A Highly Multiplexed PCR Platform for Gene Expression Profiling from FFPE Tissue

描述(申请人提供)：本申请的总体目标是提供一个高度多元化、定量和自动化的系统，用于使用福尔马林固定的石蜡包埋(FFPE)材料作为样本来源快速识别弥漫性大B细胞淋巴瘤(DLBCL)亚群；这是一项研究和临床尚未获得的技术。DLBCL约占所有非霍奇金淋巴瘤的30%，因此是美国这种癌症最常见的亚型。虽然许多患者对治疗反应良好，但也有相当一部分患者仍然难以治愈或复发。基因表达谱显示，这种反应的差异反映在肿瘤的生物学上，根据肿瘤细胞的来源定义了两个亚群。由17个基因组成的小组中的分子特征可以区分这些亚群。目前检测基因表达模式的技术主要围绕两种现有的形式：微阵列和实时聚合酶链式反应。前者虽然能够检测到成百上千个基因，但缺乏敏感性和定量能力；而聚合酶链式反应虽然是定量的，非常敏感，但其多重能力极其有限。因此，迫切需要结合多重属性和灵敏、定量分析的技术，以促进对肿瘤生物学的理解，使其转化为临床诊断实用。的档案 FFPE肿瘤是翻译癌症基因组研究的宝贵资源。然而，利用FFPE组织是具有挑战性的，因为它通常来自含有在固定和储存过程中碎裂的RNA的小活检组织，从而使其不太适合于微阵列分析。因此，缺乏能够利用FFPE作为组织来源进行遗传分析的定量、多重、灵敏和Robus分析方法。为了克服上述障碍，我们建议开发一种新的、快速的、自动化的检测方法来检测、量化和分类DLBCL的两个主要亚型，从而促进对肿瘤类型的及时诊断，以便为临床治疗方案提供信息。这样一种技术，可以检测到 并使用FFPE样本作为来源材料在单管反应中定量多个基因靶点，目前尚不存在。这项提案的目标将通过完成三个具体目标来实现。这些措施包括(I)通过采用PrimeraDx的新兴STAR技术和Iceplex仪器系统来开发名为“Iceplex Multiplex FFPE Assay”的技术；(Ii)验证所得分析的分析性能特征；以及(Iii)通过进行一项一致性研究，将新开发的分析结果与目前用于DLBCL亚型的参考微阵列技术进行比较，以验证该分析的性能。 公共卫生相关性：在过去的十年里，随着肿瘤基因表达谱分析的优势，癌症诊断发生了重大转变，这导致了癌症检测、诊断和治疗的改进。然而，为了充分发挥这种方法的潜力，需要克服技术障碍，包括在一次检测中可以检测到的基因数量，以及使用可能被降解的储存组织样本。PrimeraDx将通过从存储的肿瘤组织材料中提供单反应、多样本分析的自动化解决方案来解决这些问题，以便对类型进行分类 这是一种常见的人类淋巴瘤，从而有助于快速诊断、确定治疗方案和分析治疗反应。

DESCRIPTION (provided by applicant): The overall goal of this application is to deliver a highly multiplexed, quantitative, and automated system for the rapid identification of diffuse large B-cell lymphoma (DLBCL) subgroups using formalin-fixed paraffin-embedded (FFPE) material as the sample source; a technology that is not yet available to the research and clinical communities. DLBCL accounts for ~30% of all non-Hodgkin lymphomas and thus is the most common subtype of this cancer in the United States. While many patients respond well to treatment, there is a sizable subset that remains refractory or suffers relapse. Gene expression profiling has revealed that this difference in response is reflected in the biology of the tumor an two subgroups have been defined based on the origin of the tumor cell. Molecular signatures within a panel of 17 genes can differentiate these subgroups. Technologies to detect gene expression patterns currently center around two existing formats: microarrays and real-time PCR. The former, although able to detect hundreds to thousands of genes, suffer from their lack of sensitivity and quantitative capacity, while PCR, although quantitative and exceedingly sensitive, has extremely limited multiplexing capability. Therefore, technologies that combine attributes of multiplexing with sensitive, quantitative analysis are critically needed to advance the understanding of tumor biology toward translation into clinical diagnostic utility. Archives of FFPE tumors represent a valuable resource for translational cancer genomic research. However, utilizing FFPE tissue is challenging since it is often derived from small biopsies containing RNA that is fragmented during fixation and storage, thus rendering it less suitable for microarray analysis. As a result there is a lack of quantitative, multiplexed, sensitive, and robus assays that are able to utilize FFPE as a source of tissue for genetic analysis. In order to overcome these above obstacles we propose to develop a novel, rapid, and automated assay to detect, quantify, and classify the two main subtypes of DLBCL, thus facilitating timely diagnosis of the tumor type in order to inform clinical treatment options. Such a technology, that can detect and quantify multiple gene targets in a single-tube reaction using FFPE specimens as the source material, does not currently exist. The objective of this proposal will be met by completing three Specific Aims. These consist of (i) developing the technology, to be termed the 'ICEPlex Multiplex FFPE Assay', by adapting PrimeraDx's emerging STAR technology and ICEPlex instrument system for this purpose; (ii) validating the analytical performance characteristics of the resultant assay; and (iii) verifying performance of the assay by conducting a concordance study comparing results from the newly developed assay with a reference microarray technology currently used for DLBCL subtyping. PUBLIC HEALTH RELEVANCE: The past decade has seen a major shift in cancer diagnostics with the ascendance of gene expression profile analysis of tumors, which is leading to improvements in cancer detection, diagnosis, and treatment. However, to achieve the full potential of this approach, technology impediments involving the number of genes that can be detected in a single assay and the use of stored tissue samples that may be degraded need to be overcome. PrimeraDx will address these problems by providing an automated solution for a single-reaction, multi-sample analysis from stored tumor tissue material in order to classify types of a common human lymphoma, thereby facilitating rapid diagnosis, determination of treatment options, and analysis of response to therapy.