Cell-Free DNA Bisulfite Sequencing Reveals Epithelial-Mesenchymal Transition Signatures for Breast Cancer.

Cell-free DNA (cfDNA), shed by malignant tumor cells into extracellular fluid, provides valuable epigenetic information indicative of cancer status. Nipple aspirate fluid (NAF), a noninvasive liquid biopsy from at-risk women, contains nucleic acid and protein biomarkers from adjacent cancer cells, showing promise for breast cancer (BrC) detection. However, despite its potential, the application of cfDNA in NAF for BrC screening is still underexplored. Here, we report a proof-of-concept study for using cfDNA bisulfite sequencing (cfBS) to assess tumor DNA methylation signatures from NAF samples. For four healthy individuals and three BrC patients, cfBS achieved greater than 20× sequencing depth with an average coverage of 26.5× on the genome. A total of 7471 differentially methylated regions were identified, with significant hypermethylation in BrC samples compared to healthy controls. Gene set enrichment analysis indicated that the differentially methylated genes (DMGs) were significantly associated with epithelial-mesenchymal transition (EMT). By developing a novel EMT scoring metric, we found that BrC samples had more of a mesenchymal phenotype than samples from healthy individuals. CDH1, WNT2, and TRIM29 were hypermethylated near the promoter region, while COL5A2 was hypermethylated in the coding region. The DNA methylation and EMT changes were validated through The Cancer Genome Atlas Breast Invasive Carcinoma study, which confirmed that DMGs were associated with gene expression change and that our methylation-based EMT score reliably distinguished tumors from healthy controls. Our findings support the utilization of the NAF cfDNA cfBS methylation profile for noninvasive BrC screening and pave the way for enhanced early detection of this disease.