Supplementary Material for: BRCA Mutation-Related and Claudin-Low Breast Cancer: Blood Relatives or Stepsisters?

<br><strong><em>Background:</em></strong> BRCA mutation-associated (BRCAmut) breast cancer represents a heterogeneous group displaying certain molecular features. Claudin-low breast cancers (CLBC) overlap with characteristics of BRCAmut tumors; therefore, we have investigated whether these are identical subtypes. <b><i>Methods:</i></b> Using public gene expression data, CLDN, CDH1, 9-cell line claudin-low predictor (9CLCLP) and PAM50 expression was evaluated in BRCAmut and BRCA wild-type (BRCAwt) breast cancer cases focusing on their possible overlap with the CLBC subtype. A separate formalin-fixed, paraffin-embedded (FFPE) cohort of 22 BRCAmut and 19 BRCAwt tumor tissues was used for immunohistochemical examination of AR, CD24, CD44, CK5/6, claudin-1, -3, -4 and -7, E-cadherin, EGFR, estrogen receptor (ER), EZH2, HER2, Ki67, p53, progesterone receptor (PgR) and vimentin expression. <b><i>Results:</i></b> In the data sets, CLDN1 (ROC = 0.785, p &lt; 0.001), CDH1 (ROC = 0.785, p &lt; 0.001), CLDN7 (ROC = 0.723, p &lt; 0.001), CLDN3 (ROC = 0.696, p = 0.020) and CLDN4 (ROC = 0.685, p = 0.027) were expressed at higher level in BRCAmut than BRCAwt tumor tissue. The PAM50 subtype differed from the assigned immunohistochemistry (IHC)-based subtype in 30%. Based on accessible 9CLCLP predictor genes, BRCAmut breast cancer does not display the claudin-low phenotype. Utilizing FFPE samples, claudins were evidently expressed in both BRCAmut and BRCAwt cases. However, at the protein level, only claudin-3 expression was higher in BRCAmut tumors, while claudin-1, -4 and -7 and E-cadherin expression was lower compared to BRCAwt cases. A CD24^low/CD44^high phenotype was found in BRCAmut tumors upon comparison with BRCAwt cases (p &lt; 0.001 and p = 0.001, respectively). <b><i>Conclusions:</i></b> There is a prominent correlation between the genes under focus herein and BRCA mutation status. BRCAmut tumors bear stem cell characteristics displaying a distinct cell adhesion molecule profile characterized by high expression of CDH1 and CLDN4 according to public gene expression data set analysis, and higher claudin-3 expression as detected by IHC; thus, BRCAmut breast carcinomas are not identical with the previously identified claudin-low subtype of breast cancer.

**<em>背景：</em>** BRCA突变相关（BRCA mutation-associated, BRCAmut）乳腺癌是一类具有特定分子特征的异质性肿瘤。低紧密连接蛋白型乳腺癌（claudin-low breast cancers, CLBC）与BRCAmut肿瘤的特征存在重叠，因此本研究旨在探讨二者是否为同一亚型。
**<em>方法：</em>** 本研究借助公共基因表达数据集，对BRCAmut与BRCA野生型（BRCA wild-type, BRCAwt）乳腺癌样本中的紧密连接蛋白（claudin, CLDN）、CDH1、9细胞系低紧密连接蛋白预测模型（9-cell line claudin-low predictor, 9CLCLP）及PAM50的表达水平进行检测，重点探究二者与CLBC亚型的潜在重合性。此外，本研究纳入一组福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded, FFPE）队列，包含22例BRCAmut与19例BRCAwt肿瘤组织，用于对雄激素受体（androgen receptor, AR）、CD24、CD44、细胞角蛋白5/6（cytokeratin 5/6, CK5/6）、CLDN1、CLDN3、CLDN4、CLDN7、E-钙粘蛋白、表皮生长因子受体（epidermal growth factor receptor, EGFR）、雌激素受体（estrogen receptor, ER）、EZH2、人表皮生长因子受体2（human epidermal growth factor receptor 2, HER2）、Ki67、p53、孕激素受体（progesterone receptor, PgR）及波形蛋白（vimentin）的表达进行免疫组化（immunohistochemistry, IHC）检测。
**<em>结果：</em>** 在基因表达数据集中，BRCAmut肿瘤组织中CLDN1（受试者工作特征曲线（Receiver Operating Characteristic curve, ROC）= 0.785, p < 0.001）、CDH1（ROC=0.785, p < 0.001）、CLDN7（ROC=0.723, p < 0.001）、CLDN3（ROC=0.696, p=0.020）及CLDN4（ROC=0.685, p=0.027）的表达水平显著高于BRCAwt肿瘤组织。PAM50亚型与基于免疫组化分配的分型结果在30%的样本中存在差异。基于可获取的9CLCLP预测基因分析结果，BRCAmut乳腺癌并不具备低紧密连接蛋白型表型。利用FFPE样本检测发现，紧密连接蛋白在BRCAmut与BRCAwt样本中均有明确表达。但在蛋白层面，仅CLDN3在BRCAmut肿瘤中的表达水平更高，而CLDN1、CLDN4、CLDN7及E-钙粘蛋白的表达水平均低于BRCAwt肿瘤组织。与BRCAwt样本相比，BRCAmut肿瘤呈现CD24^低/CD44^高的表型（分别对应p<0.001与p=0.001）。
**<em>结论：</em>** 本研究聚焦的基因与BRCA突变状态存在显著相关性。通过公共基因表达数据集分析可知，BRCAmut肿瘤具有干细胞特征，其细胞黏附分子谱具有独特性，表现为CDH1与CLDN4的高表达；且经免疫组化检测证实，其CLDN3的表达水平更高。综上，BRCAmut乳腺癌与此前已明确的低紧密连接蛋白型乳腺癌亚型并非同一类别。