Jeon Minsun Stacey, Ding Zehuan, Pei Casey, Li Jing, Xie Linglin, Sauter Edward, Zhang Ke Kurt
Center for Epigenetics and Disease Prevention, Institute of Biosciences & Technology, Texas A&M Health Science Center, Houston, TX 77030, USA.
Rigor and Reproducibility Core, Institute of Biosciences & Technology, Texas A&M Health Science Center, Houston, TX 77030, USA.
Int J Mol Sci. 2025 Sep 7;26(17):8723. doi: 10.3390/ijms26178723.
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. , , and were hypermethylated near the promoter region, while 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.
游离DNA（cfDNA）由恶性肿瘤细胞释放到细胞外液中，提供了指示癌症状态的有价值的表观遗传信息。乳头抽吸液（NAF）是一种针对高危女性的非侵入性液体活检样本，含有来自相邻癌细胞的核酸和蛋白质生物标志物，在乳腺癌（BrC）检测方面显示出前景。然而，尽管具有潜力，但cfDNA在NAF中用于BrC筛查的应用仍未得到充分探索。在这里，我们报告了一项概念验证研究，该研究使用cfDNA亚硫酸氢盐测序（cfBS）来评估NAF样本中的肿瘤DNA甲基化特征。对于4名健康个体和3名BrC患者，cfBS实现了大于20倍的测序深度，基因组平均覆盖度为26.5倍。共鉴定出7471个差异甲基化区域，与健康对照相比，BrC样本中存在显著的高甲基化。基因集富集分析表明，差异甲基化基因（DMG）与上皮-间质转化（EMT）显著相关。通过开发一种新的EMT评分指标，我们发现BrC样本比健康个体的样本具有更多的间质表型。 、 和 在启动子区域附近发生高甲基化，而 在编码区域发生高甲基化。通过癌症基因组图谱乳腺浸润癌研究验证了DNA甲基化和EMT变化，该研究证实DMG与基因表达变化相关，并且我们基于甲基化的EMT评分能够可靠地区分肿瘤和健康对照。我们的研究结果支持利用NAF cfDNA cfBS甲基化谱进行非侵入性BrC筛查，并为加强该疾病的早期检测铺平了道路。
