摘要目的 分析高通量测序技术与荧光PCR技术检测肺腺癌驱动基因变异的特点.方法采用高通量测序技术检测福建省立医院2015年1月至2017年1月间372例原发性肺腺癌手术切除样本的10个驱动基因变异(仅检测点突变、插入、缺失和融合基因,未检测拷贝数变异),并采用荧光PCR技术对福建省立医院2013年4月至2015年4月间169例肺腺癌样本进行9个驱动基因热点突变检测,根据指南和OncoKB数据库对检测到突变丰度≥1.0%的突变位点进行分类,分析驱动基因变异的特点.结果 高通量测序技术检测到67种突变形式(Ⅰ类、Ⅱ类、ⅢA类、ⅢB类和Ⅳ类),突变总阳性率为86.6%(322/372),Ⅰ类突变(美国食品药品管理局或国家药品监督管理局批准用于非小细胞肺癌的用药治疗靶点)阳性率为71.2%(265/372);Ⅱ类突变(写入中外诊疗指南有明确诊断/治疗/预后意义的变异或已经写入该领域的专家共识的变异位点)阳性率3.0%(11/372);ⅢA类突变(令人信服的临床证据预测对非小细胞肺癌靶向药物的反应)阳性率为3.0%(11/372);ⅢB类突变(令人信服的临床证据用于其他肿瘤可预测疗效的基因变异)阳性率为4.3%(16/372);Ⅳ类突变(令人信服的生物学证据预测对非小细胞肺癌靶向药物的反应)阳性率为8.1%(30/372);临床意义不明的变异和可能是良性的变异阳性率为18.8%(70/372).荧光PCR技术检测169例肺腺癌样本中驱动基因热点突变(Ⅰ类、Ⅱ类、Ⅲ类和Ⅳ类)总阳性率为81.7%(138/169).对于高通量测序技术与荧光PCR技术均检测的20例原发性肺腺癌手术切除样本检测结果,18例结果一致,2例不一致是由于荧光PCR技术采用的试剂盒未涵盖这2个突变位点:EGFR基因c.2571_2573delinsTCG(p.L858R)和ALK融合基因HIP1?ALK_H19:A20 fusion.结论 肺腺癌驱动基因变异主要发生在热点区域,高通量测序技术可较全面地检测出肺腺癌驱动基因中具有显著临床意义和潜在临床意义的突变位点,需要检测多个驱动基因时,推荐使用高通量测序技术.

abstractsObjective To study the characteristics of lung adenocarcinoma driver gene variants detected by next generation sequencing (NGS) and quantitative fluorescence PCR. Methods NGS was performed on 372 surgical resections from primary lung adenocarcinoma patients to detect 10 driver gene mutations, single?nucleotide variants(SNV), insertion / deletion and gene fusions; and quantitative fluorescence PCR were performed on 169 surgical resections from primary lung adenocarcinoma patients to detect nine driver gene hotspot mutations. Variants of VAF (variant allele frequency)≥1.0% were classified into 1 of 4 levels according to the guidelines and the precision oncology knowledge base of OncoKB, and the characteristics were investigated. Results Sixty seven variants(leve1?4) were found by NGS, the positive rate of total mutations was 86.6% (322/372), in which variants at four levels were detected: levelⅠvariant, which was recognized as biomarker predictive of response to an FDA/NMPA approved drug in non?small cell lung cancer (NSCLC), was 71.2% (265/372);levelⅡvariant, which was recognized as being standard care by the NCCN or other expert panels, was 3.0% (11/372); levelⅢA, a variant with compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication 3.0% (11/372); levelⅢB, a variant with compelling clinical evidence supports the biomarker as being predictive of response to a drug in another indication, was 4.3% (16/372); and levelⅣ, a variant with compelling biological evidence supports the biomarker as being predictive of response to a drug, was 8.1% (30/372). The positive rate of unknown clinical significance and/or benign/likely benign variants was 18.8% (70/372). The positive rate of mutations detected by quantitative fluorescence PCR was 81.7% (138/169). Eighteen of the 20 samples showed concordance between NGS and quantitative fluorescence PCR. The two discordant cases could be due to the lack of coverage of two mutation sites in fluorescence PCR: EGFR c. 2571_2573delinsTCG(p. L858R), and HIP1?ALK_H19:A20 fusion. Conclusions Lung adenocarcinoma driver gene variants occur mainly in hotspot region, and NGS can comprehensively detect the driver gene variants of significant and potential clinical significance. NGS should be recommended when multiple genes need to be tested.