EXPRESSION BASED MARKERS FOR BREAST CANCER DETECTION

这项提议将试图确定乳腺癌的基于表达的标记，并开发使用这些标记来检测循环中的乳腺癌细胞的检测方法。我们的应用程序是杜克乳腺癌项目和雅培诊断乳腺癌风险集团之间的合作努力，并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正在进行的(和单独资助的)努力，使用强大的新基因组学工具。在杜克大学，作为CGAP的一部分，乳腺癌和正常乳腺上皮的SAGE(基因表达序列分析)文库正在构建中。同样在杜克大学，将使用高密度Affymetrix表达芯片(将抽取30,000个基因/EST)分析一系列20种原发乳腺癌。最后，在雅培诊断公司，已经通过使用Incell公司的表达文库识别了标记。这三个表达数据来源将被电子比较，并将确定以组织或癌症特有的方式高水平表达的最有希望的标记。在计算机分析后，将对先导标记进行验证算法，包括Northern杂交、多组织RNA斑点杂交、RT-PCR和原位细胞杂交。一组乳腺细胞株和一系列50例原发乳腺癌将被用来确定这些标记在目标组织中的普遍表达情况。在这些初步的验证步骤之后，将通过在正常全血中加入已知数量的乳腺癌细胞来进行一系列重建实验，这些细胞来自细胞系和恶性积液。上皮细胞将使用磁珠/抗体技术进行纯化，得到的纯化样本将通过流式细胞仪、组织学和定量RT-PCR进行分析。为了提高敏感性，一个独立的平行屏幕将专注于在乳腺癌子集中高度过度表达的标志物。在此背景下，将探索多路聚合酶链式反应的方法，还将包括使用端粒酶基因(TERT)的表达作为癌症特异性标记物与其他先导标记物一起使用。针对最有希望的候选基因的抗体将被开发出来，特别是针对可能分泌或呈现在细胞表面的基因产物。最后，杜克大学将收集新诊断为乳腺癌的妇女的全血和血清样本。该银行将构成已被证明是最可靠的标记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.