Prognostic Marker Analysis of Disseminated Cancer Cells

描述(由申请人提供)：在所有新诊断为乳腺癌且没有临床转移性疾病证据的妇女中，有30%的人在诊断时骨髓中可以检测到播散性肿瘤细胞(DTC)。DTC的存在与转移性疾病发展和癌症死亡的风险增加有关。化疗可以消除部分乳腺癌患者的DTC。然而，那些化疗后持续存在DTC的女性死于乳腺癌的可能性是其他女性的五倍。这一数据表明，化疗耐药的DTC可能是能够形成转移灶的前体细胞。从生物学上讲，DTC是一个异质的细胞群体，临床研究表明，只有一部分细胞具有形成转移的能力。识别与不良临床结局相关的特定DTC将导致一个新的预后标志物，并识别发生转移性疾病和乳腺癌死亡的高危女性。我们认为，化疗后出现的持续性播散性肿瘤细胞代表了所有DTC中的一个独特的亚群，是化疗反应差的预测因子，并与不良的临床结果相关。我们推测，化疗耐药的DTC可以通过他们独特的肿瘤标志物蛋白的表达来识别，这些蛋白可能与乳腺癌干细胞表达的蛋白相似。在这项建议中，我们的具体目标是：1)表征化疗后DTC表达的肿瘤标记物；2)将这些标记物的表达与化疗前检测到的DTC的表达进行比较；3)将已定义的肿瘤标记物在DTC上的表达与乳腺癌患者的临床预后相关联，以确定那些预测疾病复发的标记物；4)利用特定的AIMS 1和2中鉴定的生物标记物来分离纯化的DTC，用于进一步的分子分析。 待分析的样本来自西特曼癌症中心组织采购中心，在那里，经过处理和冷冻保存的骨髓细胞和细胞学载片是从局部晚期乳腺癌患者的新辅助化疗治疗前后制备的。DTC的肿瘤标志物表达将通过双重免疫细胞化学方法确定，方法是使用抗细胞角蛋白抗体和一组针对已知与转移潜力、化疗耐药和乳腺癌干细胞标志物相关的细胞表面蛋白的抗体。有关特定AIMS 1和2生物标记物表达的信息将被用于DTC的免疫磁性分离和进一步的分子分析。我们已经成功地使用双重免疫染色和免疫磁分离来检测含有DTC的骨髓模型系统中的乳腺癌细胞。 拟议的实验结果将确定DTC在化疗后持续表达的一组独特的肿瘤标志物，并与细胞的转移潜力有关。识别具有重要生物学意义的DTC将使我们能够将新诊断为乳腺癌的妇女分为高风险组和低风险组，以发展为转移性疾病。此外，确定DTC表达的肿瘤标记物并对这些细胞进行分子分析将使人们对播散性肿瘤细胞的生物学有新的见解，并开发新的靶向治疗方法。

DESCRIPTION (provided by applicant): Thirty percent of all women newly diagnosed with breast cancer and no clinical evidence of metastatic disease will have disseminated tumor cells (DTC) detectable in their bone marrow at the time of diagnosis. The presence of DTC is associated with an increased risk of metastatic disease development as well as cancer death. Chemotherapy can eliminate DTC in some breast cancer patients. However, those women with persistent DTC after chemotherapy are five times more likely to die from their breast cancer. This data suggests that chemotherapy-resistant DTC are likely to be precursor cells capable of forming metastatic foci. Biologically, DTC are a heterogeneous population of cells and clinical studies suggest that only a subset have the ability to form metastases. Identification of specific DTC that correlate with poor clinical outcome would result in a new prognostic marker and identify women at high risk for developing metastatic disease and breast cancer death. We propose that persistent disseminated tumor cells present after chemotherapy represent a unique subpopulation of all DTC, are predictors of a poor response to chemotherapy, and correlate with poor clinical outcome. We hypothesize that chemotherapy-resistant DTC can be identified by their expression of a unique constellation of tumor marker proteins which may be similar to those expressed by breast cancer stem cells. In this proposal, our specific aims are: 1) characterize tumor markers expressed by DTC which are present after chemotherapy, 2) compare the expression of these markers to that on DTC detected prior to chemotherapy, 3) correlate expression of the defined tumor markers on DTC with clinical outcome of breast cancer patients to identify those markers that are predictive of disease recurrence, 4) Utilize biomarkers identified in Specific Aims 1 and 2 to isolate purified DTC for further molecular analysis. Specimens to be analyzed are from the Siteman Cancer Center Tissue Procurement Center where processed and cryopreserved bone marrow cells and cytological slides have been prepared from women with locally advanced breast cancer before and after treatment with neoadjuvant chemotherapy. Tumor marker expression by DTC will be determined by double immunocytochemistry using anti-cytokeratin antibodies and a panel of antibodies directed against cell surface proteins known to be associated with metastatic potential, chemotherapy resistance, and breast cancer stem cell markers. Information on biomarker expression from Specific Aims 1 and 2 will be utilized for immunomagnetic isolation of DTC and further molecular analysis of these cells. We have successfully used both double immunostaining and immunomagnetic separation to detect breast cancer cells in model systems of bone marrow containing DTC. The results of the proposed experiments will identify a unique constellation of tumor makers expressed by DTC persistent after chemotherapy and associated with the metastatic potential of the cells. Identification of biologically important DTC will allow us to stratify women newly diagnosed with breast cancer into high and low risk groups for the development of metastatic disease. Furthermore, defining tumor markers expressed by DTC and performing molecular analysis of these cells will lead to new insights into the biology of the disseminated tumor cells and to the development of new targeted therapies.