Liquid biopsy epigenetics: establishing a molecular profile based on cell‐free <scp>DNA</scp>

Liquid biopsies containing circulating tumor DNA (ctDNA) are important biomarkers across several forms of cancer. The detection of mutations in cell-free DNA (cfDNA) indicates the presence of ctDNA. However, unsatisfactory ctDNA mutation sensitivities, issues with sequencing errors, and clonal hematopoiesis variants have limited the clinical utility of mutation-based ctDNA assays. Recently, a new avenue of cfDNA assays has been developed, focusing on cfDNA epigenetics. Here, we outline the recent advancements in cfDNA epigenetics, focusing on cfDNA methylation, fragmentomics, and post-translational modifications (PTMs) of circulating nucleosomes. We present various methylation strategies concerning ctDNA detection and tissue of origin (TOO) analyses. cfDNA fragmentomics focuses on cfDNA fragment lengths, fragment end motifs, and nucleosome positioning to infer gene expression and estimate the ctDNA fraction. Lastly, we discuss the development of cell-free chromatin immunoprecipitation of circulating nucleosomes with PTMs. This method has been implemented to detect tumor gene expression, TOO, and treatment resistance. Combining the epigenetic features of cfDNA will expand the utility of liquid biopsies to give a more comprehensive insight into tumor biology, treatment response, and resistance.