酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用  

Detection and clinical significance of EGFR and KRAS mutation in peripheral blood from tumor patients by REDE-DHPLC

作　　者：杨卓[1] 龙美娟 王斐[1] 陈倩[1] 赵保健 郭野[1] 黄媛[1] 苏秀兰[3] 张旭 崔巍[1] 

机构地区：[1]中国医学科学院北京协和医院检验科,100730 [2]北京市表观生物技术有限公司 [3]内蒙古医学院临床医学研究中心

出　　处：《中华检验医学杂志》2011年第4期327-332,共6页Chinese Journal of Laboratory Medicine

摘　　要：目的 建立一种REDE-DHPLC检测外周血肿瘤游离核酸EGFR、KRAS基因突变的方法,并探讨其临床应用价值.方法 采用限制性内切酶Mse Ⅰ、Msc Ⅰ、BstN Ⅰ和BglⅠ分别切断EGFR基因第19、21号外显子和KRAS基因第12、13号密码子的野生型片段以富集突变片段,建立检测血浆EGFR和KRAS基因突变的REDE-DHPLC法,并采用50%、10%、5%、1%和0.1%稀释度的质粒标准品评价REDE-DHPLC法和传统DHPLC法的检测灵敏度.然后,采用REDE-DHPLC法检测120例NSCLC和120例结直肠癌患者血浆和石蜡组织标本中的EGFR和KRAS基因突变.同时,用传统DHPLC法和测序法进行平行检测,以评价REDE-DHPLC法检测NSCLC和结直肠癌患者血浆中2个基因突变的诊断效能.结果 REDE-DHPLC法对EGFR和KRAS基因4个突变位点检测的灵敏度均达0.1%,传统DHPLC法检测灵敏度均为1.0%,REDE-DHPLC法对含0.1%突变的质粒标准品重复2～3次检测均为阳性.REDE-DHPLC法、传统DHPLC法和测序法检测120例NSCLC患者血浆EGFR基因总突变率分别为27.5%、16.7%和12.5%,检测120例结直肠癌患者血浆KRAS基因总突变率分别为38.3%、25.8%和16.7%.REDE-DHPLC法检测EGFR和KRAS基因总突变率均高于传统DHPLC法（x2值分别为4.092、4.301,P均＜0.05）和测序法（x2值分别为8.438、14.127,P均＜0.05）;将REDE-DHPLC法检测EGFR和KRAS基因突变与传统DHPLC法相比,敏感度均为100%（20/20,31/31）,特异度分别为87.0%（87/100）和83.2%（74/89）;与测序法相比,敏感度均为100%（15/15,20/20）,特异度分别为82.9%（87/105）和74.0%（74/100）.REDE-DHPLC法与传统DHPLC法检测EGFR、KRAS基因突变的符合率分别为89.2%（107/120,Kappa=0.690,P＜0.05）和87.5%（105/120,Kappa=0.718,P＜0.05）.REDE-DHPLC检测血浆与组织标本中EGFR和KRAS基因总突变符合率分别为91.7%（33/36,Kappa=0.939,P＜0.05）和90.2%（46/51,Kappa=0.914,P＜0.05）.结论 REDE-DHPLC法灵敏度和特异性高,结果易判读,且可有效避免纯合点突�Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC（x2 = 4. 092, 4. 301, all P ＜ 0. 05 ） and sequencing method （x2= 8. 438,14. 127,all P ＜ 0. 05 ）. In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% （20/20,31/31）, the specificities were 87. 0% （87/100）and 83. 2% （74/89）. In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%（ 15/15,20/20）, the specificities were 82.9% （87/10

关 键 词：癌 非小细胞肺 结直肠肿瘤 受体 表皮生长因子 原癌基因蛋白质类 突变 色谱法 高压液相 

分 类 号：R734.2[医药卫生—肿瘤]