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来自广泛基因文库的探针进行高度多重荧光原位杂交的技术.我们将集中在基因的拷贝数变化代表可能的可操作的治疗目标。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，构建、测试和验证最佳的检测平台。这一目标将通过两个目标实现：目标一。开发一种稳健、可重复的检测方法，用于构建至少50个基因座特异性DNA序列探针的文库。我们将使用一种组合标记方法，其中每个探针用每个探针两个或三个荧光团的组合进行条形码化，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，量化和分析所获得的数据。Aim II.我们将开始通过在手术活检标本和分离的循环肿瘤细胞（CTC）中筛选可操作的基因拷贝数改变来测试该测定法的临床实用性。这项技术的发展将使我们能够更接近解决以下问题：肿瘤异质性的患者特异性动力学是否是治疗反应变化的基础，以及治疗随访中CTC拷贝数谱的评估是否可以预测对治疗的反应。该项目将作为诊断的开发和临床实施的模式，以造福患者，并将用于传播知识和专业知识，以临床癌症诊断领域的一般。

 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.