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技术，允许使用广泛基因文库中的探针进行高复用荧光原位杂交。我们将重点关注拷贝数变异代表可能可行的治疗靶点的基因。我们将利用我们在癌症染色体异常诊断测试方面的长期经验，建立、测试和验证一个最佳的分析平台。这一目标将在两个目标中实现：目标1 .开发一个强大的，可重复的试验，用于构建至少50个位点特异性DNA序列探针的文库。我们将使用组合标记方法，其中每个探针与每个探针的两个或三个荧光团的组合条形码，允许多达120个DNA探针同时杂交。我们将开发一个成像系统来解码组合标签，记录，量化和分析获得的数据。目的二世。我们将通过筛选手术活检标本和分离的循环肿瘤细胞（ctc）中可操作的基因拷贝数改变来开始测试该分析的临床应用。这项技术的发展将使我们更接近于解决以下问题：肿瘤异质性的患者特异性动态是否构成对治疗反应变化的基础，以及在治疗随访中对ctc拷贝数谱的评估是否可以预测对治疗的反应。该项目将作为发展和临床实施诊断的典范，以造福患者，并将用于向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.