乳腺癌组织中EGFR/KRAS/BRAF基因突变检测与分析

Detection and analysis of EGFR/KRAS/BRAF gene mutation status in breast cancer tissues

[目的]检测乳腺癌组织中表皮生长因子受体(EGFR)以及其下游KRAS BRAF基因的突变类型和突变率,分析其各突变类型之间有无相关性,以期为乳腺癌药物靶点筛选和乳腺癌患者的分子靶向治疗提供更多依据。[方法]取自2016~2017年间宜昌市中心人民医院病理科收集的乳腺癌患者术后冰冻肿瘤组织样本60例以及健康人群DNA样本30例,分别提取基因组DNA,采用PCR扩增和基因测序的方法对基因组中的EGFR外显子18-21,KRAS外显子2.3以及BRAF外显子15进行基因突变分析。[结果]在检测的60例乳腺癌组织样本中,总计有8例样本存在错义突变。其中EGFR突变率为11.7%(7/60例),均为外显子18和20突变。包括第18号外显子P699A突变3例,突变率为5.0%;第20号外显子L778M、V819A突变各4例,突变率6.7%。并包含3例L778M合并V819A突变,另有2例中分别出现了I780M合并S783C和L788V合并L815F突变。BRAF外显子15仅出现了1例T599P突变,突变率为1.7%(1/60例)。30例正常人群对照组未检测到错义突变。[结论]EGFR在乳腺癌中的突变主要以外显子18和20为主,存在P699A.L778M,V819A突变且突变率高达11.7%。BRAF在乳腺癌中的突变率极低,仅为1.7%。未检测到乳腺癌组织中KRAS突变。与正常组织相比较,EGFR外显子18和20的高突变率提示其与乳腺癌的发病存在密切关系,EGFR突变是潜在的抗乳腺癌治疗靶点。

[Objective]Detection of epidermal growth factor receptor(EGFR)and its downstream KRAS,BRAF gene mutations incidence and mutation type in breast cancer tissues can be used to analyze the correlation between their mutation types and find the new drug target that canl provide a basis for individualized molecular targeted therapy for breast cancer patients.[Meth-od]60 cases of frozen tumor tssue samples and 30 normal blood samples were collected from the pathology department of Yichang Central Peoples Hospital from 2016 to 2017.Genomic DNA was extracted and PCR amplifcation and gene sequencing were used.Gene mutation analysis was performed on EGFR exon 18-21,KRAS exons 2 and 3,and BRAF exon 15 in genomie DNA.[Result]Of the 60 breast cancer tssue samples tested,a total of 8 samples had missense mutations,of which EGFR muta-tion rate was 11.7%(7/60 cases),all of which were exon 18 and 20 mutations,including the 3 cases of P699A mutation in exon 18 and 4 cases of L778M and V819A mutations in exon 20,the mutation rates were 5.0%and 6.7%,respectively;and in exon 20,there were 3 cases of L778M combined with V819A mutation and 1 case of I780M combined with S783C and L788 V combined with L815F mutation.There was only one T599P mutation in BRAF exon 15 with a mutation rate of 1.7%(1/60 ca-ses);no missense mutation was detected in the control group of 30 normal controls.[Conclusion]The mutations of EGFR in breast cancer were mainly exon 18 and 20,with P699A,L778M and V819A mutations,and the mutation rate was as high as 11.7%.The mutation rate of BRAF in breast cancer is extremely low,only 1.7%.KRAS mutations were not detected in breast cancer tissues.Compared with normal tssues,the high mutation rates of ECFR exons 18 and 20 suggest a close relationship with the incidence of breast cancer,and EGFR mutations are potential anti-breast cancer treatment targets.