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目的探讨直接测序法和肽核酸钳制PCR(PNA-PCR)法检测K-Ras基因突变状态与转移性结直肠癌患者疗效及预后的相关性。方法收集110例转移性结直肠癌患者的石蜡包埋肿瘤组织,采用直接测序法和PNA-PCR法分别检测肿瘤组织的K-Ras基因第2外显子第12、13密码子的突变状态,并分析其与患者预后的关系。结果直接测序法检测到43例K-Ras基因突变,PNA-PCR法除了检测出这些突变之外,还在直接测序法检测的野生型中发现了10例突变。对K-Ras突变状态与患者的预后分析发现,直接测序法检测的K-Ras野生型及突变型患者的中位生存时间(OS)分别为20.5个月和15.6个月(P=0.067)。PNA-PCR法检测的野生型和突变型患者的中位OS分别为21.3个月和15.8个月(P=0.014)。两种方法检测的野生型与突变型的有效率和无病进展时间(PFS)差异均无统计学意义。按照这两种方法的检测结果分为3组,高突变组、低突变组和野生型组,仅高突变组与野生型组的OS差异有统计学意义(15.6个月vs.21.3个月,P=0.040)。Cox多因素分析显示,ECOG评分(HR=2.70,95%CI:1.39～5.25,P=0.003)和K-Ras丰度(HR=1.52,95%CI:1.52～2.19,P=0.026)与患者的预后相关。结论 K-Ras突变不是以伊立替康或奥沙利铂为主方案的疗效预测因子。PNA-PCR法检测的K-Ras突变状态与患者的预后有关。建议用PNA-PCR法确定野生型患者,而突变型患者则用直接测序法来确定。
Objective To investigate the relationship between the mutation status of K-Ras gene and the efficacy and prognosis of patients with metastatic colorectal cancer by direct sequencing and peptide-nucleic acid-clamp PCR (PNA-PCR). Methods Totally 110 paraffin-embedded tumor tissues of patients with metastatic colorectal cancer were collected. The mutation status of codon 12 and 13 in K-Ras gene exon 2 of tumor tissues was detected by direct sequencing and PNA-PCR. And analyze its relationship with the prognosis of patients. Results 43 K-Ras gene mutations were detected by direct sequencing. In addition to these mutations detected by PNA-PCR, 10 mutations were found in the wild type detected by direct sequencing. The prognostic analysis of K-Ras mutation status and patients found that the median survival time (OS) of K-Ras wild-type and mutant patients detected by direct sequencing were 20.5 months and 15.6 months (P = 0.067), respectively. The median OS of wild-type and mutant patients detected by PNA-PCR was 21.3 months and 15.8 months (P = 0.014), respectively. There was no significant difference between the two methods in detecting the effective rate and the progression free time (PFS) of wild type and mutant type. The results of these two methods were divided into three groups: high mutation group, low mutation group and wild type group, and there was significant difference in OS only between high mutation group and wild type group (15.6 months vs.21.3 months, P = 0.040). Cox multivariate analysis showed that patients with ECOG score (HR = 2.70, 95% CI: 1.39-5.25, P = 0.003) and K-Ras abundance (HR = 1.52, 95% CI: 1.52-2.19, P = 0.026) The prognosis. Conclusions K-Ras mutations are not predictors of response to either irinotecan or oxaliplatin-based regimens. PNA-PCR assay K-Ras mutation status and prognosis of patients. Proposed PNA-PCR method to determine the wild-type patients, while the mutant patients were determined by direct sequencing.