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

用于 FFPE 组织基因表达谱分析的高度多重 PCR 平台

• 批准号: 8432665
• 负责人: Jork N Nolling
• 金额: $ 33.52万
• 依托单位: PRIMERADX, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-19 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432665
• 关键词: Accounting; Address; Archives; B-Lymphocytes; Biological Assay; Biological Markers; Biological Preservation; Biopsy; Blood capillaries; Cancer Detection; Cancer Diagnostics; Cells; Characteristics; Classification; Clinical; Clinical Treatment; Communities; Detection; Diagnosis; Diagnostic; Formalin; Freezing; Future; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Goals; Human; Length; Lymphoma; Malignant Neoplasms; Methodology; Methods; Microarray Analysis; Molecular; Molecular Profiling; Non-Hodgkin' s Lymphoma; Outcome; Paraffin Embedding; Patients; Performance; Proteins; Protocols documentation; RNA; Reaction; Refractory; Regimen; Relapse; Research; Resources; Sampling; Solid Neoplasm; Solutions; Source; Specimen; Structure of germinal center of lymph node; Subgroup; System; Technology; Testing; Time; Tissue Sample; Tissues; Translations; Tube; Tumor Biology; Tumor Tissue; United States; Work; base; cancer genomics; capillary; clinical application; clinical practice; cost; flexibility; genetic analysis; innovation; instrument; large cell Diffuse non-Hodgkin' s lymphoma; meetings; neoplastic cell; novel; rapid diagnosis; response; sample fixation; tumor

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.

描述（由申请人提供）：本申请的总体目标是提供一个高度多重、定量和自动化的系统，用于使用福尔马林固定石蜡包埋（FFPE）材料作为样品来源快速识别弥漫性大 B 细胞淋巴瘤（DLBCL）亚组；研究和临床界尚无法使用的技术。 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，但仍有相当一部分患者仍然难治或复发。基因表达谱显示，这种反应差异反映在肿瘤的生物学中，并且根据肿瘤细胞的起源定义了两个亚组。一组 17 个基因内的分子特征可以区分这些亚组。目前检测基因表达模式的技术主要围绕两种现有形式：微阵列和实时 PCR。前者虽然能够检测数百到数千个基因，但缺乏敏感性和定量能力，而PCR虽然定量且非常敏感，但其多重能力极其有限。因此，迫切需要将多重属性与灵敏的定量分析相结合的技术，以促进对肿瘤生物学的理解，并将其转化为临床诊断用途。档案 FFPE 肿瘤是转化癌症基因组研究的宝贵资源。然而，利用 FFPE 组织具有挑战性，因为它通常源自含有 RNA 的小活检，这些 RNA 在固定和储存过程中会碎裂，从而使其不太适合微阵列分析。因此，缺乏能够利用 FFPE 作为基因分析组织来源的定量、多重、灵敏且稳健的检测方法。 为了克服上述障碍，我们建议开发一种新颖、快速和自动化的检测方法来检测、量化和分类 DLBCL 的两种主要亚型，从而促进肿瘤类型的及时诊断，从而为临床治疗选择提供信息。这样的技术，可以检测 并使用 FFPE 样本作为源材料在单管反应中量化多个基因靶标，目前还不存在。 该提案的目标将通过完成三个具体目标来实现。其中包括 (i) 通过采用 PrimeraDx 的新兴 STAR 技术和 ICEPlex 仪器系统来开发称为“ICEPlex Multiplex FFPE Assay”的技术； (ii) 验证所得测定的分析性能特征； (iii) 通过将新开发的测定结果与目前用于 DLBCL 亚型分型的参考微阵列技术进行比较，来验证测定的性能。 公共健康相关性：过去十年，随着肿瘤基因表达谱分析的兴起，癌症诊断发生了重大转变，这导致癌症检测、诊断和治疗方面的改进。然而，为了充分发挥这种方法的潜力，需要克服涉及单次测定中可检测的基因数量以及使用可能降解的储存组织样本的技术障碍。 PrimeraDx 将通过提供自动化解决方案来解决这些问题，对存储的肿瘤组织材料进行单反应、多样本分析，以便对类型进行分类 一种常见的人类淋巴瘤，从而有助于快速诊断、确定治疗方案以及分析治疗反应。