Zhang Rui, Peng Rongxue, Li Ziyang, Gao Peng, Jia Shiyu, Yang Xin, Ding Jiansheng, Han Yanxi, Xie Jiehong, Li Jinming
National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China.
Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China.
Clin Chem. 2017 Sep;63(9):1465-1475. doi: 10.1373/clinchem.2017.272559. Epub 2017 Jul 18.
Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently.
We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories.
The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with T790M, L858R, G12D, and a deletion in exon 19, as well as with variant 2.
The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.
由于血浆中肿瘤来源的游离DNA（cfDNA）浓度低、检测方法多样且工作流程复杂，检测其中的体细胞基因组改变具有挑战性。此外，目前尚无合适的质量控制材料。
我们基于微球菌核酸酶消化法开发了一套合成cfDNA质量控制材料（SCQCMs），其中包含掺入的cfDNA，该cfDNA带有体细胞突变作为模拟cfDNA，并以匹配的基因组DNA作为遗传背景，以在实际临床检测中模拟配对的肿瘤-正常样本。将含有1500-2000个碱基且带有单核苷酸变体或插入缺失的定点诱变DNA以及来自经CRISPR/Cas9编辑且具有重排的细胞的基因组DNA进行片段化、定量，并添加到源自HEK293T细胞的经微球菌核酸酶消化的DNA中。为证明其适用性，将SCQCMs与患者来源的血浆样本进行比较，并在一项涵盖11个实验室的合作研究中进行验证。
通过下一代测序对SCQCMs进行分析的结果与患者来源的血浆样本结果高度一致，包括cfDNA的大小分布和测序数据的质量控制指标。超过95%的实验室能够正确检测出含有T790M、L858R、G12D以及外显子19缺失的SCQCMs，以及变体2。
SCQCMs已成功应用于广泛的环境、方法和信息学技术中。我们得出结论，SCQCMs可作为循环肿瘤DNA体细胞突变检测测试性能评估中的最佳质量控制材料。
