Prognostic Marker Analysis of Disseminated Cancer Cells

描述（由申请人提供）：30%的新诊断为乳腺癌且没有临床转移性疾病证据的女性在诊断时骨髓中可检测到弥散性肿瘤细胞（DTC）。DTC的存在与转移性疾病发展的风险增加以及癌症死亡有关。化疗可以消除部分乳腺癌患者的DTC。然而，那些化疗后持续DTC的妇女死于乳腺癌的可能性要高出5倍。这些数据表明，耐化疗的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，临床研究表明，只有一小部分具有形成转移的能力。识别与不良临床结果相关的特异性DTC将产生新的预后标志物，并识别发生转移性疾病和乳腺癌死亡的高风险妇女。我们认为化疗后持续播散的肿瘤细胞代表了所有DTC的一个独特亚群，是化疗不良反应的预测因子，并且与不良的临床结果相关。我们假设耐化疗的DTC可以通过表达一组独特的肿瘤标记蛋白来识别，这些标记蛋白可能与乳腺癌干细胞表达的相似。在这项建议中，我们的具体目标是：1)鉴定化疗后DTC表达的肿瘤标志物，2)将这些标志物的表达与化疗前检测到的DTC的表达进行比较，3)将DTC上定义的肿瘤标志物的表达与乳腺癌患者的临床结局相关联，以确定那些预测疾病复发的标志物，4)利用特异性目标1和2中鉴定的生物标志物分离纯化的DTC进行进一步的分子分析。

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.