The Early Detection Research Network: Biomarker Developmental Laboratories

早期检测研究网络：生物标记发育实验室

• 批准号: 7982287
• 负责人: ALVIN Y LIU
• 金额: $ 43.71万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-22 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7982287
• 关键词: AGR2 gene; Antibodies; Biological Markers; CD Antigens; Cancer Detection; Cell Separation; Cell surface; Cells; DNA; Development; Diagnosis; Disease; Early Detection Research Network; Gene Expression; Genes; Heterogeneity; Laboratories; MME gene; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Microarray Analysis; Monoclonal Antibodies; Mus; Outcome; PC3 cell line; Pattern; Procedures; Prostatic Neoplasms; Proteins; Recombinants; Risk Assessment; Specimen; Stratification; Stromal Cells; Testing; Tissue Microarray; Tissues; Transcript; Tumor Tissue; Urine; Western Blotting; assay development; cancer cell; cell type; extracellular; human AGR2 protein; men; multiple reaction monitoring; novel; prognostic; tissue preparation; tool; tumor

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.

描述（由申请人提供）：我们使用靶向方法来发现用于检测开发的生物标志物。首先，通过将肿瘤内细胞类型的转录组或基因表达与其正常对应物的转录组或基因表达进行比较来鉴定用于癌症检测和疾病分层的标志物。细胞类型特异性转录组通过使用细胞表面CD抗原的抗体从组织样本中分离细胞，然后使用Affyssin DNA阵列进行表达分析来确定。第二，选择编码细胞外/分泌蛋白的肿瘤上调基因。基因AGR 2在前列腺癌细胞中表现出相对高水平的表达，并且使用商业小鼠抗体1C 3通过Western印迹分析在肿瘤组织制备物中检测到17-kDa AGR 2蛋白。第三，组织微阵列分析验证了AGR 2在前列腺肿瘤中的表达，而非癌组织中几乎没有AGR 2的表达。第四，新的单克隆抗体正在产生对AGR 2的尿液测试，以筛选男性前列腺癌，其中肿瘤分泌的AGR 2蛋白可能存在于排尿？ng/ml水平。针对细菌产生的重组AGR 2蛋白产生的1C 3抗体似乎不识别由AGR 2-I-前列腺癌细胞系CL 1分泌的天然AGR 2。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。肿瘤异质性可归因于多种癌细胞类型：Gleason模式3 vs. Gleason模式4; CD 10- vs. CD 10+。这些细胞类型中的差异表达基因可用作风险评估标记物，因为模式4和CD 10+癌细胞与不良结局相关。此外，标记物可来自肿瘤相关基质细胞的差异表达基因。一个例子是发现释放到肿瘤组织制备物中的~29 kDa CD 90/THY 1蛋白。正在开发一种多标记分析工具，多反应监测（MRM）质谱法，以同时测量尿液中的多种信息蛋白。细胞转录组允许开发癌细胞类型特异性签名，其由基因及其丰度（在转录物计数中）组成。这些可以应用于诊断肿瘤是否存在特定的癌细胞类型，然后提供预后信息。