肺癌相关基因突变二代测序检测试剂参考品的建立

Establishment of reference materials for in vitro diagnostics of lung cancer relevant mutations based on next-generation sequencing

目的:建立肺癌相关基因突变二代测序(next-generation sequencing,NGS)检测试剂参考品并制定其质量标准,用于NGS技术以细胞游离脱氧核糖核酸(cell free DNA,cfDNA)为待测样本的肺癌相关基因突变检测试剂的质量控制和评价。方法:选择携带EGFR基因G719S,E746_A750del,S768I,T790M,L858R,EML4-ALK以及ROS1-SLC34A2基因融合突变的细胞系进行培养,并对各细胞系携带的基因突变进行鉴定和突变频率测定。将细胞系DNA按适当比例混合后超声打断,模拟自然状态cfDNA,作为阳性、重复性和最低检出限参考品候选样本;不携带上述基因突变的阴性细胞系DNA打断后作为阴性参考品候选样本。参考品候选样本的基因突变的频率经过定量后应用8家公司的试剂进行协作标定。根据作标定结果确定参考品的质量标准,并考察其稳定性。结果:本参考品为包含8种肺癌相关基因突变的混合参考品,由1份阳性参考品、4份阴性参考品、2份重复性参考品以及4份最低检出限参考品(L1～L4)组成,质量标准规定对最低检出限参考品L1～L3检测各基因突变的最低突变频率在0. 7%～1. 2%时必须检出。稳定性考核结果表明反复冻融3次不影响参考品的稳定性。结论:本研究建立了肺癌相关基因突变二代测序检测试剂参考品并制定了质量标准,能够为相关试剂的质量控制和评价提供依据。

Objective： To establish reference materials for in vitro diagnostics of lung cancer relevant mutations in cell-free deoxyribose nucleic acid （cfDNA） based on next-generation sequencing （NGS） and related quality standards. Methods： Cell lines harboring EGFR mutations of G719S, E746_A750del, S7681, T790M, L858R, EMK4-ALK fusion and SLC34A2-ROSI fusion were selected for the further culture enrichment, and the validation of mutations and quantification of mutant allelie frequencies were performed for those ceil lines. For preparation of the positive, precision and limit of detection of the reference material candidates, the DNA of the above cell lines were proportionally pooled and sheared by using Covaris uhrasonicator to simulate natural efDNA; for preparation of the negative candidates, the DNA of negative cell lines without the above mutations were sheared using the same ultrasonication condition. All candidates were validated and quantified and then distributed to eight domestic manufacturers for the collaborative study. The stability of all candidates were well studied. Results： The reference materials were composed of 1 positive sample, 4 negative samples, 2 precision samples, and 4 limit of detection samples （ L1 - L4）. The quality standard for limit of detection samples, all 8 kinds of mutations harbored in L1 - L3 must be detected, the lowest mutant allelic frequencies range 0.7% - 1.2% for each mutations, respectively. The three times freeze-thawing cycle did not impact the stability the reference panel. Conclusion： The reference materials and the related quality standard have been established, which could be used for the quality control and evaluation of in vitro diagnostics of lung cancer relevant mutations in cfDNA based on NGS.