TMEM, MENAcalc, and MENAINV as Prognostic and Predictive Markers for Breast Cancer Metastasis

摘要 乳腺癌的死亡在很大程度上可归因于全身性、血源性播散性转移疾病。 鉴于目前预后标准的局限性，识别可能转移的肿瘤和 对治疗有反应是需要的。基于多光子的活体成像显示，侵袭性癌细胞 在啮齿动物乳腺肿瘤中，血管周围结构含有网膜高表达的肿瘤 细胞、巨噬细胞和内皮细胞，都是相互联系的。这种细胞的三分式排列 促进癌细胞进入血管。我们已经在人类体内发现了这种微环境 对福尔马林固定的石蜡包埋组织进行三重免疫染色，以模拟... 自发地标记3种细胞类型。我们将这三种细胞类型的直接结合称为TMEM，即肿瘤微- 转移的环境。最近，在Kaiser Permanente(KP)的一组患者中，我们表明 TMEM与ER+/HER2阳性乳腺癌的远处转移风险呈正相关，独立于 IHC4，提供预后的综合免疫组织化学评分(基于ER、PR、HER2、Ki67) 与Oncotype Dx®复发评分(RS)相当的信息。将这项工作扩展到包括 与其他新近开发的预后标志物(例如，PAM50)进行比较现在是有必要的。对于TMEM来说， 侵袭性癌细胞的鉴定使用侵袭性和迁移性癌细胞的蛋白质标记物 “Mena”，有多种剪接变体：Mena11a是一种抗转移异构体，表达于上皮样 但不是间充质样瘤细胞，而Mena侵袭性(MenaINV)具有强大的促转移表型。 键入间充质样瘤细胞。最近，我们使用多重定量免疫荧光技术 估计缺乏抗转移的Mena11a亚型的Mena的丰度。我们展示了这个标记， 它反映了肿瘤上皮-间充质转化(EMT)的相对量 使肿瘤细胞可以参与TMEM组装并与TMEM相互作用，是积极的 与乳腺癌死亡的风险有关。我们现在建议：检查这些标记之间的关联 在一项包含600对病例对照的病例对照研究中，两组患者均存在远处转移的风险 嵌套在8769例乳腺癌患者的扩展KP队列中，以及从 B28试验(评估紫杉醇作为辅助化疗的价值)；检查与 与和相比，最强的TMEM相关标记或标记组合的远处转移风险 除了IHC4和PAM50(KP研究)和Oncotype Dx®RS和PAM50(B28研究)[~B28研究的2/3 样本将有可用的Oncotype Dx®RS结果])；检查是否与TMEM相关或标记 联合预测治疗反应(在B28)；并在B28研究人群中进行外部验证 TMEM/MENA评分(TMEM、MENECALC、MenaINV的组合与风险关系最强) 在KP研究人群中发展起来的。

ABSTRACT Breast cancer mortality is largely attributable to systemic, hematogenously-disseminated metastatic disease. Given the limitations of current prognostic criteria, new methods to identify tumors likely to metastasize and respond to therapy are needed. Multiphoton-based intravital imaging has shown that invasive carcinoma cells in rodent mammary tumors intravasate via peri-vascular structures containing a Mena over-expressing tumor cell, a macrophage, and an endothelial cell, all contacting each other. This tri-partite arrangement of cells facilitates entry of carcinoma cells into the blood vessel. We have identified this microenvironment in human breast cancer samples using a triple immunostain for formalin-fixed paraffin-embedded tissue that simultan- eously labels the 3 cell types. We call the direct apposition of these 3 cell types “TMEM”, for Tumor Micro- Environment of Metastasis. Recently, in a cohort of patients at Kaiser Permanente (KP), we showed that TMEM was positively associated with risk of distant metastasis in ER+/HER2- breast cancer independently of IHC4, a composite immunohistochemical score (based on ER, PR, HER2, Ki67) that provides prognostic information comparable to the Oncotype Dx® Recurrence Score (RS). Extension of this work to encompass comparison to other recently developed prognostic markers (e.g., PAM50) is now warranted. For TMEM, the invasive carcinoma cells are identified using a protein marker for invasive and migratory cancer cells called “Mena”, which has multiple splice variants: Mena11a is an anti-metastatic isoform expressed in epithelial-like but not mesenchymal-like tumor cells, while Mena invasive (MenaINV) confers a potent pro-metastatic pheno- type in mesenchymal-like tumor cells. Recently, we used multiplex quantitative immunofluorescence to estimate the abundance of Mena lacking its anti-metastatic Mena11a isoform. We showed that this marker, Menacalc, which reflects the relative amount of epithelial-mesenchymal transition (EMT) that a tumor has undergone so that tumor cells can participate in TMEM assembly and interact with TMEMs, was positively associated with risk of breast cancer death. We now propose to: examine the association of these markers (Menacalc/MenaINV/TMEM) with risk of distant metastasis both in a case-control study of 600 case-control pairs nested in an expanded KP cohort of 8769 breast cancer cases, and in 1000 breast cancer cases sampled from the B28 trial (which assessed the value of paclitaxel as adjuvant chemotherapy); examine the association with risk of distant metastasis of the strongest TMEM-related marker or marker combination in comparison to and in addition to IHC4 and PAM50 (KP study) and Oncotype Dx® RS and PAM50 (B28 study) [~2/3 of the B28 study sample will have Oncotype Dx® RS results available]); examine whether TMEM-related marker or marker combination predicts response to therapy (in B28); and externally validate in the B28 study population the TMEM/Mena score (the combination of TMEM, Menacalc, MenaINV most strongly associated with risk) developed in the KP study population.