Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial
Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial
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DOI：
10.1016/j.jmoldx.2015.07.006
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发表时间：
2016-01-01
影响因子：
4.1
通讯作者：
Williams, Paul M.
中科院分区：
文献类型：
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作者：
Lih, Chih-Jian;Sims, David J.;Williams, Paul M.
稳健且经过分析验证的检测方法对于临床研究至关重要。我们概述了一项针对下一代测序突变检测试验的分析验证研究，该试验用于国家癌症研究所基于分子特征的癌症治疗分配（NCI-MPACT）试验（NCT 01827384）中的患者选择。使用来自正常或肿瘤细胞系和具有已知变体的异种移植物的DNA样品，我们评估了NCI-MPACT测定在五种变体类型中的灵敏度、特异性和再现性：单核苷酸变体（SNV）、均聚（HP）区域的SNV（>= 3个相同碱基）、小插入/缺失（indel）、大indel（缺口>= 4 bp）和HP区域的indel。该方法对64个SNV、9个HP区域的SNV和11个大的indel的灵敏度为100%，对6个indel的灵敏度为83.33%，对15个HP区域的indel的灵敏度为93.33%。在96次单型图谱细胞运行中，在380个可操作的突变位点中发现零假阳性（100%特异性）。再现性分析显示，在检测到的变异中，操作者内和操作者间的平均一致性为96.3%至100%，治疗选择的再现性为100%。到目前为止，已经筛选了38个肿瘤，34个通过了分析前质量控制，18个具有可操作的治疗分配突变。NCI-MPACT检测非常适合其预期的研究用途，并可作为开发下一代测序检测的模板，用于其他癌症临床试验应用。
Robust and analytically validated assays are essential for clinical studies. We outline an analytical validation study of a targeted next-generation sequencing mutation-detection assay used for patient selection in the National Cancer Institute Molecular Profiling Based Assignment of Cancer Therapy (NCI-MPACT) trial (NCT01827384). Using DNA samples from normal or tumor cell lines and xenografts with known variants, we assessed the sensitivity, specificity, and reproducibility of the NCI-MPACT assay in five variant types: single-nucleotide variants (SNVs), SNVs at homopolymeric (HP) regions (>= 3 identical bases), small insertions/deletions (indels), large indeLs (gap >= 4 bp), and indels at HP regions. The assay achieved sensitivities of 100% for 64 SNVs, nine SNVs at HP regions, and 11 large indels, 83.33% for six indels, and 93.33% for 15 indels at HP regions. Zero false positives (100% specificity) were found in 380 actionable mutation loci in 96 runs of hapLotype map cells. Reproducibility analysis showed 96.3% to 100% intraoperator and 98.1% to 100% interoperator mean concordance in detected variants and 100% reproducibility in treatment selection. To date, 38 tumors have been screened, 34 passed preanalytical quality control, and 18 had actionable mutations for treatment assignment. The NCI-MPACT assay is well suited for its intended investigational use and can serve as a template for developing next-generation sequencing assays for other cancer clinical trial applications.