Highly Multiplexed FISH for In Situ Genomics

 描述(由申请人提供)：本申请的总体目标是设计和开发一种允许使用广泛基因库中的探针进行高度多重荧光原位杂交的技术。我们将重点关注其拷贝数变化代表可能的治疗靶点的基因。我们将利用我们实施癌症染色体异常诊断测试的长期经验，建立、测试和验证一个最佳的分析平台。这一目标将通过两个目标来实现：目标I.建立一种健壮的、可重复性的方法，用于构建至少50个位点特异性DNA序列探针的文库。我们将使用一种组合标记方法，在这种方法中，每个探针都用两个或三个荧光团的组合进行条形码编码，允许同时杂交多达120个DNA探针。我们将开发一种成像系统来解码组合标签，记录、量化和分析所获得的数据。目的II.我们将开始通过筛选外科活检标本和分离的循环肿瘤细胞(CTCs)中可操作的基因拷贝数变化来测试该检测的临床实用性。这项技术的发展将使我们能够更近地解决以下问题：患者特定的肿瘤异质性动力学是否是治疗反应变化的基础，以及在治疗随访中评估CTCs拷贝数分布是否可以预测治疗反应。该项目将作为开发和临床实施有利于患者的诊断学的模式，并将用于向一般临床癌症诊断领域传播知识和专业知识。

 DESCRIPTION (provided by applicant): The overall objective of this application is to design and develop a technology permitting highly- multiplexed fluorescence in situ hybridization using probes from a broad library of genes. We will focus on genes whose copy number variation represents possible actionable therapeutic targets. We will build, test and validate an optimal assay platform leveraging our long-standing experience implementing diagnostic tests for chromosomal abnormalities in cancer. This objective will be achieved in two aims: Aim I. Develop a robust, reproducible assay for constructing a library of at least 50 locus-specific DNA sequence probes. We will use a combinatorial labeling approach in which each probe is bar-coded with a combination of two or three fluorophores per probe, allowing for up to 120 DNA probes to be simultaneously hybridized. We will develop an imaging system to decode the combinatorial label, record, quantify and analyze the obtained data. Aim II. We will begin to test the clinical utility of the assay by screening for actionable gene copy number alterations in surgical biopsy specimens and in isolated circulating tumor cells (CTCs). The development of this technology will allow us to get closer to address the question of whether patient-specific dynamics of tumor heterogeneity underlie variation in response to treatment and whether the evaluation of CTCs copy number profile in the follow up of treatment can predict response to therapy. This project will serve as a model for development and clinical implementation of diagnostics for the benefit of patients, and will be used to disseminate knowledge and expertise to the clinical cancer diagnostic field in general.