采用液滴数字聚合酶链反应检测肺癌中的表皮生长因子受体突变
Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction.
作者信息
Xu Qing, Zhu Yazhen, Bai Yali, Wei Xiumin, Zheng Xirun, Mao Mao, Zheng Guangjuan
机构信息
Translational Bioscience and Diagnostics, WuXi AppTec, Shanghai, People's Republic of China.
Department of Pathology, Guangdong Provincial Hospital of TCM, Guangzhou University of Chinese Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, Guangzhou, People's Republic of China.
出版信息
Onco Targets Ther. 2015 Jun 22;8:1533-41. doi: 10.2147/OTT.S84938. eCollection 2015.
BACKGROUND
Two types of epidermal growth factor receptor (EGFR) mutations in exon 19 and exon 21 (ex19del and L858R) are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR)-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR) method in detecting the three EGFR mutations in patients with lung cancer.
METHODS
Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR.
RESULTS
The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect. About 49% of this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M, 6.4%). Two patients with the ex19del mutation also had a naïve T790M mutation.
CONCLUSION
These data suggest that the ddPCR method could be useful in the personalized treatment of patients with lung cancer.
背景
外显子19和外显子21中的两种表皮生长因子受体（EGFR）突变（外显子19缺失和L858R）在肺癌患者中很常见，且对EGFR靶向抑制敏感。当患者复发时，已发现外显子20中的一种耐药突变（T790M）与药物治疗相伴出现。这三种突变是指导个性化治疗的有价值的伴随诊断生物标志物。基于定量聚合酶链反应（qPCR）的方法已被临床医生广泛用于指导治疗决策。本研究的目的是评估液滴数字聚合酶链反应（ddPCR）方法在检测肺癌患者三种EGFR突变中的技术和临床敏感性及特异性。
方法
使用来自H1975和PC-9细胞的基因组DNA以及92份正常人血液标本，来确定ddPCR检测的技术敏感性和特异性。对78例中国肺腺癌患者的福尔马林固定、石蜡包埋标本的基因组DNA同时采用qPCR和ddPCR进行检测。
结果
三种ddPCR检测的检测限为0.02%，测量的动态范围从1到20,000拷贝很宽。L858R和外显子19缺失检测在技术和临床环境中的背景水平为0%。T790M检测似乎有0.03%的技术背景。ddPCR检测在正确确定患者的EGFR突变状态方面很可靠，且动态范围似乎优于qPCR方法。T790M的ddPCR检测能够检测出qPCR方法未能检测出的患者样本。该患者队列中约49%有EGFR突变（L858R，15.4%；外显子19缺失，29.5%；T790M，6.4%）。两名有外显子19缺失突变的患者也有原始的T790M突变。
结论
这些数据表明ddPCR方法可能对肺癌患者的个性化治疗有用。
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