结直肠癌KRAS、NRAS和BRAF基因罕见突变类型及其临床意义(附1513例)

Infrequent gene mutations of KRAS,NRAS and BRAF in colorectal cancer and their clinical significance:a report of 1513 cases

目的探讨结直肠癌KRAS、NRAS和BRAF基因罕见突变类型及其临床意义。方法采用回顾性描述性研究方法。收集2013年12月至2018年11月北京大学肿瘤医院收治的1513例结直肠癌患者的临床病理资料;男921例,女592例;平均年龄为59岁,年龄范围为15~97岁。提取肿瘤组织基因组DNA,采用Sanger测序法进行KRAS(第2、3外显子),NRAS(第2、3外显子)和BRAF(第15外显子)基因突变检测。观察指标:(1)KRAS、NRAS和BRAF基因突变类型情况。(2)KRAS、NRAS和BRAF基因突变类型与临床病理特征的关系。(3)单一基因罕见突变及其临床病理特征。(4)同时2种基因突变及其临床病理特征。计数资料采用绝对数和百分比表示,组间比较采用χ^2检验。结果(1)KRAS、NRAS和BRAF基因突变类型情况:1513例结直肠癌患者中,KRAS、NRAS和BRAF的基因突变率分别为37.806%(572/1513)、3.173%(48/1513)和5.486%(83/1513)。KRAS外显子2突变率为35.889%(543/1513),外显子3突变率为1.917%(29/1513)。NRAS外显子2突变率为1.322%(20/1513),外显子3突变率为1.851%(28/1513)。BRAF外显子15突变率为5.486%(83/1513)。KRAS基因突变类型主要为外显子2第12、13密码子和外显子3第61密码子突变,突变率分别为27.759%(420/1513)、7.733%(117/1513)和1.586%(24/1513)。7例结直肠癌患者发生KRAS基因第14、59及60密码子区域的罕见突变,突变率为0.463%(7/1513),其中2例[0.132%(2/1513)]V14I突变、2例[0.132%(2/1513)]A59T突变、2例[0.132%(2/1513)]A59E及1例[0.066%(1/1513)]G60D突变。NRAS基因突变类型主要为外显子2第12、13密码子及外显子3第61密码子突变,突变类型主要为Q61K,突变率为0.925%(14/1513),其次为G12D突变,突变率为0.727%(11/1513)。Q61R、Q61H、Q61L、G13R、G12C、G12V、G12S、G13D及G13C的突变率均较低。BRAF基因突变类型主要为外显子15第600密码子突变,突变类型主要为V600E突变,突变率为4.957%(75/1513)。8例结直肠癌患者发生BRAF基因罕见突变,突变率为0.529%(8/1513),其中5例[0.330%(5/1513)]D594G突变、1例[0.066%(1/1513)]D594H突变、1例[0.066%(1/1513)]S607T突变及1例[0.066%(1/1513)]599~600密码子插入突变AGA(T599 V600insAGA)。1513例患者中,4例[(0.264%,4/1513)]发生KRAS基因第12及13密码子同时突变,其中2例[(0.132%,2/1513)]发生G12V与G13D同时突变;1例[(0.066%,1/1513)]发生G12D与G13A同时突变;1例[(0.066%,1/1513)]发生G12V与G13F同时突变。此外,1例[(0.066%,1/1513)]发生KRAS G13D与NRAS G12S同时突变,1例[(0.066%,1/1513)]发生KRAS G12C与NRAS Q61H同时突变。(2)KRAS、NRAS和BRAF基因突变类型与临床病理特征的关系:KRAS基因突变中肿瘤部位、肿瘤分化程度比较,差异均有统计学意义(χ^2=25.317,4.166,P<0.05)。BRAF基因突变中性别、肿瘤部位、肿瘤分化程度、淋巴结转移比较,差异均有统计学意义(χ^2=11.290,22.317,38.035,12.611,P<0.05)。进一步研究结果显示:NRAS基因突变类型中Q61K突变和Q61R突变发生在年龄<65岁、≥65岁比例分别为12/18、2/10和1/18、5/10,两者比较,差异均有统计学意义(χ^2=5.600,7.542,P<0.05)。(3)单一基因罕见突变及其临床病理特征:1513例患者中,15例发生单一基因罕见突变。7例发生KRAS基因第14、59及60密码子罕见突变患者中,男6例,女1例;年龄<65岁6例,年龄≥65岁1例;肿瘤位于左半结肠3例,右半结肠3例,直肠1例;肿瘤组织学类型及分化程度为高、中分化腺癌6例,低分化腺癌1例;TNM分期Ⅳ期6例,Ⅱ期1例;有远处转移6例,无远处转移1例;有淋巴结转移3例,无淋巴结转移4例;术后均无复发。8例发生BRAF基因罕见突变患者中,男4例,女4例;年龄<65岁4例,年龄≥65岁4例;肿瘤位于左半结肠5例,右半结肠1例,直肠2例;肿瘤组织学类型及分化程度为中分化腺癌7例,低分化腺癌1例;TNM分期Ⅳ期5例,Ⅲ期2例,Ⅱ期1例;有远处转移6例,无远处转移2例;有淋巴结转移3例,无淋巴结转移5例;术后复发1例。(4)同时2种基因突变及其临床病理特征:1513例患者中,6例发生同时2种基因突变,男5例,女1例;年龄<65岁2例,年龄≥65岁4例;肿瘤位于左半结肠1例,右半结肠4例,直肠1例;肿瘤组织学类型及分化程度为高、中分化腺癌5例,低分化腺癌1例;TNM分期Ⅳ期5例,Ⅱ期1例;有远处转移4例,无远处转移2例;有淋巴结转移3例,无淋巴结转移3例;术后复发1例。结论结直肠癌患者的KRAS和BRAF基因罕见突变常发生在罕见的密码子区域,并以点突变为主,KRAS、NRAS和BRAF基因突变与临床病理特征相关,可为结直肠癌的治疗提供重要依据。

Objective To investigate the infrequent gene mutations of KRAS,NRAS and BRAF in colorectal cancer and their clinical significance.Methods The retrospective and descriptive study was conducted.The clinicopathological data of 1513 patients with colorectal cancer who were admitted to the Peking University Cancer Hospital from December 2013 to November 2018 were collected.There were 921 males and 592 females,aged from 15 to 97 years,with an average age of 59 years.The genomic DNA of tumor tissue was extracted,and the mutation status of KRAS(exon 2,3),NRAS(exon 2,3)and BRAF(exon 15)was detected by the Sanger sequencing.Observation indicators:(1)mutation status of KRAS,NRAS and BRAF;(2)relationship of different mutation status of KRAS,NRAS and BRAF with clinicopathological characteristics;(3)infrequent mutation status of single gene and its clinicopathological characteristics;(4)simultaneous mutations of two genes and their clinicopathological characteristics.Count data were expressed by absolute numbers or percentages,and comparison between groups was analyzed by the chi-square test.Results (1)Mutation status of KRAS,NRAS and BRAF:the mutation rates of KRAS,NRAS and BRAF were 37.806%(572/1513),3.173%(48/1513)and 5.486%(83/1513)of the 1513 patients with colorectal cancer,respectively.The mutation rates of exon 2 and exon 3 in KRAS were 35.889%(543/1513)and 1.917%(29/1513),respectively.The mutation rates of exon 2 and exon 3 in NRAS were 1.322%(20/1513)and 1.851%(28/1513),respectively.The mutation rate of exon 15 in BRAF was 5.486%(83/1513).The mutation of KRAS mainly occurred in codon 12,13 of exon 2 and codon 61 of exon 3,with a mutation rate of 27.759%(420/1513),7.733%(117/1513),and 1.586%(24/1513),respectively.Infrequent mutation in codon 14,59,60 of KRAS were found in 7 patients with colorectal cancer[0.463%(7/1513)],including V14I mutation in 2 cases[0.132%(2/1513)],A59T mutation in 2 cases[0.132%(2/1513)],A59E mutation in 2 cases[0.132%(2/1513)]and G60D mutation in 1 case[0.066%(1/1513)].The mutation of NRAS mainly occurred in codon 12,13 of exon 2 and codon 61 of exon 3,including Q61K with a mutation rate of 0.925%(14/1513),followed by G12D with a mutation rate of 0.727%(11/1513).The mutation rates of Q61R,Q61H,Q61L,G13R,G12C,G12V,G12S,G13D,and G13C were relatively low.The mutation of BRAF mainly occurred in codon 600 of exon 15 as V600E mutation,with a mutation rate of 4.957%(75/1513).Infrequent mutation in BRAF were found in 8 patients with colorectal cancer,with a mutation rate of 0.529%(8/1513),including D594G mutation in 5 cases[0.330%(5/1513)],D594H mutation in 1 case[0.066%(1/1513)],S607T mutation in 1 case[0.066%(1/1513)],and 599-600 codon insertion AGA in 1 case[0.066%(1/1513)].Of the 1513 patients,4[0.264%(4/1513)]had simultaneous mutations at codon 12 and 13 of KRAS,including 2[0.132%(2/1513)]with simultaneous mutations at G12V and G13D,1[0.066%(1/1513)]with simultaneous mutations at G12D and G13A,and 1[0.066%(1/1513)]with simultaneous mutations at G12V and G13F.In addition,1 patient[0.066%(1/1513)]had simultaneous mutations at G13D of KRAS and G12S of NRAS,and 1 patient[0.066%(1/1513)]had simultaneous mutations at G12C of KRAS and Q61H of NRAS.(2)Relationship of different mutation status of KRAS,NRAS and BRAF with clinicopathological characteristics:patients with different tumor location and tumor differentiation degree had significantly different KRAS mutation status(χ^2=25.317,4.166,P<0.05);patients with different gender,tumor location,tumor differentiation degree,and lymph node metastasis had significantly different BRAF mutation status(χ^2=11.290,22.317,38.035,12.611,P<0.05).The proportion of Q61K mutation and Q61R mutation of NRAS in the patients with age of<65 and≥65 years was 12/18,2/10 and 1/18,5/10,respectively,showing significant differences between the two groups(χ^2=5.600,7.542,P<0.05).(3)Infrequent mutation status of single gene and its clinicopathological characteristics:15 of the 1513 patients had single gene mutation.Of the 7 patients with infrequent mutations in codon 14,59 and 60 of KRAS,6 were males and 1 was female;6 were<65 years old and 1 was≥65 years old;3 had tumors located in the left colon,3 in the right colon and 1 in the rectum;6 had highly or moderately differentiated adenocarcinoma and 1 had poorly differentiated adenocarcinoma;6 were in stageⅣand 1 was in stageⅡof TNM staging;6 had distant metastasis and 1 had no distant metastasis;3 had lymph node metastasis and 4 had no lymph node metastasis;there was no postoperative recurrence.Of the 8 patients with infrequent gene mutation of BRAF,4 were males and 4 were females;4 were<65 years old and 4 were≥65 years old;5 had tumors located in the left colon,1 in the right colon and 2 in the rectum;7 had moderately differentiated adenocarcinoma and 1 had poorly differentiated adenocarcinoma;5 were in stageⅣ,2 in stageⅢ,and 1 in stageⅡof TNM staging;6 had distant metastasis and 2 had no distant metastasis;3 had lymph node metastasis and 5 had no lymph node metastasis;1 had postoperative recurrence.(4)Simultaneous mutations of two genes and their clinicopathological characteristics:6 of the 1513 patients had simultaneous mutations of two genes.Of 6 patients with simultaneous mutations of two genes,5 were males and 1 was female;2 were<65 years old and 4 were≥65 years old;1 had tumor located in the left colon,4 in the right colon and 1 in the rectum;5 had highly or moderately differentiated adenocarcinoma and 1 had poorly differentiated adenocarcinoma;5 were in stageⅣand 1 was in stageⅡof TNM staging;4 had distant metastasis and 2 had no distant metastasis;3 had lymph node metastasis and 3 had no lymph node metastasis;1 had postoperative recurrence.Conclusions The infrequent mutations of KRAS and BRAF in colorectal cancer often occur in the rare codon region and mainly are point mutations.Different mutations of KRAS,NRAS and BRAF are related to clinicopathological features,which provide an important basis for treatment of colorectal cancer.