasma cfDNA methylome profiling identifies a cardiac fibroblast-derived signal epigenetic changes associated with cardiotoxicity. These data support targeted cfDNA methylation as a promising non-invasive approach for monitoring and biomarker discovery in cardio-oncology and warrant validation in larger cohorts.