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检测开发的生物标志物。首先，通过比较肿瘤细胞类型与正常细胞类型的转录组或基因表达，确定癌症检测和疾病分层的标志物。细胞类型特异性转录组是通过使用细胞表面CD抗原抗体从组织标本中分离细胞，然后使用Affymetrix DNA阵列进行表达分析来确定的。其次，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表现出较高的表达水平，使用商业小鼠抗体1C3进行Western blot分析，在肿瘤组织制剂中检测到17kda的AGR2蛋白。第三，组织芯片分析证实了前列腺肿瘤中AGR2的表达，而非癌组织中AGR2的表达很少。第四，针对AGR2的新单克隆抗体正在产生，用于开发前列腺癌患者的尿液检测，其中肿瘤分泌的AGR2蛋白可能存在于前列腺癌患者的尿中。ng / ml的水平。针对细菌产生的重组AGR2蛋白提出的1C3抗体似乎不能识别由AGR2- 1 -前列腺癌细胞系CL1分泌的天然AGR2。本文还提出了一种新的方法来获得有用的抗体，用肿瘤组织制剂中的蛋白质免疫小鼠。肿瘤异质性可归因于多种癌细胞类型：Gleason 3型与Gleason 4型；CD10-和CD10+。这些细胞类型之间的差异表达基因是有用的风险评估标记，因为模式4和CD10+癌细胞与不良预后相关。此外，还可以从肿瘤相关基质细胞的差异表达基因中获得标记物。其中一个例子是发现释放到肿瘤组织制剂中的~29 kDa CD90/THY1蛋白。一种多标记分析工具，多反应监测（MRM）质谱法，正在开发中，以测量多种信息蛋白同时在尿液中。细胞转录组允许癌细胞类型特异性特征的发展，这些特征由基因和它们的丰度（在转录计数中）组成。这些可以用于诊断肿瘤是否存在特定的癌细胞类型，从而提供预后信息。

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.