最近的研究强调了游离 DNA (cfDNA) 甲基化谱在检测乳腺癌 (BC) 及其不同亚型中的重要性。我们使用无细胞甲基化 DNA 免疫沉淀和高通量测序 (cfMeDIP-seq) 来研究血浆 cfDNA 甲基化是否可以为 BRCA1/2 种系突变患者的乳腺癌特征分析提供信息，从而实现早期癌症检测和治疗反应。纳入了 23 名具有 BRCA1 和 BRCA2 基因种系突变的 BC 患者、19 名没有 BRCA1/2 突变的健康对照者以及两名携带 BRCA1/2 突变的健康个体。在诊断时收集所有研究对象的血样，并通过离心分离血浆。从 1 mL 血浆中提取游离 DNA，并对每个样本进行 cfMeDIP-seq。对免疫沉淀样品进行浅层全基因组测序。然后，鉴定了 25 名 BRCA 种系突变携带者和 19 名非携带者之间的差异甲基化 300 bp 区域 (DMR)。将 DMR 与公共数据集中的肿瘤特异性区域进行比较，以进行公正的分析。最后，基于 GLMnet 和随机森林模型训练了两个统计分类器，以评估所识别的 DMR 是否能够区分 BRCA 阳性和健康样本。与 19 个对照相比，我们在 25 名 BRCA 种系突变携带者中确定了 7,095 个高甲基化区域和 212 个低甲基化区域。这些区域能够以高精度和灵敏度区分肿瘤和健康样本。我们发现 BRCA1/2 突变乳腺癌的循环肿瘤 DNA 的特征是参与 DNA 修复和细胞周期的基因低甲基化。我们发现了与这些 DRM 相关的 TF，并确定了成红细胞转化特异性 (ETS) 家族的蛋白质在高甲基化区域特别活跃。最后，我们评估这些区域可以区分健康样本中的 BRCA 阳性，AUC 为 0.95，敏感性为 88%，特异性为 94.74%。我们的研究强调了肿瘤细胞衍生的 DNA 甲基化在 BC 中的重要性，报告携带 BRCA1、BRCA2 突变的患者和野生型对照患者之间的甲基化谱不同。我们的微创方法可以实现早期癌症诊断、微小残留病评估以及治疗反应监测。© 2024。作者。

Recent studies have highlighted the importance of the cell-free DNA (cfDNA) methylation profile in detecting breast cancer (BC) and its different subtypes. We investigated whether plasma cfDNA methylation, using cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq), may be informative in characterizing breast cancer in patients with BRCA1/2 germline mutations for early cancer detection and response to therapy.We enrolled 23 BC patients with germline mutation of BRCA1 and BRCA2 genes, 19 healthy controls without BRCA1/2 mutation, and two healthy individuals who carried BRCA1/2 mutations. Blood samples were collected for all study subjects at the diagnosis, and plasma was isolated by centrifugation. Cell-free DNA was extracted from 1 mL of plasma, and cfMeDIP-seq was performed for each sample. Shallow whole genome sequencing was performed on the immuno-precipitated samples. Then, the differentially methylated 300-bp regions (DMRs) between 25 BRCA germline mutation carriers and 19 non-carriers were identified. DMRs were compared with tumor-specific regions from public datasets to perform an unbiased analysis. Finally, two statistical classifiers were trained based on the GLMnet and random forest model to evaluate if the identified DMRs could discriminate BRCA-positive from healthy samples.We identified 7,095 hypermethylated and 212 hypomethylated regions in 25 BRCA germline mutation carriers compared to 19 controls. These regions discriminate tumors from healthy samples with high accuracy and sensitivity. We show that the circulating tumor DNA of BRCA1/2 mutant breast cancers is characterized by the hypomethylation of genes involved in DNA repair and cell cycle. We uncovered the TFs associated with these DRMs and identified that proteins of the Erythroblast Transformation Specific (ETS) family are particularly active in the hypermethylated regions. Finally, we assessed that these regions could discriminate between BRCA positives from healthy samples with an AUC of 0.95, a sensitivity of 88%, and a specificity of 94.74%.Our study emphasizes the importance of tumor cell-derived DNA methylation in BC, reporting a different methylation profile between patients carrying mutations in BRCA1, BRCA2, and wild-type controls. Our minimally invasive approach could allow early cancer diagnosis, assessment of minimal residual disease, and monitoring of response to therapy.© 2024. The Author(s).