结直肠肿瘤中KRAS和BRAF基因突变特点及检测方法对比分析的研究

发布时间：2017-12-31 00:35

本文关键词：结直肠肿瘤中KRAS和BRAF基因突变特点及检测方法对比分析的研究　出处：《上海交通大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:研究远端结直肠腺瘤和腺癌病变中KRAS和BRAF基因突变的特点,初步了解分子学诊断在指导结直肠癌用药中的临床价值;同时,对比分析Cast PCR法和DNA直接测序法检测肠镜活检标本KRAS G12D和BRAF V600E突变的差异。方法:在肠镜下,采用活检钳收集远端结直肠腺瘤(腺瘤组,n=32)和腺癌(腺癌组,n=20)病变组织;抽提病变组织基因组DNA;并采用DNA直接测序法测定病变组织DNA的KRAS和BRAF基因序列。同时,使用Cast PCR法检测KRAS G12D和BRAF V600E突变情况。结果:1、采用DNA直接测序法在32例腺瘤组患者中,KRAS基因突变7例,突变率21.9%;20例腺癌组患者中,KRAS基因突变7例,突变率35%。两组患者KRAS基因第12密码子和第13密码子较常见突变类型均为G12D和G13D。统计学结果显示腺瘤组和腺癌组KRAS基因阳性突变率无明显统计学意义(P0.05),而且两组患者KRAS基因阳性突变率与性别、腺瘤分化程度、腺癌分化类型之间均无明显统计学差异。两组患者均未见BRAF V600E突变。2、结直肠腺瘤组Cast PCR法检测KRAS G12D突变率28.1%,比DNA直接测序法高12.5%;结直肠癌组Cast PCR法检测KRAS G12D突变率30%,比DNA直接测序法高15%。两种方法阴性符合率100%。Mutation Detector分析结果显示Cast PCR法能够检测出突变量1%的突变。从检测突变率分析,两种方法无统计学意义(P0.05),结直肠腺瘤组总体符合率87.5%(Kappa值0.6429),结直肠癌组总体符合率85%(Kappa值0.5833)。两组患者均未见BRAF V600E突变。结论:1、远端结直肠腺瘤和腺癌中BRAF V600E突变率较西方国家低;KRAS基因突变率较BRAF V600E基因突变率高,与西方国家基本一致,提示检测KRAS基因突变在指导结直肠癌用药中的临床价值可能更大,可在野生型KRAS基因型结直肠癌抗EGFR单克隆抗体耐药的基础上进一步检测BRAF基因突变情况。2、Cast PCR法灵敏度高达0.1%,能够高效的检测肠镜活检标本中低突变量的KRAS G12D,而且操作简单、耗时短、可重复性强,比DNA直接测序法临床实用价值可能更高。
[Abstract]:Objective: to study the characteristics of KRAS and BRAF gene mutations in distal colorectal adenoma and adenocarcinoma, and to explore the clinical value of molecular diagnosis in guiding drug use of colorectal cancer. At the same time, the differences of KRAS G12D and BRAF V600E mutations between Cast PCR method and DNA direct sequencing method were analyzed. The lesions of distal colorectal adenoma (adenoma group) and adenocarcinoma (adenocarcinoma group) were collected by biopsy forceps. Genomic DNA was extracted from diseased tissues. The KRAS and BRAF gene sequences of DNA were determined by DNA direct sequencing. The mutation of KRAS G12D and BRAF V600E was detected by Cast PCR method. Results: 1. DNA direct sequencing was used in 32 patients with adenoma. There were 7 cases of KRAS gene mutation, the mutation rate was 21. 9%. There were 7 cases of KRAS gene mutation in 20 patients with adenocarcinoma. G12D and G13D were the most common mutations in codon 12 and codon 13 of KRAS gene in both groups. Statistical results showed that KRAS gene was positive in adenoma group and adenocarcinoma group. There was no significant difference in the rate of sexual mutation (. P0.05). Moreover, there was no significant difference between the two groups in KRAS gene mutation rate and sex, adenoma differentiation, adenocarcinoma differentiation type. There was no significant difference in BRAF V600E mutation between the two groups. The mutation rate of KRAS G12D was detected by Cast PCR in colorectal adenoma group, which was higher than that by DNA direct sequencing method. The mutation rate of KRAS G12D was detected by Cast PCR method in colorectal cancer group (30%%). The negative coincidence rate of the two methods was 100% higher than that of DNA direct sequencing. The results of Detector analysis showed that Cast. PCR method can detect 1% mutation. There was no significant difference between the two methods (P 0.05). The overall coincidence rate of colorectal adenoma group was 87.5% and the Kappa value was 0.6429). There was no BRAF V600E mutation in both groups. Conclusion: 1. The mutation rate of BRAF V600E in distal colorectal adenoma and adenocarcinoma was lower than that in western countries. The mutation rate of KRAS gene is higher than that of BRAF V600E gene, which is consistent with that of western countries, suggesting that detection of KRAS gene mutation may be of more clinical value in guiding drug use in colorectal cancer. The sensitivity of BRAF gene mutation. 2Cast PCR assay can be as high as 0.1% on the basis of the resistance to EGFR monoclonal antibody of wild-type KRAS genotype colorectal cancer. KRAS G12D, which can be used to detect the low mutation quantity of enteroscopy biopsy samples, is simple, time consuming and reproducible, which may be more valuable than DNA direct sequencing method in clinical practice.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.34

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