The Early Detection Research Network: Biomarker Developmental Laboratories

描述(由申请人提供)：我们使用有针对性的方法来发现用于分析开发的生物标记物。首先，癌症检测和疾病分层的标志是通过比较肿瘤内细胞类型的转录本或基因表达与正常对应的细胞类型的转录本或基因表达来确定的。通过使用细胞表面CD抗原抗体从组织标本中分离细胞，然后使用Affymetrix DNA阵列进行表达分析，确定细胞类型特定的转录本。第二，选择编码细胞外/分泌蛋白的肿瘤上调基因。AGR2基因在前列腺癌细胞中有较高水平的表达，使用商品化的小鼠抗体1C3进行Western印迹分析，在肿瘤组织标本中检测到17-kDa AGR2蛋白。第三，组织芯片分析证实了前列腺癌组织中AGR2的表达，而非肿瘤组织中AGR2的表达很少。第四，针对AGR2的新的单克隆抗体正在产生，以开发一种尿液检测方法来筛查前列腺癌患者，前列腺癌患者的排尿中可能存在肿瘤分泌的AGR2蛋白，水平为1ng/ml。针对细菌产生的重组AGR2蛋白而产生的1C3抗体似乎不识别由AGR2-I-前列腺癌CL1细胞分泌的天然AGR2。还提出了一种获得有用抗体的新方法，用肿瘤组织制剂中的蛋白质免疫小鼠。肿瘤的异质性可以归因于多种癌细胞类型：Gleason 3型与Gleason 4型；CD10-vs.CD10。这些细胞类型中差异表达的基因作为风险评估标记物是有用的，因为模式4和CD10癌细胞与不良预后相关。此外，标记还可以从肿瘤相关基质细胞的差异表达基因中获得。一个例子是~29 kDa CD90/THY1蛋白被发现释放到肿瘤组织制剂中。一种多标记分析工具--多反应监测(MRM)质谱仪正在开发中，用于同时检测尿液中的多个信息蛋白质。细胞转录本允许发展出癌细胞类型特异的特征，包括基因及其丰度(在转录本计数中)。这些可以应用于诊断肿瘤是否存在特定类型的癌细胞，然后提供预后信息。

DESCRIPTION (provided by applicant): We use a targeted approach to discover biomarkers for assay development. First, markers for cancer detection and disease stratification are identified by comparing the transcriptomes, or gene expression, of cell types within tumors to those of their normal counterpart. Cell type-specific transcriptomes are determined via cell isolation from tissue specimens using antibodies to cell surface CD antigens followed by expression analysis using Affymetrix DNA arrays. Second, tumor upregulated genes encoding extracellular/secreted proteins are selected. The gene AGR2 showed a comparatively high level of expression in prostate cancer cells, and the 17-kDa AGR2 protein was detected in tumor tissue preparations by Western blot analysis using a commercial mouse antibody 1C3. Third, tissue microarray analysis validated prostate tumor expression of AGR2, and non-cancer tissue showed little AGR2 expression. Fourth, new monoclonal antibodies are being generated against AGR2 for the development of a urine test to screen men with prostate cancer, where tumor secreted AGR2 protein might be present in voided urine at ?ng/ml levels. The 1C3 antibody, which was raised against a bacterially produced recombinant AGR2 protein, appeared not to recognize native AGR2 as secreted by the AGR2-I- prostate cancer cell line CL1. A novel procedure to obtain useful antibodies is also proposed to immunize mice with proteins in tumor tissue preparations. Tumor heterogeneity could be attributed to multiple cancer cell types: Gleason pattern 3 vs. Gleason pattern 4; CD10- vs. CD10+. Differentially expressed genes among these cell types are useful as risk assessment markers because pattern 4 and CD10+ cancer cells are associated with poor outcomes. In addition, markers could be derived from differentially expressed genes of tumor-associated stromal cells. One example is the ~29 kDa CD90/THY1 protein found released into tumor tissue preparations. A multi-marker analysis tool, multiple reactions monitoring (MRM) mass spectrometry, is being developed to measure multiple informative proteins simultaneously in urine. Cell transcriptomes allow the development of cancer cell type- specific signatures that consist of genes and their abundance (in transcript counts). These can be applied to diagnose tumors for the presence of specific cancer cell types, which will then provide prognostic information.