特异引物双扩增即时PCR与传统测序法检测肠癌、肺癌患者K-ras基因突变的比较

Screening for K-ras mutations in colorectal and lung cancers by using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing

目的 以特异引物双扩增即时PCR技术K-ras检测试剂盒（下称ADx-K-ras即时PCR试剂盒）和Sanger DNA测序法同时检测结直肠癌和肺癌患者K-ras基因,以了解K-ras基因突变频率和突变类型,比较ADx-K-ras即时PCR试剂盒和Sanger DNA测序法用于肿瘤K-ras基因体细胞突变检测的临床价值.方法 收集临床肿瘤病理石蜡切片样品827例,其中肠癌样品583例,肺癌样品244例,提取DNA后,对K-ras基因第12和13密码子进行PCR扩增后,使用ADx-K-ras即时PCR试剂盒进行检测,与此同时,将PCR扩增后的产物进行Sanger DNA测序.两种方法对K-ras基因第12和13密码子的检测结果进一步进行各个突变类型的数目和突变率的统计对比.结果 ADx-K-ras即时PCR试剂盒对827例样品检测都得到了明确的结果,检测成功率为100%,Sanger DNA测序成功检测了677例,成功率为81.9%.583例肠癌样品中ADx-K-ras即时PCR试剂盒检测出突变192例,突变检出率为32.9%,Sanger DNA测序成功的样品533例,检出突变160例,突变检出率为30.0%.244例肺癌样品中ADx-K-ras即时PCR试剂盒检出突变26例,突变检出率为10.7%,Sanger DNA测序成功的样品144例,检出突变12例,突变检出率为8.3%.肠癌中第12密码子第2位的GGT→GAT最常见,占全部突变的35.1%（66/188）,其次是第13密码子第2位的GGC→GAC,26.6%（50/188）,第12密码子第2位的GGT→GTT,18.6%（35/188）,第12密码子第1位的GGT→GCT最少见,1.6%（3/188）.肺癌中第12密码子第1位的GGT→GTT最常见,占全部突变的40.9%（9/22）,同样第12密码子第1位的GGT→GCT最少见,占全部突变的4.5%（1/22）.结论 肠癌中K-ras突变率明显高于肺癌.对于甲醛固定石蜡包埋样品而言,ADx-K-ras即时PCR试剂盒对样品的DNA质量的耐受性较好,检测成功率高于Sanger DNA测序,可替代Sanger DNA测序法成为临床上对肿瘤K-ras基因体细胞突变检测的实用方法.

Objective To map out the frequency and types of K-ras gene mutations present incolorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes. Methods A total of 827 formalin-fixed paraffin-embedded （FFPE） blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx＇s K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed. Results 533 out of 583 （91.4%） colorectal cancer samples and 144 out of 244 lung cancer samples（59.0%） were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583（100.0%） colorectal plus 244 out of 244（100.0%） lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 （32.9%） showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples （27.4%） with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26（10.7%） showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples （8.3%） showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% （66/188） mutations, followed by GGC→GAC at codon 13 with 26.6% （50/188） and GGT→GTT at codon 12 with 18.6% （35/188）,while GGT→GCT at codon12 was the most rare with only 1.6% （3/188） of the total mutation cases. In patients with lung cancer analyzed,GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% （9/22）, and GGT→GCT at codon 12 the most rare with only about 4.5% （1/22） of the total mutation cases. Conclusions K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods.ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.