RASL-Seq Expression Profiling of FFPE Tissues

 描述(申请人提供)：该项目将开发和验证RASL-Seq，一种多重靶向表达谱技术，用于分析固定的肿瘤标本。这项技术使用寡聚连接、扩增和下一代测序进行定量，一次检测细胞裂解产物中的数百个RNA靶标。这种高度多元化的分析将允许单一的、固定的组织样本到答案分析协议，该协议可以应用于不同的肿瘤类型，同时提供针对样本量身定做的简单报告。这项技术将 还通过在RNA中进行体细胞突变和基因融合检测，以及在DNA水平上测量突变，以及测量DNA的可变区，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，可能在单个检测或两个平行检测中，可以向研究人员提供改进的信息，在诊断检测的情况下，可以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将提供冗余，还将提供特定基因组突变是否真正导致癌症的指示， 或者如果是不同的突变在驱动它，这将通过更好地将治疗集中在可能的治疗靶点上，从而改善患者的治疗决策。体细胞突变的测量可能比单独进行基因组DNA分析的灵敏度更高，因为每个细胞中通常存在更多的靶分子。自最初应用以来，一些拟议的工作已经完成，并证明了测量固定组织的可行性，从而允许将范围扩大到DNA测量。为了证明商业化的准备情况，我们将建立突变检测，优化方案，引入额外的阳性对照和质量评估步骤，评估重复性、敏感性和特异性。 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(重复性、动态范围、突变的检测极限和对照的行为)，并进行验证和验证研究，以确定可对突变进行独立评估的突变检测的阳性预测值和阴性预测值。该计划将建立一组突变和约1,500个基因和突变的泛癌分析作为商业分析。它将建立一个通过验证程度合格的癌症相关基因表达和突变数据库，研究人员可以从中选择定制的RASL-Seq分析内容。如果成功，将通过识别区分高级别前列腺上皮内瘤变(HGPN)和侵袭性前列腺癌(PCA)的生物标记物和突变来验证有效性，并可能通过证明存在生物标记物来区分无前列腺癌患者的HGPIN和与PCa相关的HGPIN，以此作为确定导致进展的机制的一步，并提供可用于诊断的生物标记物，以识别可能进展为PCa的HGPIN患者。总体而言，该项目将提供一种可商业化的技术和特定的商业试剂盒分析，允许同时测量数百个对常见癌症具有临床意义的基因的表达和突变，所有这些都将在一个满足临床研究人员需求的单一流程中完成，从FFPE组织切片开始，以一份简单的报告结束。

 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.