结直肠癌患者血清外泌体代替肿瘤组织检测K-Ras基因突变的研究

Detection of K-Ras mutation in exosomes instead of tumor tissues from patients with colorectal cancer

目的探讨结直肠癌患者血清外泌体代替肿瘤组织进行DNA中K-Ras基因12密码子突变检测的价值。方法收集2014年5月至2015年9月于我院病理组织学诊断为局部晚期或转移性结直肠癌患者的外周血标本90例,使用Exo Quick试剂提取血清外泌体,盐析法提取外泌体及血细胞DNA,采用PCR方法扩增K-Ras基因12密码子并通过高通量测序法检测突变,比较血清外泌体DNA与血细胞DNA突变的特异性,比较外泌体与组织学检测的K-Ras基因12密码子突变情况。结果 90例结直肠癌患者中检测出外泌体K-Ras基因12密码子突变43例,突变率为47.8%。全组共检出4种突变类型,以G12D突变率为最高。与组织学检测比较,灵敏度为90.6%,一致性为81.1%(Kappa值=0.62,P<0.05)。结论血清外泌体DNA用于检测肿瘤相关突变,与肿瘤组织相比一致性较高,可作为液体活检的来源指导肿瘤的个体化治疗。

Objective To investigate the value of the 12th codon of K-Ras gene mutation detection in the serum exosomes instead of the tumor tissues in patients with colorectal cancer.Methods The peripheral blood samples of 90 patients pathologically diagnosed as locally advanced or metastatic colorectal cancer from May 2014 to September 2015 in our hospital were collected.The serum exosomes were extracted by ExoQuick agent.Exosomes and blood cells were extracted by salting out method.The 12th codon of K-Ras gene was amplified by PCR, and the mutation was detected by next generation sequencing.The specificity of serum exosomes DNA and blood cell DNA mutations was compared.Mutations of 12th codon of K-Ras gene were compared between exosomes and histological detection.Results Among 90 cases with colorectal cancer, 43 cases of the 12th codon mutation in K-Ras gene were detected with the mutation rate of 47.8%.Four kinds of mutation were detected, and the G12D mutation rate was the highest.Compared with histological examination, the sensitivity was 90.6% and the consistency was 81.1%（Kappa=0.62,P〈0.05）.Conclusion The consistent of serum exosome DNA used for detection of tumor-related mutation was higher than that of tumor tissues, which can be used as a source of liquid biopsy to guide the individualized treatment of tumors.