Project 1: Identification and Validation of Panel of Early Cell Surface Gene Targets

摘要-本研究项目福尔斯属于肿瘤异质性的基因组学及其对肿瘤生长的影响。 进展为EAC，作为RFA-CA-16-006中定义的可能区域之一。我们先前已经 使用基因表达谱鉴定EGFR和ErbB 2作为有希望的细胞表面靶点， 频率基因扩增用于EAC成像，并已开发出对这些特异性的肽 目标的我们现在的目标是确定BE向EAC进展中出现的早期靶点。我们希望能找到更大的 与晚期靶标相比，早期靶标的可变性和较低的表达水平，因此已经制备了一种更 严格的方法。我们将使用AffysseTM阵列和RNAseq分析来识别早期基因靶点， 在细胞表面上过表达，并且可以显影用于成像。我们将使用这些复杂的基因组 工具，提供基因改变，突变和DNA拷贝数变异的全面分析， 人类食道标本，以确定一组有前途的候选人。我们将严格验证 使用定量RT-PCR和免疫组织化学在组织微阵列上检测候选靶点， 一组独立的样本这种方法需要大量的HGD， 在我们的大型组织生物储存库中获得n = 780例人食管标本，包括n = 560例EAC， 重新采集（无新辅助化疗或放疗）。对于每个经过验证的早期目标，我们将 转染端粒酶永生化的BE细胞以在细胞表面上过表达靶标，用于开发 项目2中的单体和二聚体肽。 这些目标的成功完成将导致一组早期基因的鉴定和验证， 在BE到EAC进展中出现的靶点，将用于项目2，以开发新的肽成像 剂.

Abstract – This research project falls under genomics of tumor heterogeneity and their impact on the progression toward EAC as one of the possible areas defined in RFA-CA-16-006. We have previously identified EGFR and ErbB2 using gene expression profiles as promising cell surface targets based on high- frequency gene amplification for imaging of EAC, and have developed peptides that are specific for these targets. We now aim to identify early targets that arise in progression of BE to EAC. We expect to find greater variability and lower levels of expression for early compared with late targets, thus have prepared a more rigorous approach. We will use Affymetrix arrays and RNAseq analysis to identify early gene targets that are overexpressed on the cell surface and can be developed for imaging. We will use these sophisticated genomic tools to provide a comprehensive analysis of gene alterations, mutations, and DNA copy number variations in human esophagus specimens to identify a panel of promising candidates. We will rigorously validate candidate targets using quantitative RT-PCR and immunohistochemistry on tissue microarrays prepared from an independent cohort of specimens. This approach requires a large number of HGD that are uniquely available in our large tissue biorepository of n = 780 human esophageal specimens, including n = 560 EAC, collected de novo (no neo-adjuvant chemo or radiation therapy). For each early target validated, we will transfect telomerase-immortalized BE cells to overexpress the target on the cell surface for use in developing the monomer and dimer peptides in Project 2. Successful completion of these aims will result in identification and validation of a panel of early gene targets that arise in progression of BE to EAC that will be used in Project 2 to develop novel peptide imaging agents.