Assay Validation of Targeted RNA sequencing to Detect Kinase Gene Fusions

项目摘要 针对DNA水平改变（如点突变和拷贝）的靶向下一代测序检测 考虑到临床试验，数字改变常规用于晚期癌症患者， 研究药物。RNA测序，虽然成功地应用于发现基因融合， 基因表达的定量评估尚未转化为患者护理的临床级测试 部分是由于有限的专业知识和在临床上创建准确和精确的测定的困难 等级实验室。我们假设靶向RNA测序可以作为一种通用的临床分级方法， 用于晚期癌症患者治疗试验的分子合格性工具。在本提案中，我们 将开发包括分析验证的临床级靶向RNA测序测定（目标1：准确性， 精度目标2：性能范围）和临床真实的世界样本的性能（目标3：临床 验证），最后，我们将通过提供探针/方案广泛分发该测定（目标4： 试剂和方案）。靶向RNA测序是一种无偏的策略相比，传统的FISH或 PCR检测，而是因为它专注于可操作的基因。对于病理学中的常规使用，它也具有成本效益 台式测序仪器上的任何实体瘤或血液恶性肿瘤的实验室。在UH 2 在分析验证阶段，我们将确定靶向RNAseq检测的灵敏度和特异性 利用连续稀释的基因融合。这将包括控制的可报告范围和间隔的描述 样品对不同数量的RNA、降解RNA、冷冻和福尔马林固定石蜡的性能- 将对包埋样本进行评估。重现性（运行之间）和重复性（运行内）将 评估以定义测定精密度。在临床验证的UH 3阶段，该检测试剂盒将应用于 包含已知基因融合体的样品的多样化集合，所述基因融合体包括不同的激酶和融合体结构。 此外，UH 3将包括对从活性临床试验中采集的未知肿瘤样本的性能评估。 肿瘤测序研究。最后，该检测将出口到另一个癌症中心，以扩大临床应用。 验证检测试剂盒，并证明如何将检测试剂盒导出以更广泛地用于患者护理。我们 非常适合通过已建立的CLIA认证的癌症基因组学实验室来实施这一提议 和一个多学科的团队，包括医学肿瘤学家，生物信息学家，病理学家，临床 实验室该提案将通过证明 临床有效性和实用性的RNA测序临床决策。

PROJECT SUMMARY Targeted next generation sequencing assays for DNA level alterations such as point mutations and copy number alterations are routinely used for patients with advanced cancer considering clinical trials for investigational agents. RNA sequencing, while successfully applied for the discovery of gene fusions and quantitative assessment of gene expression, has yet to be translated into clinical grade testing for patient care in part due to limited expertise and perceived difficulty in creating an accurate and precise assay in a clinical grade laboratory. We hypothesize that targeted RNA sequencing can serve as a versatile clinical grade tool for molecular eligibility of patients with advanced cancer for therapeutic trials. In this proposal, we will develop a clinical grade targeted RNA sequencing assay including analytic validation (Aim 1: Accuracy, Precision Aim 2: Performance Range) and performance on clinical real world samples (Aim 3: Clinical Validation), and finally we will broadly distribute this assay by providing probes/protocols (Aim 4: Distribution of reagents and protocols). Targeted RNA sequencing is an unbiased strategy compared to traditional FISH or PCR assays, but because it is focused on actionable genes. It is also cost-effective for routine use in pathology labs on desktop sequencing instruments for any solid tumor or hematologic malignancy. During the UH2 Phase of Analytic Validation, we will determine the sensitivity and specificity of targeted RNAseq to detect gene fusions utilizing serial dilutions. This will include delineation of reportable ranges and intervals on control samples. Performance on various quantities of RNA, degraded RNA, frozen and formalin-fixed paraffin- embedded samples will be assessed. Reproducibility (between runs) and repeatability (within runs) will be assessed to define assay precision. During the UH3 Phase of Clinical Validation, the assay will be applied to a diverse collection of samples comprising known gene fusions including varied kinases and fusion structures. Further, UH3 will include performance assessment on unknown tumor samples collected from an active clinical tumor sequencing study. Lastly, the assay will be exported to another cancer center to expand clinical validation of the assay and to demonstrate how the assay can be exported for broader use in patient care. We are well suited to carry out this proposal through an established CLIA-certified Cancer Genomics Laboratory and a multi-disciplinary team including medical oncologists, bioinformaticians, pathologists, clinical laboratorians. This proposal will have a broader impact in clinical trials for oncology by demonstrating the clinical validity and utility of RNA sequencing for clinical decision-making.