A comparison of methods for analyzing DNA methylation from clinical archival macrodissected formalin-fixed paraffin-embedded tissue

DNA methylation at the 5′ position of cytosine is a classical epigenetic mark in the human genome, and alterations in DNA methylation are frequently observed in cancer. Formalin-fixed, paraffin-embedded (FFPE) tissue is widely used in next-generation sequencing (NGS) for retrospective molecular studies due to preserved tissue architecture and extensive clinically annotated archives. However, fragmented and degraded nucleic acids often limit its utility. Although whole-genome bisulfite sequencing (WGBS) has long been the gold standard for assessing DNA copy-number and methylation, newer enzymatic approaches such as enzymatic-methylation sequencing (EM-seq) and the biomodal duet multiomics solution evoC kit have emerged. Here, we characterized these three methods in FFPE residual triple-negative breast cancer (TNBC) tissue. TNBC is the most aggressive breast cancer subtype, lacks targetable alterations, and is associated with a high risk of local and distant recurrence, with most metastatic progression occurring within three years of diagnosis. Our findings demonstrate the feasibility of applying all three sequencing approaches to low-input FFPE residual TNBC samples and highlight the method-specific trade-offs that should be considered when selecting a sequencing strategy. These findings support a streamlined framework for genetic and epigenetic profiling of archival FFPE residual TNBC samples and future cohort expansion.