Endobronchial ultrasound-guided transbronchial needle aspiration for identifying epidermal growth factor receptor mutations

目的 探讨经气管超声引导内镜穿刺(EBUS-TBNA)纵膈淋巴结穿刺标本中表皮生长因子受体(EGFR)突变检测的可行性.方法 收集2010年2月至2010年7月全部26例经EBUS-TBNA纵膈淋巴结穿刺诊断为肺腺癌的标本.穿刺针为EBUS标准22G针,待涂片快速细胞学病理诊断腺癌后,在同一部位穿刺,将穿刺标本推入生理盐水中.经离心后首先提取组织DNA,采用胸外科优化的突变体富集法检测外显子19及21的突变.首先经过特异性消化野生型EGFR基因的限制性内切酶切割,再进行聚合酶链反应(PCR)扩增.因绝大部分野生型基因已断裂,无法作为PCR反应模板,使得扩增产物中突变型基因的比例大大增加.结果 全部26例患者均成功提取DNA,男16例,女10例,平均年龄62.1岁(34～ 79岁).经EBUS穿刺2组、4组、7组或10组淋巴结,诊断为肺腺癌,涂片标本均显示淋巴细胞背景中可见多量腺癌细胞.经改良突变体富集法检测示其中12例存在EGFR突变,突变率达到46.2％,7例为外显子19突变,5例为外显子21突变,余14例阴性全部标本均进行测序验证,14例阴性者测序也为阴性,12例突变者测序仅7例为阳性,余5例测序为阴性.全部12例EGFR突变患者中4例患者通过新辅助化疗后进行了根治性手术,术后对肿瘤标本再次检测EGFR基因突变,检测结果与术前相同.结论 采用EBUS-TBNA穿刺纵膈淋巴结组织标本来检测EGFR突变是可行的,通过高灵敏度的检测方法可获得更高的检出率.Objective To assess the usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to detect epidermal growth factor receptor (EGFR) mutations in cells recovered fiom malignant mediastinal nodes from patients with non-small-cell lung carcinoma (NSCLC).Methods From February 2010 to July 2010,26 cases of mediastinal lymph node biopsy specimens were diagnosed as lung adenocarcinoma by EBUS.Needle puncture site for the EBUS was standard 22G needle,once cytology pathology diagnosed adenocarcinoma,at the same site with the conventional needle puncture method to obtain the biopsy specimen which would be save in the saline solution.After centrifugation,the specimen was extracted DNA throuugh TIANamp Micro DNA Kit,and to detect EGFR exon 19 and 21 in use of mutation enriched method improved by our department.First of all,through specific digestion of wildtype EGFR gene restriction enzyme cutting,and then amplificated by polymerase chain reaction (PCR).For most wild-type gene had been broken as the PCR reaction template can not be made,the mutant gene amplification product increased substantially.Results All 26 patients were successfully extracted DNA,16 were male and 10 female,mean age of 62.1 years (34 to 79 years).EBUS puncture mediastinal lymph nodes,cytology pathology have shown that large numbers of adenocarcinoma cells with lymphocytes visible in the background.Somatic mutations of the EGFR gene were detected in 12 of 26 cases (46.2％ ),7 cases of exon 19 mutation,5 cases of exon 21 mutation.All samples were verified by sequencing,14 negative cases were negative in DNA sequencing,7 of 12 cases were positive for DNA sequencing,the other 5 cases were negative.4 patients of 12 cases of patients with EGFR mutations had a radical surgery after neoadjuvant chemotherapy,tumor specimens were tested again with EGFR gene mutation,the same result as the preoperative test results.Conclusion EBUS-TBNA mediastinal lymph node biopsy in the detection of EGFR mutations in tissue samples was feasible,through the highly sensitive methods,higher detection rate was availabie,the result can be used as evidence of EGFR inhibitor therapy.首都医学发展科研基金资助项目中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)081469-14712