非小细胞肺癌EGFR突变检测技术的研究进展

针对表皮生长因子受体(epidermal growth factor receptor,EGFR)突变基因的检测是肺癌靶向治疗的重要基石.目前临床中主要针对肺癌组织、脱落细胞以及液态活检标本开展检测工作,此外尿液标本近期也展现出良好的检测效率.针对上述标本,临床中开展多种检测技术,如以特定靶点为目的的技术,包括数字PCR、楔形探针扩增阻滞突变系统等;以筛查为目的的检测技术,包括二代测序(next generation sequencing,NGS)、Sanger测序等,这些技术在特定的标本类型和检测目的中具有独特的优势.一些新兴的技术,如拉曼光谱,也在临床检测中展现出良好的应用前景.     

Value of serum tumor markers for predicting EGFR mutations in non-small cell lung cancer patients

ObjectivesWe investigated whether serum tumor markers (STMs) represent a valuable noninvasive tool to predict epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients.MethodsA retrospective analysis was performed for 143 NSCLC patients at the Peking University International Hospital from December 2014 to December 2019. EGFR mutations in the tumor tissues were identified by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and next generation sequencing (NGS). The relationships between EGFR mutation and several clinicopathological features were analyzed.ResultEGFR mutation were found more frequently in female (56.67%, P = 0.01), never-smokers (55.26%, P = 0.004), and those with lung adenocarcinoma (ADC) (52.17%, P < 0.001). The positive mutation rate for the EGFR gene were higher in the squamous cell carcinoma antigen (SCCA)group (≤1.5 ng/ml) and in the gastrin-releasing peptide precursor (preGRP) increased group (≥69.2 pg/ml), and this difference was statistically significant (P < 0.05). Univariate logistic regression analysis demonstrated that females (Odd ratio [OR]: 2.435, 95% confidence interval [CI]: 1.232, 4.813, P = 0.01) and never-smokers (OR = 0.370; CI = 0.186, 0.734; P = 0.004), lung adenocarcinoma patients (OR = 9.091; CI = 2.599, 21.800; P = 0.001), the SCC group (≤1.5 ng/ml) (OR = 0.331, CI = 0.120, 0.914; P = 0.033), and the preGRP group (≥69.2 pg/ml) (OR = 5.478, CI = 1.462, 20.528; P = 0.012) patients were risk factors for EGFR gene mutation. Multivariate logistic regression analysis demonstrated that lung ADC and proGRP elevation were independent risk factors for predicting EGFR gene positivity (P < 0.05).ConclusionSTMs are associated with mutant EGFR status and could be integrated with other clinical factors to facilitate the classification of EGFR mutation status among NSCLC patients.     

非小细胞肺癌患者胸腔积液中EGFR基因突变的检测及临床意义

目的:探讨采用非小细胞肺癌患者胸腔积液标本肿瘤细胞进行表皮生长因子(epidermal growth factor receptor,EGFR)基因突变检测的可行性及其临床意义.方法:采用Sanger测序法检测17例非小细胞肺癌患者胸腔积液及对应的17例手术或肺部穿刺组织标本EGFR基因18～21外显子基因突变,并进行统计分析.结果:胸腔积液标本17例共检出5例突变,检出率29.41％.手术或穿刺组织标本17例共检出7例突变,检出率41.18％.胸腔积液标本EGFR基因突变检出率略低于手术或穿刺组织标本.结论:采用Sanger测序法进行非小细胞肺癌患者胸腔积液中EGFR基因突变的检测,方法可行,尤其适用于无法获取手术或肺部穿刺组织标本的患者.     

Detection of EML4-ALK fusion genes in non-small cell lung cancer patients with clinical features associated with EGFR mutations

EML4‐ALK fusion genes have been recognized as novel “driver mutations” in a small subset of non‐small cell lung cancers (NSCLC). The frequency of EML4‐ALK fusions in NSCLC patients who have clinical characteristics related to EGFR mutation remains unknown. We screened 102 Chinese patients with NSCLC based on one or more of the following characteristics: female, no or light smoking history, and adenocarcinoma histology. EML4‐ALK fusion genes were identified by RT‐PCR, whereas EGFR (Exons 18–21) and KRAS (Exons 1 and 2) mutations were detected by DNA sequencing. Eight specimens (8%) were positive for EML4‐ALK fusions, with seven being Variant 1 and one Variant 2. There were 44 (43%) and 17 (16%) patients harboring EGFR and KRAS mutations, respectively. Thirty‐one (31%) cases were wild type for EML4‐ALK, EGFR, and KRAS mutations. Of the eight patients with EML4‐ALK, none had an EGFR mutation, whereas a KRAS mutation was detected in one patient. Histologically, five of the EML4‐ALK positive tumors were adenocarcinoma and two were mixed adenosquamous carcinoma; only one was a squamous carcinoma. Our data support the conclusion that the EML4‐ALK fusion gene defines a new molecular subset of NSCLC with distinct pathologic features. © 2012 Wiley Peiodicals, Inc.     

EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients

The epidermal growth factor receptor (EGFR), and its family members play an important role in the development and progression of lung cancers. It has been reported that somatic mutations in the tyrosine kinase domain of the EGFR or ERBB2 genes occur in a subset of patients with lung cancer. We searched for mutations of the EGFR, ERBB2, and KRAS genes in surgically resected non-small cell lung cancers (NSCLCs) to determine the prevalence of these mutations in Korean lung cancer patients. In addition, we examined the relationship between the mutations and clinicopathologic features of lung cancers. Mutations of the EGFR, ERBB2, and KRAS genes were determined by polymerase chain reaction-based direct sequencing in 115 surgically resected non-small cell lung cancers. EGFR mutations were present in 20 patients (17.4%). The EGFR mutations were found only in adenocarcinomas (20 of 55 adenocarcinomas, 36.4%). The ERBB2 mutation was found in 1 adenocarcinoma of the 115 NSCLCs (0.9% overall; 1.8% of the 55 adenocarcinomas). KRAS mutations were found in 6 (5.2%) of the 115 NSCLCs (2 of 60 squamous cell carcinomas, or 3.3%, and 4 of 55 adenocarcinomas, or 7.3%). EGFR mutations in adenocarcinomas were more frequent in women (P = 0.02) and in never-smokers (P = 0.004). EGFR mutations in adenocarcinomas were not associated with pathologic stage in never-smokers, but were more frequent in pathologic stage II-IV than in stage I in ever-smokers (P = 0.01). Of the 55 adenocarcinomas, 25 (45.5%) had mutations of one or another of the three genes; EGFR mutations were never found in adenocarcinomas together with ERBB2 or KRAS mutations. These findings suggest that the EGFR mutation is frequent in Korean lung cancer patients, and that the ERBB2 mutation is rare. Further studies are needed to investigate the role of EGFR mutations in the carcinogenesis of adenocarcinoma among smokers.     

ARMS法与下一代测序法检测非小细胞肺癌EGFR基因突变的比较

目的本研究旨在通过对突变扩增阻滞系统(ARMS)法与下一代测序法(NGS)检测非小细胞肺癌(NSCLC)EGFR基因突变的比较分析,来探索更适合我国临床肺癌患者EGFR基因突变的检测方法。方法收集22例NSCLC患者标本,分别用ARMS法及二代测序法对标本进行EGFR基因突变检测,分析比较两种方法的优劣。结果本次实验的22例标本其中17例EGFR基因突变结果两种检测方法一致,结果为EGFR单突变6例,双突变1例,阴性10例;2例ARMS法检测为单突变,二代测序法检测为双突变;3例ARMS法未检测到突变,二代测序法检测到突变。结论 ARMS法与二代测序法检测EGFR基因突变结果基本一致,但各有优缺点,两种方法结合检测效果更好。     

Detection of EGFR mutation in supernatant,cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing

To determine epidermal growth factor receptor (EGFR) mutation in advanced non-small cell lung cancer (NSCLC) patients and compare the detection efficiency between different sample resources, both high resolution melting (HRM) analysis and direct sequencing method were used to analyze 36 pleural effusion samples and 22 matched biopsy tumor tissues collected from NSCLC patients. For each pleural effusion sample, the supernatant and the cell pellets were examined separately. Among all the 36 cases of pleural effusion samples, 18 mutations of EGFR were found in cell-free supernatant while 13 mutations were found in the cell pellets as detected by HRM analysis. In the 22 matched samples, 13 cases of EGFR mutations were identified in paraffin-embedded biopsy tissue samples, 12 cases in the cell-free supernatant and 9 cases in the cell pellets of pleural effusion. EGFR mutations in 15 cases out of the total 36 pleural effusion samples detected by direct sequencing were also identified by HRM analysis, giving 100% efficiency for HRM method. The results established the important role of HRM as a reliable and efficient method to determine EGFR mutation status and indicated the feasibility of using pleural effusion in replacement of biopsy tissues in particular clinical cases. Furthermore, the cell-free supernatant of pleural effusion might be a better resource for mutation detection than cell pellets.     

中国人非小细胞肺癌EGFR和K-RAS基因突变情况的研究

目的研究中国人非小细胞肺癌表皮生长因子受体(epidermal growth factor receptor,EGFR)基因和K-RAS基因突变情况。方法通过PCR扩增和基因测序的方法检测了101例中国人非小细胞肺癌(non-small cell lung cancers,NSCLCs)EGFR第18、19和21外显子及K-RAS密码子12、13的突变情况,并观察分析了其突变与肺癌临床特征及吉非替尼(gefitinib,商品名:易瑞沙/Iressa)药物治疗肺癌的疗效间的关系。结果共检测到26例EGFR突变(25.7%),3例K-RAS突变(2.9%),并发现腺癌患者、非吸烟患者和女性患者EGFR突变率较高(分别为44.2%、65.7%和48.3%)。10例服用吉非替尼有效的患者9例伴有EGFR突变。结论中国人肺癌的EGFR突变率高于西方人,吉非替尼疗效与EGFR突变有关。     

埃克替尼对非小细胞肺癌EGFR 18外显子突变的临床疗效

目的:探讨埃克替尼对非小细胞肺癌(non-small cell lung cancer,NSCLC)EGFR 18外显子G719X/E709X/G724S的临床疗效.方法:回顾性分析24例埃克替尼治疗EGFR 18外显子少见突变的NSCLC患者,服用至病情进展或出现不可耐受的毒副作用,比较疗效.结果:24例G719X/E709X/G724S突变患者中G719X突变19例,中位无进展生存时间2.8个月,E709X突变3例,中位无进展生存时间3.1个月,G724S突变2例,中位无进展生存时间3.5个月.G724S突变患者生存时间稍长.复合突变与单纯突变相比,复合突变中位无进展生存时间更长(G719X突变3.3个月Vs.2.6个月,P=0.029;E709X突变7.2个月vs.2.7个月,P=0.225).结论:埃克替尼在EGFR基因18外显子少见突变的疗效上比传统敏感突变未见明显优势,但复合突变比单纯突变临床获益更多.     

埃克替尼对非小细胞肺癌EGFR 20外显子突变的临床疗效分析

目的:探讨埃克替尼对非小细胞肺癌(non-small cell lung cancer,NSCLC)EGFR 20外显子S768I/20-ins/T790M/V769M突变的临床疗效.方法:回顾性分析58例埃克替尼治疗EGFR 20外显子少见突变的NSCLC,服用至病情进展或出现不可耐受的毒副反应,并观察疗效.结果:58例S768I/20-ins/T790M/V769M突变患者中S768I突变20例,中位生存时间3.2个月,20-ins突变18例,中位生存时间1.6个月,T790M突变21例,中位生存时间1.6个月,V769M突变1例,生存时间3.2个月.S768I突变和V769M突变患者生存时间稍长.单纯突变与复合突变相比,复合突变中位生存时间更长(S768I突变2.2个月vs.3.3个月,P=0.174;T790M突变2.45个月vs.2.9个月,P=0.845).结论:埃克替尼在EGFR基因20外显子少见突变的疗效上比传统敏感突变未见明显优势,但复合突变比单纯突变临床获益更多.     

Detection of c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis

High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.     

非小细胞肺癌患者ALK,EGFR及KRAS基因的检测及临床病理特征

目的:研究汉族非小细胞肺癌患者中间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)、表皮生长因子受体(epidermal growth factor receptor,EGFR)及Kirsten鼠肉瘤基因(Kirstenrat sarcoma,KRAS)突变的阳性率及其与临床病理特征的关系.方法:采用免疫组织化学(immunohistochemistry,IHC)的方法检测ALK融合基因异常表达,采用PCR检测EGFR基因和KRAS基因突变,采用x2检验及Fisher精确概率法进行数据分析.结果:共2 267例进行了ALK融合基因检测,其中1 655例同时进行了EGFR突变检测,951例同时进行了KRAS检测.ALK融合基因、EGFR基因及KRAS基因突变阳性率分别为7.28％(165/2 267)、48.58％(804/1 655)、11.40％(108/947).ALK基因突变多见于年轻、腺癌患者;EGFR基因突变多见于女性、腺癌患者;KRAS基因突变多见于老年、男性、腺癌患者.1 655例同时进行了ALK与EGFR检测的病例中,共6例存在双基因突变(0.36％);947例同时进行了ALK与KRAS检测,共4例存在双基因突变(0.42％);943例同时进行了EGFR与KRAS突变检测,未发现双突变病例.结论:非小细胞肺癌患者ALK,EGFR和KRAS基因的突变与患者的年龄、性别、组织学类型均存在相应的联系,个别病例可以出现ALK融合基因与EGFR或KRAS突变共存.     

埃克替尼对非小细胞肺癌EGFR 21外显子少见突变的临床疗效

目的:探讨埃克替尼对非小细胞肺癌(non-small cell lung cancer,NSCLC)EGFR 21外显子L861Q/L833F突变的临床疗效.方法:回顾性分析17例埃克替尼治疗EGFR 21外显子少见突变的NSCLC,服用至病情进展或出现不可耐受的毒副作用,并观察疗效.结果:17例L861Q/L833F突变患者中L861Q突变17例,中位生存时间2.2个月,L833F突变1例,中位生存时间4.2个月.L833F突变患者生存时间稍长.复合突变与单纯突变相比,复合突变中位生存时间更长(L861Q突变2.1个月vs.5.6个月,P=0.065).结论:埃克替尼在EGFR基因21外显子少见突变的疗效上比传统敏感突变未见明显优势,但复合突变比单纯突变临床获益更多.     

非小细胞肺癌表皮生长因子受体、间变性淋巴瘤激酶、ROS1基因突变及突变共存的临床病理学意义

目的:探讨非小细胞肺癌表皮生长因子受体（EGFR）基因突变、间变性淋巴瘤激酶（ALK）及ROS1基因融合突变情况与临床病理特征的关系,并分析驱动基因共突变的病例。方法:收集福建医科大学附属协和医院1 508例非小细胞肺癌患者临床病理资料,采用荧光PCR法检测EGFR基因突变及ALK、ROS1基因融合突变情况,统计分析驱...     

非小细胞肺癌患者肿瘤组织中EGFR和KARS基因突变的分子病理检测分析

目的：探讨非小细胞肺癌患者肿瘤组织中EGFR和KRAS基因各亚型突变情况。方法：应用直接测序方法检测非小细胞肺癌石蜡组织中1273例EGFR基因和1062例KRAS基因突变情况。结果：非小细胞肺癌肿瘤组织中EGFR基因总突变率为36.68%(467/1273),外显子18、19、20和21的突变率分别为1.02%(13/1273)、18.93%(241/1273)、2.59%(33/1273)和15.95%(203/1273);EGFR基因各外显子之间双重突变共17例(1.34%),其中18外显子与20外显子双重突变3例(0.24%),19外显子与20外显子双重突变7例(0.55%),19外显子与21外显子双重突变4例(0.31%)和20外显子与21外显子双重突变3例(0.24%);EGFR基因各外显子内双重突变共2例(2.18%),均为21外显子双重突变。KRAS基因总突变率为3.01%(32/1062),外显子2的密码子5、12、13和25的突变率分别为0.09%(1/1062)、2.64%(28/1062)、0.18%(2/1062)和0.09%(1/1062),外显子3密码子61的突变率为0.09%(1/1062)。结论：非小细胞肺癌患者中EGFR基因存在较高的突变率,尤其为19和21外显子突变,其基因突变亚型分类能指导EGFR-TKI的肿瘤靶向治疗,KRAS基因突变率虽低但不容忽视,其基因突变预示着EGFR-TKI原发耐药。     

双环探针特异引物荧光聚合酶链反应法检测非小细胞肺癌表皮生长因子受体基因突变

目的探讨双环探针特异引物荧光聚合酶链反应法(BPSP-qPCR)检测非小细胞肺癌( NSCLC)表皮生长因子受体(EGFR)基因突变的敏感性和稳定性。方法采用BPSP-qPCR法检测NSCLC中EGFR基因第18～21号外显子突变,分析突变与临床病理特征、不同检测样本间的关系。结果265例NSCLC样本中19-del和（或）L858R突变占30.2％( 80/265)。资料完整的184例中,女性、不吸烟者、腺癌有更高的19-del和（或）L858R突变率,分别为39.7％ (31/78)、41.0％ (43/105)、37.8％ (51/135) (P ＜0.05);T790M合并19-del和（或）L858R占3.3％(6/184);男性转移灶、女性和不吸烟者胸腔积液样本有较高的19-del和（或）L858R阳性突变率,分别为29.6％( 8/27)、42.9％(9/21)和40.7％(11/27),而非腺癌转移灶为0(P＞0.05)。结论BPSP-qPCR法检测EGFR基因突变稳定、敏感。EGFR基因敏感突变多见于女性、非吸烟、腺癌,胸腔积液样本和转移灶少量样本能够检出EGFR突变,指导临床治疗。     

应用高分辨熔解曲线技术检测经典型苯丙酮尿症患者PAH基因第7外显子基因突变

目的 建立操作简便、快速,使用成本低,结果准确的鉴定PAH基因第7外显子c.728G>A突变热点的基因诊断方法.方法 应用高分辨熔解曲线(high resolution melting,HRM)技术,对山西省临床确诊的88例经典型苯丙酮尿症患儿第7外显子c.728G>A突变热点进行检测,并将检测结果进行测序验证.结果 受检患儿PAH基因第7外显子c.728G>A突变位点HRM技术检测结果和测序结果完全相符.结论 高分辨熔解曲线技术操作简便、快速,使用成本低,结果准确,可作为PAH基因第7外显子热点突变的快速、灵敏检测方法.     

Detection of epidermal growth factor receptor (EGFR) exon 19 E746-A750 deletion and EGFR exon 21 point mutations in lung adenocarcinoma by immunohistochemistry: a comparative study to EGFR exons 19 and 21 mutations analysis using PCR followed by high-resolution melting and pyrosequencing

Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) with adenocarcinoma histology confers susceptibility to treatment with EGFR tyrosine kinase inhibitors (TKI). Analysis of these activating mutations is typically performed by molecular techniques including polymerase chain reaction (PCR) amplification followed by deoxyribonucleic acid (DNA) melting curve analysis and/or direct sequencing. Recently developed mutation-specific immunohistochemical stains for the L858R point mutation in exon 21 and an E746-A750 deletion in exon 19 of the EGFR gene may offer a fast, cost-effective alternative to molecular testing. In this study, EGFR exons 19 and 21 mutations were analyzed using EGFR E746-A750 del and EGFR L585R point mutation-specific antibodies and the results were compared with the exon 19 deletions and exon 21 L588R point mutation as detected by molecular testing. The findings demonstrate a high concordance rate (100% in 12 cases) between the two methodologies for EGFR exon 21 L585R point mutation but a low concordance rate (54% in 22 cases) for exon 19 deletions when using EGFR E746-A750 mutation-specific antibody compared to molecular testing. This low concordance rate between the two methods is due to the fact that EGFR exon 19 mutations include deletions other than E746-A750 as well as insertion/deletions that will not be covered by exon 19 E746-A750 del mutation-specific antibody. Despite this limitation, the current EGFR mutation-specific antibodies can prove useful as an alternative to molecular tests for exon 21 L585R mutations as well as a subset of EGFR exon 19 deletion mutations, particularly in cases with low percentage of tumor concentration and in decalcified tissue specimens that are not suitable for DNA-based molecular assays.