EXPRESSION BASED MARKERS FOR BREAST CANCER DETECTION

该提案将尝试识别乳腺癌的基于表达的标记物，并开发使用这些标记物检测循环乳腺癌细胞的测定法。 我们的应用程序是由杜克乳腺癌项目和雅培诊断乳腺癌风险投资集团合作开发的，将利用每个机构的相当大的优势。标记识别的基本方法利用了正在进行的（和单独资助的）使用强大的新基因组学工具的努力。 在杜克，作为CGAP的一部分，正在构建乳腺癌和正常乳腺上皮细胞的SAGE（基因表达系列分析）文库。 同样在杜克，一系列的20个原发性乳腺癌将使用高密度的Affyssin表达芯片进行分析（30,000个基因/EST将被采样）。 最后，在Abbott Diagnostics，已经通过使用Incyte Corporation表达文库鉴定了标记物。 这三种表达数据来源将进行电子比较，并将鉴定以组织或癌症特异性方式高水平表达的最有希望的标志物。 在计算机模拟分析后，将对先导标记物进行验证算法，包括北方印迹、多组织RNA斑点印迹、RT-PCR和原位细胞杂交。 一组乳腺细胞系和一系列50种原发性乳腺癌将用于确定这些标志物在靶组织中的表达情况。在这些初始验证步骤之后，将通过在正常全血中掺入已知数量的乳腺癌细胞（来源于细胞系和恶性渗出液）来进行一系列复溶实验。 将使用磁珠/抗体技术纯化上皮细胞，并通过流式细胞术、组织学和定量RT-PCR分析所得纯化样品。 为了提高敏感性，一个单独的和平行的屏幕将集中在标记物，是高度过表达的一个子集的乳腺癌。 在此背景下，将探索PCR多重方法，并且还将包括使用端粒酶基因（TERT）表达作为与其他先导标记物结合的癌症特异性标记物。 将开发针对最有希望的候选物的抗体，特别是针对可能分泌或呈递在细胞表面上的基因产物的抗体。 最后，将从杜克新诊断为乳腺癌的妇女中收集全血和血清样本。 该数据库将构成已被证明最稳健的标记物和方法的主要验证集。

This proposal will attempt to identify expression-based markers for breast cancer and develop assays using these markers to detect circulating breast cancer cells. Our application has been developed as a collaborative effort between the Duke Breast Cancer Program and Abbott Diagnostic Breast Cancer Venture Group and will utilize the considerable strengths of each institution. The basic approach for marker identification takes advantage of ongoing (and separately funded) efforts using powerful new genomics tools. At Duke, SAGE (serial analysis of gene expression) libraries of breast cancer and normal breast epithelium are being constructed as part of CGAP. Also at Duke, a series of 20 primary breast cancers will be analyzed using high-density Affymetrix expression chips (30,000 genes/ESTs will be sampled). Finally, at Abbott Diagnostics, markers have already been identified by using the Incyte Corporation expression libraries. These three sources of expression data will be compared electronically and the most promising markers that are expressed at high levels in a tissue or cancer specific manner will be identified. After the in silico analysis, the lead markers will be put through a validation algorithm that will include northern blotting, multiple tissue RNA dot blots, RT-PCR, and in situ cytohybridization. A panel of breast cell lines and a series of 50 primary breast cancers will be used to determine how commonly expressed these markers are in the target tissue. After these initial validation steps, a series of reconstitution experiments will be performed by spiking normal whole blood with known numbers of breast cancer cells, derived from cell lines and malignant effusions. Epithelial cells will be purified using magnetic bead/antibody technology and the resulting purified samples will be analyzed by flow cytometry, histology, and quantitative RT-PCR. Towards increasing sensitivity, a separate and parallel screen will focus on markers that are highly overexpressed in a subset of breast cancers. PCR multiplexing approaches will be explored in this context and will also include using telomerase gene (TERT) expression as a cancer specific marker in conjunction with other lead markers. Antibodies to the most promising candidates will be developed, particularly for gene products that may be secreted or presented on the cell surface. Finally, a collection of whole blood and serum samples will be obtained from women newly diagnosed with breast cancer at Duke. This bank will constitute a primary validation set for markers and approaches that have proven to be the most robust.