Assay Validation of Targeted RNA sequencing to Detect Kinase Gene Fusions

项目总结 靶向下一代测序分析DNA水平变化，如点突变和复制 对于考虑进行临床试验的晚期癌症患者，数字改变是常规使用的 调查人员。RNA测序，虽然成功地应用于发现基因融合和 基因表达的定量评估尚未转化为患者护理的临床分级测试 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分析的困难 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。 晚期癌症患者参加治疗试验的分子资格的工具。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/协议来广泛分发该分析(目标4：分发 试剂和方案)。靶向RNA测序是一种与传统的鱼类或 聚合酶链式反应分析，但因为它关注的是可操作的基因。对于病理学的常规使用，它也具有成本效益。 台式测序仪器上的实验室可检测任何实体肿瘤或血液系统恶性肿瘤。在UH2期间 分析验证阶段，我们将确定靶向RNAseq检测的敏感性和特异性 利用系列稀释进行基因融合。这将包括描述可报告的范围和控制间隔 样本。对不同数量的RNA、降解的RNA、冷冻和福尔马林固定的石蜡- 嵌入的样本将进行评估。重复性(运行之间)和重复性(运行内)将是 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将包括对从活跃临床收集的未知肿瘤样本进行的性能评估 肿瘤测序研究。最后，化验结果将被出口到另一个癌症中心，以扩大临床应用。 验证化验，并演示如何将化验输出，以便在病人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来实施这项建议 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 实验室的人。这项提议将在肿瘤学的临床试验中产生更广泛的影响，因为它展示了 RNA测序在临床决策中的临床有效性和实用性。

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.