循环肿瘤细胞的磁分离法建立及在肺癌EGFR-TKI治疗中的应用  

Magnetic isolation of circulating tumor cells and its application in EGFR-TKI therapy of lung cancer

作　　者：阴建[1] 尹寒露 王毅[2] 陈文萍[2] 陈立文[1] 刘志安[1] 胡志斌[1] 沈洪兵[1] 

机构地区：[1]南京医科大学公共卫生学院流行病学系,江苏省恶性肿瘤生物标志物与防治重点实验室,南京211166 [2]南京市胸科医院呼吸科,南京210009

出　　处：《临床检验杂志》2015年第3期170-174,共5页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省自然科学基金(BK20141435);南京医科大学归国人员启动资金(2011RC11);南京市医学科技发展项目(YKK13088)

摘　　要：目的探索磁分离循环肿瘤细胞(circulating tumor cells,CTCs)联合竞争性等位基因特异性Taq ManPCR(Cast PCR)检测表皮生长因子受体(EGFR)基因突变的可行性。方法收集12例晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者血液样本各7.5 m L,两步法磁分离CTCs,免疫荧光染色检测上皮细胞黏附分子(Ep CAM)和细胞角蛋白(Cytokeratin)后,流式细胞术检测CTCs数量及纯度;Cast PCR检测CTCs及循环DNA中EGFR基因del EGFR19、T790M和L858R突变;用EGFR基因突变检测试剂盒(ADx-ARMS法)检测癌组织的上述位点突变。结果高表达与不表达Ep CAM的CTCs分别在6例和12例NSCLC患者中被检测出;del EGFR19、T790M和L858R突变例数分别在2例、8例和5例NSCLC患者的CTCs中被检测出,其总检出率为83.3%(10/12);2例患者的循环DNA中检出L858R突变;ADx-ARMS法检测12例NSCLC患者癌组织中上述3种突变分别是2例、5例和3例,总检出率为75.0%(9/12)。CTCs与癌组织的突变具有一致性(Kappa=0.75,P=0.007),循环DNA与癌组织的突变一致性无统计意义(Kappa=0.06,P=0.546)。结论磁分离CTCs联合Cast PCR能有效检测EGFR基因突变,值得在NSCLC患者的EGFR酪氨酸激酶抑制剂(EGFR-TKI)治疗中推广应用。Objective To evaluate the feasibility of detecting epidermal growth factor receptor gene（ EGFR） mutations in the patients with non-small cell lung cancer（ NSCLC） by magnetically isolated circulating tumor cells（ CTCs） combined with competitive allele specific Taq ManPCR（ Cast PCR）. Methods Twelve blood samples（ 7. 5 m L for each sample） and tumor tissues from NSCLC patients were detected. A 2-step protocol was developed for CTCs enrichment by leukocyte depletion. The CTCs were stained with the antibodies of epithelial cell adhesion molecule（ Ep CAM） and cytokeratin and detected by flow cytometry. The mutations of del EGFR19,L858 R and T790 M in CTCs and circulating DNA were detected by Cast PCR,and the mutations in tumor tissues were detected by EGFR gene mutation detection kit（ ADx-ARMS）. Results CTCs with and without Ep CAM expression were detectable in 6 and all of the 12 blood samples respectively. The mutations of del EGFR19,T790 M and L858 R in CTCs were detectable in 2,8 and 5 blood samples respectively and the mutations were detectable in totaling 10 blood samples（ 83. 3%）. Only L858 R mutation was detectable in circulating DNA of 2 blood samples. The 3 mutations were detectable in 2,5 and 3 tumor tissues respectively,and the 3 mutations were detectable in totaling 9 blood samples（ 75. 0%）. Statistical analysis revealed that the mutations in CTCs were consistent with those in tumor tissues（ Kappa = 0. 75,P = 0. 007）. There was no significant consistence of these mutations between circulating DNA and tumor tissues（ Kappa = 0. 06,P = 0. 546）. Conclusion The magnetically isolated CTCs enrichment combined with Cast PCR should be effective for detecting EGFR mutations and worthy to be applied in EGFR-tyrosine kinase inhibitor（ TKI） therapy for NSCLC patients.

关 键 词：循环肿瘤细胞 非小细胞肺癌 磁分离 表皮生长因子受体 基因突变 酪氨酸激酶抑制剂 

分 类 号：R734.2[医药卫生—肿瘤]