K-ras基因突变与结直肠癌临床病理因素的关系

目的 建立K-ras基因简单、快捷、经济的检测方法,并将检测结果与临床病理因素进行相关性分析.方法 采用PCR-直接测序法和聚合酶链式反应-限制性片段长度多态性测序法(PCR-RFLP)同时检测40例结直肠癌肿瘤组织标本K-ras基因第12、13密码子突变情况;另113例患者仅用PCR-RFLP-测序法检测K-ras基因第12、13密码子突变情况.将K-ras基因突变检测结果与临床病理因素进行相关性分析.结果 40例结直肠癌患者的肿瘤组织经常规PCR扩增后直接测序无一例发现K-ras基因第12、13密码子的突变;而这40例标本经PCR-RFLP-测序法检测发现:8例含有K-ras基因第12密码子突变,3例含有K-ras基因第13密码子突变,总突变检出率为27.5%(11/40).153例结直肠癌肿瘤组织标本经PCR-RFLP-测序法检测共发现突变58例,突变率为37.9%(58/153),其中第12密码子突变46例,第13密码子突变12例.G→A是K-ras基因突变最常见的突变形式(25/58,43.1%).K-ras基因第12和13密码子突变与患者性别、肿瘤浸润深度、分化程度、与淋巴结转移、远处转移及分期无明显相关性(P＞0.05),与年龄、肿瘤发生部位密切相关(P＜0.05),年龄越大突变概率越低,突变最常发生的肿瘤部位为升结肠.结论 PCR-RFLP-测序法能够快捷、灵敏地检测出肿瘤组织中K-ras基因的突变,适合作为K-ras基因突变常规检测方法.K-ras基因第12和13密码子突变是结直肠癌中一个常见的分子事件,与患者年龄、肿瘤发生部位有相关性.

K-ras gene mutation in colorectal cancer and its clinicopathologic significance

Objective To establish a simple, rapid and economical method in detecting mutations of oncogene K-ras and to investigate its mutations in colorectal cancer tissues and its relationship with clinicopathologic characteristics of colorectal carcinoma. Methods Forty colorectal cancer tissues were tested for K-ras mutations at codon 12 and codon 13 using polymerase chain reaction (PCR) followed by direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)followed by sequence analysis. The other 113 colorectal cancer tissues were tested for K-ras mutations at codon 12 and codon 13 using PCR-RFLP followed by sequence analysis only. The mutation results were analyzed with the corresponding clinical pathological data. Results Among 40 colorectal cancer cases,none of K-ras mutations at codon 12 and codon 13 was detected by PCR followed by direct sequencing.However, K-ras mutations were found in 11 cases (11/40, 27. 5% ) by PCR-RFLP followed by sequence analysis, including 8 cases at codon 12 and 3 cases at codon 13 respectively. Among 153 colorectal cancer cases, point mutations were detected by PCR-RFLP followed by sequence analysis in 58 cases (37.9%).Point mutations at codon 12 were found in 46 cases and 12 cases at codon 13. Mutations with the highest frequency were G→A transitions (25/58,43.1% ) at codon 12. No significant correlation was observed between mutations of K-ras and gender, invasive depth, tumor differentiation, number of invaded lymph nodes, distant metastasis and clinical stage (P＞0.05). Mutation of oncogene K-ras at codon 12 and codon 13 was closely related with age and tumor location (P＜0.05). The incidence of K-ras mutation was significantly higher in younger patients and in patients with ascending colon cancer. Conclusions PCRRFLP followed by sequence analysis is a rapid, simple, sensitive and low-cost method. It is a suitable technology for detecting hot-spot mutations in the K-ras oncogene. Mutation of oncogene K-ras at codon 12 and codon 13 is a common molecular event in colorectal carcinogenesis, which might be related with age and tumor location.