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

Area of Science:

• Epigenetics
• Oncology
• Biomarker Discovery

Background:

• Cell-free DNA (cfDNA) in extracellular fluid offers epigenetic insights into cancer status.
• Nipple aspirate fluid (NAF) is a noninvasive liquid biopsy source containing biomarkers for breast cancer (BrC).
• The use of cfDNA in NAF for BrC screening remains largely unexplored.

Purpose of the Study:

• To investigate the utility of cfDNA bisulfite sequencing (cfBS) for analyzing DNA methylation signatures in NAF for BrC detection.
• To establish a proof-of-concept for cfDNA methylation profiling in NAF for noninvasive breast cancer screening.

Main Methods:

• Collected NAF samples from healthy individuals and BrC patients.
• Performed cfDNA bisulfite sequencing (cfBS) to assess genome-wide DNA methylation.
• Identified differentially methylated regions (DMRs) and performed gene set enrichment analysis.
• Developed and validated a novel epithelial-mesenchymal transition (EMT) scoring metric based on methylation data.

Main Results:

• cfBS achieved high sequencing depth and coverage in NAF samples.
• Identified 7471 DMRs, with significant hypermethylation observed in BrC samples compared to controls.
• Differentially methylated genes were strongly associated with EMT.
• BrC samples exhibited a more mesenchymal phenotype based on the novel EMT score.
• Specific genes (e.g., CDH1, WNT2, TRIM29, COL5A2) showed differential methylation patterns.

Conclusions:

• NAF cfDNA cfBS methylation profiling is a viable approach for noninvasive BrC screening.
• The identified methylation signatures and EMT score can aid in distinguishing BrC from healthy controls.
• This study provides a foundation for developing enhanced early detection strategies for breast cancer using liquid biopsies.