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渐进性癌前肿瘤，以便干预的成本和风险可以集中在有可能进展为癌症的患者身上，而进展风险较低的患者可以得到保证，不再经常接受监测。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记物，直接测量进化过程。我们先前已经证明，Barrett‘s食道克隆多样性的测量可以预测进展为食管腺癌。然而，这些方法依赖于通过从每个肿瘤中提取多个空间分离的活组织检查来检测不同的克隆。这对许多肿瘤来说是不可行的。为了推广使用遗传多样性作为其他肿瘤进展的生物标记物，我们建议开发一种在单细胞或单隐窝水平上测量遗传异质性的遗传指纹技术。我们将把单个细胞流动分选到96孔板中，并使用荧光简单重复序列聚合酶链式反应(FISSR-PCR)来检测每个细胞基因组中的插入、缺失和易位。如果单细胞分析被证明是不可靠的，我们可以将单个完整的上皮隐窝分离到单独的孔中，以在隐窝水平上表征遗传多样性。我们将对来自19个基因图谱的食道切除标本的Barrett‘s食道活检组织进行测试，以确定P53野生型上皮的遗传多样性是否低于P53杂合性缺失的上皮，后者又不如非整倍体或四倍体上皮的多样性。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不断增加的遗传多样性有关。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基因多样性是否与恶变有关。2)测试基因多样性是否与癌症预防干预或治疗的抗药性的演变有关。3)测试肿瘤进展的临床前模型的遗传多样性，以便我们能够开发出忠实地概括散发性人类肿瘤的遗传多样性的模型。这些模型对于估计抗癌药物产生抗药性的可能性，并为研究抗药性的管理和预防提供了工具。为了解决这些问题，我们利用了癌症生物学、进化生物学和计算生物学方面的专业知识，以及从1989年以来前瞻性收集的巴雷特食道队列的活检组织库。这提供了一个机会，可以将有证书的生物标记物快速转化为第四阶段生物标记物研究，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.