Hypermethylation in CpG-rich cfDNA fragments enables accurate detection of circulating tumor DNA in BRCA1- and BRCA2-associated breast and ovarian cancer

Abstract Pathogenic BRCA 1 and BRCA2 germline variants confer an increased risk of hereditary breast and ovarian cancer (HBOC). In this high-risk cohort, novel biomarkers are needed to support patient management. While circulating cell-free DNA-based conventional CpG-level averaging is promising, it dilutes tumor-specific signals in samples with low circulating tumor DNA (ctDNA) fractions. Here, we developed a targeted ultra-deep NGS assay for detecting tumor-derived ctDNA based on fragment-level hypermethylation of CpG-rich cfDNA. A panel of 360 cancer-specific CpG-rich regions derived from 450k methylation array datasets from TCGA breast cancer and GEO ovarian cancer, was applied to plasma from 31 breast cancer, 16 ovarian cancer patients, and 26 healthy controls using enzymatic methylation sequencing. Hypermethylated ctDNA (hm-ctDNA) fragments, were significantly enriched in cancer cases, identifying 62.5% of ovarian and 38.7% of breast cancers at 100% specificity. Elevated hm-ctDNA was observed across different tumor stages, with higher burden in BRCA 1 -associated cases. This approach enables highly specific, non-invasive hypermethylated ctDNA detection in HBOC populations.