Assay Validation of Targeted RNA sequencing to Detect Kinase Gene Fusions

 描述（由申请方提供）：考虑到研究药物的临床试验，常规将针对DNA水平改变（如点突变和拷贝数改变）的靶向下一代测序检测用于晚期癌症患者。RNA测序虽然成功地应用于发现基因融合和定量评估基因表达，但尚未转化为用于患者护理的临床级测试，部分原因是有限的专业知识和在临床级实验室中创建准确和精确测定的感知困难。我们假设靶向RNA测序可以作为一种多功能的临床级工具，用于晚期癌症患者的治疗试验的分子资格。在本提案中，我们将开发临床级靶向RNA测序检测试剂盒，包括分析验证（目标1：准确度，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（目标4：试剂和方案的分布）广泛分布该检测试剂盒。与传统的FISH或PCR检测相比，靶向RNA测序是一种无偏倚的策略，但因为它专注于可操作的基因。它也是具有成本效益的常规使用在病理实验室的台式测序仪器上的任何实体瘤或血液恶性肿瘤。在分析验证的UH 2阶段，我们将确定靶向RNAseq的灵敏度和特异性，以利用连续稀释检测基因融合。这将包括 对照样品可报告范围和区间的描述。将评估不同数量RNA、降解RNA、冷冻和福尔马林固定石蜡包埋样本的性能。将评估重现性（运行之间）和重复性（运行内），以确定试验精密度。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（包括各种激酶和融合体结构）的不同样本集合。此外，UH 3将包括对从活跃的临床肿瘤测序研究中收集的未知肿瘤样本的性能评估。最后，该检测试剂盒将被出口到另一个癌症中心，以扩大该检测试剂盒的临床验证，并证明如何将该检测试剂盒出口，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和包括医学肿瘤学家，生物信息学家，病理学家，临床实验室人员在内的多学科团队来实施这一建议。该提案将通过证明RNA测序在临床决策中的临床有效性和实用性，对肿瘤学临床试验产生更广泛的影响。

 DESCRIPTION (provided by applicant): 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.