Cell-free DNA fragmentomics and epigenomic profiling in hematologic malignancies: current evidence, translational limitations, and future directions

Mutation-based liquid biopsy supports diagnosis and disease monitoring in hematologic malignancies, but interpretation of plasma variants may be complicated by clonal hematopoiesis, treatment-related changes, and nonmalignant blood-cell turnover. This structured narrative review evaluates whether cell-free DNA (cfDNA) fragmentomics, DNA methylation, and 5-hydroxymethylcytosine (5hmC) provide complementary information beyond mutation detection. Direct plasma evidence is most developed for selected fragmentomic and 5hmC applications, although most studies remain retrospective, internally validated, or proof of concept. Fragmentomic studies have reported associations with lymphoma classification, outcome assessment, and longitudinal tumor-effector profiling during chimeric antigen receptor T-cell therapy, but routine clinical utility and decision thresholds are not established. Plasma 5hmC studies have reported associations with lymphoma classification, treatment response, prognosis, and measurable residual disease in acute myeloid leukemia, whereas pharmacodynamic monitoring during Bruton's tyrosine kinase inhibition remains hypothesis-generating. DNA methylation may support source-cell or lineage inference, but lineage assignment does not establish malignant fraction, and key disease-assessment studies remain largely tissue-based, cellular-DNA-based, cross-cancer, or methodological. The novelty of this review is a structured evidence map that distinguishes direct plasma evidence from indirect support and compares the three modalities by biological information, assay compatibility, and validation maturity. We also propose a hypothesis-generating framework for sample-level integration using separate assay-specific aliquots and computational combination of outputs. No universal analytical order or clinically validated integrated workflow has been established. Fragmentomic and epigenomic profiling should therefore be regarded as investigational complements to mutation-based testing, requiring independent prospective validation against established genomic, cellular, imaging, and tissue-based methods.