Assay Validation of Targeted RNA sequencing to Detect Kinase Gene Fusions

 描述(由申请人提供)：针对DNA水平改变(如点突变和拷贝数改变)的定向下一代测序分析通常用于正在考虑研究试剂临床试验的晚期癌症患者。RNA测序虽然成功地应用于基因融合的发现和基因表达的定量评估，但尚未转化为临床级别的测试，用于患者护理，部分原因是专业知识有限，而且在临床级别的实验室中创建准确和精确的测试是困难的。我们假设靶向RNA测序可以作为晚期癌症患者进行治疗试验的分子资格的通用临床分级工具。在这项提案中，我们将开发一种临床级别的靶向RNA测序分析，包括分析验证(目标1：准确性，精确度目标2：性能范围)和对临床真实样本的性能(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)来广泛分发该分析。与传统的FISH或PCR分析相比，靶向RNA测序是一种公正的策略，但因为它专注于可操作的基因。对于病理实验室中用于任何实体肿瘤或血液恶性肿瘤的桌面测序仪器的常规使用，它也是具有成本效益的。在分析验证的UH2阶段，我们将确定靶向RNAseq的敏感性和特异性，以利用系列稀释法检测基因融合。这将包括 对照样品可报告范围和间隔的划定。将评估不同数量的RNA、降解的RNA、冷冻和福尔马林固定的石蜡包埋样品的性能。将对重复性(批间)和重复性(批内)进行评估，以确定分析的精密度。在UH3临床验证阶段，该分析将应用于包括已知基因融合的不同样本集合，包括不同的激酶和融合结构。此外，UH3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进行的性能评估。最后，该化验将被出口到另一个癌症中心，以扩大该化验的临床验证，并展示该化验如何出口，以便在患者护理中更广泛地使用。我们非常适合通过CLIA认证的癌症基因组实验室和包括医学肿瘤学家、生物信息学家、病理学家和临床实验室在内的多学科团队来实施这项建议。这一建议将在肿瘤学的临床试验中产生更广泛的影响，因为它证明了RNA测序在临床决策中的临床有效性和实用性。

 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.