即时定量PCR-Sanger测序与TaqMan探针法检测结直肠癌KRAS、BRAF基因突变的对比分析

Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas

目的 对比分析即时定量PCR-Sanger测序与TaqMan探针法检测KRAS、BRAF基因突变的应用价值；探讨结直肠癌KRAS和BRAF基因突变与临床及病理的相关关系.方法 收集2008至2012年344例结直肠癌标本,经4％中性甲醛液固定、石蜡包埋组织后切片、刮取富集肿瘤细胞后提取DNA,应用即时定量PCR-Sanger测序与TaqMan探针两种方法检测分析KRAS、BRAF基因的突变阳性率、突变类型及其与临床病理特征和生存时间的相关性等.结果 即时定量PCR-Sanger测序与TaqMan探针法检出KRAS基因突变率分别为39.8％ （137/344）和38.7％（133/344）,BRAF基因突变阳性率分别为4.7％ （16/344）和4.1％（14/344）,两种方法结果差异无统计学意义（P＞0.05）.KRAS基因突变阳性率女性高于男性；BRAF基因突变阳性率结肠高于直肠,Ⅲ～Ⅳ期高于Ⅰ～Ⅱ期,印戒细胞癌高于黏液癌,非特殊类型腺癌最低,Ⅲ级高于Ⅱ级,差异均有统计学意义（P＜0.05）.两种方法总符合率为98.8％（Kappa值0.976）.BRAF基因突变型与KRAS基因突变型的患者生存时间差异有统计学意义（P =0.039）,BRAF基因突变型与BRAF/KRAS野生型的患者生存时间差异无统计学意义（P=0.058）.结论 （1）与Sanger测序法相比,TaqMan探针法具有操作简便、耗时短、效率高、重复性好、费用低以及不需特别仪器设备等优点,具有更高的临床应用价值；（2）结直肠癌中KRAS基因突变率与性别具有相关性,而BRAF基因突变率与原发部位、临床分期、组织学类型及组织学分级具有相关性；（3） BRAF基因突变是结直肠癌患者的独立预后因素.

Objective To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations,and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas.Methods Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded （FFPE） tissue samples of 344 colorectal carcinomas by microdissection.Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations.The frequency and types of KRAS/BRAF mutations,clinicopathological characteristics and survival time were analyzed.Results KRAS mutations were detected in 39.8％ （137/344） and 38.7％ （133/344） of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method,respectively.BRAF mutation was detected in 4.7％ （16/344） and 4.1％ （14/344）,respectively.There was no significant correlation between the two methods.The frequency of the KRAS mutation in female was higher than that in male （P ＜0.05）.The frequency of the BRAF mutation in colon was higher than that in rectum.The frequency of the BRAF mutation in stage Ⅲ-Ⅳ cases was higher than that in stage Ⅰ-Ⅱ cases.The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate.The frequency of the BRAF mutation in grade Ⅲ cases was higher than that in grade Ⅱ cases （P ＜ 0.05）.The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8％ （kappa =0.976）.There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas （P =0.039）.There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type （P =0.058）.Conclusions （1） Compared with real-time quantitative PCR-Sanger sequencing,TaqMan probe method is better with regard to handling time,efficiency,repeatability,cost and equipment.（2） The frequency of the KRAS mutation is correlated with gender.BRAF mutation is correlated with primary tumor site,TNM stage,histological types and histological grades.（3） BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.