An Intra-Neoplasm Genetic Diversity Assay

描述（由申请人提供）： 有效的癌症预防取决于区分惰性和进展性癌前肿瘤，以便干预措施的成本和风险可以集中在可能进展为癌症的患者身上，而低进展风险的患者可以放心并从频繁的监测中删除。由于肿瘤进展是一个克隆进化的过程，我们正在开发一类新的生物标志物，直接测量进化过程。我们以前已经表明，巴雷特食管克隆多样性的措施预测食管腺癌的进展。然而，这些方法依赖于通过从每个肿瘤中采集多个空间分离的活检来检测不同的克隆。这对于许多肿瘤是不可行的。为了推广使用遗传多样性作为其他肿瘤进展的生物标志物，我们建议开发一种遗传指纹技术，用于测量单细胞或单隐窝水平的遗传异质性。我们将流式分选单个细胞到96孔板中，并使用荧光简单重复序列间PCR（FISSR-PCR）来检测每个细胞基因组中的插入、缺失和易位。如果单细胞分析证明不可靠，我们可以将单个完整的上皮隐窝分离到单独的威尔斯孔中，以表征隐窝水平的遗传多样性。我们将在来自19个基因定位的食管切除术标本的Barrett食管活检上测试该测定，以确定p53野生型上皮的遗传多样性是否低于具有p53杂合性缺失的上皮，而具有p53杂合性缺失的上皮的遗传多样性又低于非整倍体或四倍体上皮。这将确定表征巴雷特肿瘤进展的分子事件是否与单细胞水平上遗传多样性的增加相关。该项目的成功将开启三个重要的未来研究：1）测试各种上皮内肿瘤的遗传多样性是否与恶性进展相关。2)测试遗传多样性是否与癌症预防干预或治疗的耐药性演变有关。3)对肿瘤进展的临床前模型的遗传多样性进行测试，以便我们能够开发出忠实地概括散发性人类肿瘤遗传多样性的模型。这些模型对于估计癌症预防药物产生耐药性的可能性非常重要，并为研究这种耐药性的管理和预防提供了工具。为了解决这些问题，我们利用癌症生物学，进化生物学和计算生物学的专业知识，以及从1989年以来前瞻性收集的巴雷特食管队列活检组织库的访问。这提供了一个机会，可以快速将认证的生物标志物转化为IV期生物标志物研究，并从那里进入临床。

DESCRIPTION (provided by applicant): Effective cancer prevention depends on distinguishing between indolent and progressive pre-malignant neoplasms, so that the cost and risks of interventions can be focused on patients likely to progress to cancer, while patients at low risk of progression can be reassured and removed from frequent surveillance. Because neoplastic progression is a process of clonal evolution, we are developing a novel class of biomarkers that directly measure the evolutionary process. We have previously shown that measures of clonal diversity in Barrett's esophagus predict progression to esophageal adenocarcinoma. However, those methods depended on detecting distinct clones by taking multiple, spatially separated biopsies from each neoplasm. This is not feasible for many neoplasms. In order to generalize the use of genetic diversity as a biomarker of progression to other neoplasms, we propose to develop a genetic fingerprinting technique for measuring genetic heterogeneity at the single cell, or single crypt, level. We will flow sort single cells into a 96 well plate and use fluorescent inter-simple sequence repeat PCR (FISSR-PCR) to detect insertions, deletions and translocations in each cell's genome. If single cell assays prove unreliable, we can isolate single whole epithelial crypts into separate wells to characterize genetic diversity at the crypt level. We will test this assay on Barrett's esophagus biopsies from 19 genetically mapped esophagectomy specimens to determine if p53 wildtype epithelium is less genetically diverse than epithelium with p53 loss of heterozygosity, which in turn is less diverse than aneuploid or tetraploid epithelium. This will determine if the molecular events that characterize Barrett's neoplastic progression are associated with increasing genetic diversity at the single cell level. The success of this project will open up three important future studies: 1) Tests of whether genetic diversity in a variety of intraepithelial neoplasms is associated with progression to malignancy. 2) Tests of whether genetic diversity is associated with the evolution of resistance to cancer prevention interventions or therapy. 3) Tests of the genetic diversity of pre-clinical models of neoplastic progression so that we can develop models that faithfully recapitulate the genetic diversity of sporadic human neoplasms. Such models will be important for estimating the likelihood that resistance will develop to cancer prevention agents and provide tools for studying the management and prevention of such resistance. To address these problems we utilize expertise in caner biology, evolutionary biology and computational biology, as well as access to a tissue bank of biopsies from a Barrett's esophagus cohort that has been collected prospectively since 1989. This provides an opportunity to rapidly translate credentialed biomarkers into phase IV biomarker studies and from there into the clinic.