Ultradeep targeted methylation sequencing of BRCA1 promoter in paired tumor-normal samples from breast cancer patients. Ultradeep targeted methylation sequencing of BRCA1 promoter in paired tumor-normal samples from breast cancer patients

Background: Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance patterns of BRCA1 epimutations established. Methods: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 408 patients with 411 primary breast cancers, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls and 200 newborn boys. Finally, we assessed BRCA1 methylation status among 575 mothers and 531 fathers of girls with (n = 102) and without (n = 473) BRCA1 methylation. Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (<10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1–30.5). In contrast, we found concordance in only three out of 220 patients with 221 ER≥10% tumors and zero out of 114 patients with 116 HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals, but no correlation to BRCA1 methylation status either in mothers or fathers. A significantly lower fraction of newborn boys carried BRCA1 methylation (9 / 200; 4.5%) as compared to girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46 / 575; 8.0%; p = 0.0003). Conclusions: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expression breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance. Overall design: Study includes 408 patients enrolled in three neoadjuvant breast cancer studies (EPITAX, DDP and PETREMAC) from which pretreatment tumor tissue and white blood cells DNA samples were available for analysis.

背景：正常细胞中的BRCA1表观突变（BRCA1 epimutation）已被证实与三阴性乳腺癌（triple-negative breast cancer, TNBC）风险升高相关。然而，以BRCA1表观突变为潜在病因的三阴性乳腺癌占比仍不明确；BRCA1表观突变的发生时间与潜在遗传模式亦尚未明确。
方法：为解答上述问题，本研究纳入408名共罹患411例原发性乳腺癌的患者（其中66例为三阴性乳腺癌），采集其乳腺癌组织及配对白细胞（white blood cells, WBC）样本，采用可实现等位基因分辨率甲基化分析的高灵敏度测序检测技术，分析BRCA1甲基化状态。为进一步探究BRCA1甲基化的起源时间与正常细胞中该表观修饰的特征，本研究分析了1260名新生女婴与200名新生男婴的脐带血样本。最后，本研究对102例存在BRCA1甲基化与473例不存在BRCA1甲基化的女婴的575名母亲及531名父亲的BRCA1甲基化状态进行了检测。
结果：本研究在66例三阴性乳腺癌患者中的10例，以及6例雌激素受体（estrogen receptor, ER）低表达（<10%）乳腺癌患者中的4例中，发现了与肿瘤组织一致的嵌合型白细胞BRCA1表观突变（合计：72例患者中的14例，占比19.4%；95%置信区间11.1%~30.5%）。与之形成对比的是，在220例共罹患221例ER≥10%乳腺癌的患者中仅3例存在该一致性突变，而114例共罹患116例人表皮生长因子受体2（HER2）阳性乳腺癌的患者中未发现此类一致性突变。个体内分析显示，正常细胞与肿瘤细胞中的BRCA1表观突变靶向同一等位基因。对新生女婴脐带血白细胞的BRCA1甲基化分析显示，113/1260（9.0%）的个体存在嵌合型、以单等位基因为主的BRCA1表观突变，其定性特征与成年个体相似，但该突变与父母的BRCA1甲基化状态均无相关性。新生男婴携带BRCA1甲基化的比例（9/200，4.5%）显著低于新生女婴（p=0.038）。类似地，父亲群体中白细胞BRCA1甲基化的检出率（16/531，3.0%）低于母亲群体（46/575，8.0%；p=0.0003）。
结论：本研究结果提示，产前BRCA1表观突变可能是约20%的三阴性乳腺癌与低ER表达乳腺癌的潜在病因。此类先天性嵌合型BRCA1甲基化大概率通过宫内性别相关机制产生，与孟德尔遗传无关。
整体研究设计：本研究纳入了三项新辅助乳腺癌研究（EPITAX、DDP及PETREMAC）中的408名患者，均获取了其治疗前的肿瘤组织与白细胞DNA样本用于后续分析。