RASL-Seq Expression Profiling of FFPE Tissues

FFPE 组织的 RASL-Seq 表达谱

• 批准号: 8851374
• 负责人: Joanne Mulligan Yeakley
• 金额: $ 36.37万
• 依托单位: BIOSPYDER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-04 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8851374
• 关键词: Archives; Area; Automobile Driving; Behavior Control; Biological Assay; Biological Markers; Cancer Patient; Caring; Cells; Clinical; Clinical Investigator; Clinical Research; Complex; Computer software; Confidential Information; Cultured Cells; Cytolysis; DNA; DNA Sequence; DNA Sequence Alteration; DNA analysis; Data; Data Analyses; Databases; Detection; Development; Diagnostic; Formalin; Freezing; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Fusion; Gene Mutation; Genes; Genetic Transcription; Genomic DNA; Genomics; Hour; Immunoglobulin Variable Region; Informatics; Libraries; Licensing; Ligation; Malignant Neoplasms; Malignant neoplasm of prostate; Measurable; Measurement; Measures; Mediating; Methods; Molecular Profiling; Monitor; Mutation; Mutation Detection; Nucleotides; Oligonucleotides; Output; Pathology; Patients; Performance; Poly A; Predictive Value; Preparation; Process; Prostatic Intraepithelial Neoplasias; Protocols documentation; Qualifying; Quality Control; RNA; RNA Degradation; RNA analysis; Readiness; Reading; Reagent; Reporting; Reproducibility; Research Personnel; Running; Sampling; Sensitivity and Specificity; Somatic Mutation; Specific qualifier value; Specimen; Technology; Testing; Text; Thick; Time; Tissue Sample; Tissues; Titrations; Translational Research; Validation; Variant; Work; Writing; clinically significant; cohort; crosslink; design; diagnostic assay; differential expression; drug discovery; improved; insertion/deletion mutation; interest; magnetic beads; meetings; next generation sequencing; novel; novel diagnostics; novel therapeutics; performance tests; process optimization; programs; public health relevance; sample fixation; screening; success; targeted sequencing; therapeutic target; tissue fixing; tool; transcriptome sequencing; tumor; user-friendly; validation studies; verification and validation

 DESCRIPTION (provided by applicant): This project will develop and validate the application of RASL-Seq, a multiplexed targeted expression profiling technology, to analysis of fixed tumor specimens. The technology detects hundreds of RNA targets at once in cell lysates, using oligo ligation, amplification, and next-generation sequencing for quantitation. This highly multiplexed assay will allow a single, fixed tissue sample-to-answer assay protocol that can be applied to different tumor types, while delivering a simple report tailored to the sample. The technology will also promote clinical research by enabling somatic mutation and gene fusion detection in RNA, as well as measurement of mutations at the level of DNA, and the measurement of variable regions of DNA. By measuring DNA mutations, and gene expression levels on a single platform, potentially in a single assay or two parallel assays, improved information can be provided to the investigator, and in the case of a diagnostic assay, to the patient. It is conceivable that measuring mutations at the level of RNA and not just DNA will not only provide redundancy, but will provide an indication of whether a particular genomic mutation is actually driving the cancer, or if a different mutation is driving it, which will result in improved treatment decisions for patients, by better focusing treatment to likely therapeutic targets. Measurement of somatic mutations may result in better sensitivity than genomic DNA analysis alone, due to the typically larger number of target molecules present per cell. Since the original application, some of the proposed work has been completed, and feasibility for measuring fixed tissue demonstrated, permitting scope to be expanded into DNA measurement. To demonstrate commercial readiness, we will establish the mutation assays, optimize the protocols, introduce additional positive controls and quality assessment steps, assess reproducibility, sensitivity and specificity using matched frozen and FFPE samples, and develop data analysis tools. We will establish performance measures (reproducibility, dynamic range, limit of detection for mutations, and the behavior of controls), and conduct verification and validation studies to establish the positive predictive value and negative predictive value for mutation assays where independent assessment of mutation is available. This program will establish as commercial assays a panel of mutations and a pan-cancer assay of ~1,500 genes and mutations. It will establish a cancer-related gene expression and mutation database qualified by extent of validation from which investigators can select content for customized RASL-Seq assays. If successful, utility will be validated by identifying biomarkers and mutations that differentiate between high grade prostatic intraepithelial neoplasia (HGPN) and invasive prostate cancer (PCa), and potentially by demonstrating that there are biomarkers that differentiate HGPIN in patients without PCa from HGPIN associated morphologically with PCa as a step toward identifying mechanisms leading to progression as well as providing biomarkers that can be used as a diagnostic to identify patients with HGPIN who are likely to progress to PCa. Overall the project will deliver a commercializable technology and specific commercial kit assays that will allow simultaneous measurement of expression and mutations from hundreds of genes with clinical significance for common cancers, all in a single process flow that meets the needs of clinical investigators by starting with FFPE tissue sections and ending with a simple report.

 描述（由申请人提供）：本项目将开发和验证RASL-Seq（一种多重靶向表达谱分析技术）在固定肿瘤标本分析中的应用。该技术使用寡核苷酸连接、扩增和下一代测序进行定量，同时检测细胞裂解物中的数百种RNA靶标。这种高度多路复用的检测将允许单一的、固定的组织样本到答案的检测方案，该方案可以应用于不同的肿瘤类型，同时提供针对样本定制的简单报告。该技术将 还通过实现RNA中的体细胞突变和基因融合检测，以及DNA水平的突变测量和DNA可变区的测量来促进临床研究。通过在单个平台上测量DNA突变和基因表达水平，可能在单个测定或两个平行测定中，可以向研究者提供改进的信息，并且在诊断测定的情况下，向患者提供改进的信息。可以想象，在RNA而不仅仅是DNA水平上测量突变不仅会提供冗余，而且会提供特定基因组突变是否实际上驱动癌症的指示， 或者是否是不同的突变在驱动它，这将通过更好地将治疗集中于可能的治疗靶点来改善患者的治疗决策。体细胞突变的测量可能导致比单独的基因组DNA分析更好的灵敏度，这是由于每个细胞存在的靶分子的数量通常更大。自最初的申请以来，一些拟议的工作已经完成，并且证明了测量固定组织的可行性，允许范围扩展到DNA测量。为了证明商业准备，我们将建立突变试验，优化方案，引入额外的阳性对照和质量评估步骤，评估重现性，灵敏度和特异性 使用匹配的冷冻和FFPE样本，并开发数据分析工具。我们将建立性能指标（重现性、动态范围、突变检测限和对照品的行为），并进行验证和确认研究，以确定突变检测的阳性预测值和阴性预测值，其中突变检测可进行独立评估。该计划将建立一组突变和一个约1，500个基因和突变的泛癌测定作为商业测定。它将建立一个癌症相关基因表达和突变数据库，该数据库通过验证程度进行认证，研究人员可以从中选择定制RASL-Seq检测的内容。如果成功，将通过鉴定区分高级别前列腺上皮内瘤变（HGPN）和浸润性前列腺癌（PCa）的生物标志物和突变来验证效用，并可能通过证明存在区分无PCa患者中的HGPIN与形态学上与PCa相关的HGPIN的生物标志物，作为鉴定导致进展的机制以及提供可用作PCa的生物标志物的步骤。一种诊断方法，用于识别可能进展为PCa的HGPIN患者。总的来说，该项目将提供一种可商业化的技术和特定的商业试剂盒检测，允许同时测量数百种对常见癌症具有临床意义的基因的表达和突变，所有这些都在一个单一的过程流程中，通过从FFPE组织切片开始，以简单的报告结束，满足临床研究者的需求。