结肠直肠癌KRAS、NRAS、BRAF基因突变及其与临床病理特征、p53蛋白表达间相关性研究

doi:10.16150/j.1671-2870.2018.06.012

摘要/Abstract

摘要： 目的：筛查肠直肠癌患者的KRAS、NRAS和BRAF基因亚型突变状态, 并分析其与患者临床病理特征及p53蛋白表达情况间的相关性。方法：收集110例手术切除的结肠直肠癌病理资料及组织学样本, 总结临床病理特征;采用突变阻滞扩增系统法检测KRAS、NRAS基因2、3、4号外显子第12、13、61、117、146密码子及BRAF基因15号外显子第600密码子的突变情况;采用免疫组织化学（免疫组化）法检测p53蛋白的表达情况。结果：KRAS基因突变率为45.4%（50/110）, 其中49例患者检测到2号外显子第12和13密码子突变, 1例检测到4号外显子第117和146密码子突变;NRAS基因突变率为6.3%（7/110）, 其中3例检测到2号外显子第12和13密码子突变, 3例检测到3号外显子第61密码子突变, 1例检测到4号外显子第146密码子突变, 且与KRAS基因存在共突变;BRAF基因15号外显子第600密码子突变率为2.7%（3/110）;p53蛋白阳性表达率为57.3%（63/110）, 其中27例患者存在KRAS基因突变, 7例存在NRAS基因突变, 2例存在BRAF基因突变。KRAS基因突变率与患者的淋巴结转移显著相关（P<0.05）;而NRAS基因突变与其组织分型及p53蛋白表达显著相关（P<0.05）;BRAF基因突变率与右半结肠癌显著相关（P<0.05）。结论：KRAS与结肠直肠癌发生、发展的分子机制关系密切;BRAF基因突变仅见于右半结肠, 提示右半结肠癌发生机制可能有其特殊性;p53蛋白表达与NRAS基因突变之间是否存在相互作用关系值得进一步研究。

关键词: 结肠直肠癌, KRAS, NRAS, BRAF, P53

Abstract: Objective: To examine the mutation frequencies of KRAS, NRAS and BRAF in colorectal cancer (CRC) among Chinese population, and to investigate their correlation with clinicopathological parameters and p53 protein expression. Methods: Tissue samples and pathological data of 110 CRC patients undergoing surgical excision were collected, and the clinical pathological features were summarized. Mutations in KRAS (codon 12/13 at exon 2, codon 61 at exon 3 and codon 117/146 at exon 4), NRAS (codon 12/13 at exon 2, codon 61 at exon 3 and codon 146 at exon 4) and BRAF (codon 600 at exon 15) genes were examined with ARMS-PCR. p53 protein expression was detected by immunohistochemistry. Results: 45.4% (50/110) patients carried KRAS mutations; among them, almost all individuals had mutation in KRAS codon 12/13 at exon 2, except one individual had codon 117/146 at exon 4 mutated. 6.3% (7/110) patients carried NRAS mutations; among them, 3/110 patients had mutation in NRAS exon 2 (condon 12/13), 3/110 patients had mutation in NRAS exon 3 (condon 61), 1/110 patient had mutations in both NRAS exon 4 (condon 146) and KRAS. BRAF mutations were detected in 3 patients (2.7%), and were found to have V600E mutations. p53 expression was observed in 63 (57.3%) of 110 CRC patients. Within these 63 positive samples, 27 cases showed KRAS mutations, 7 cases showed NRAS mutations and 2 cases showed BRAF mutations. KRAS mutation was significantly associated with lymph node metastasis (P<0.05) while NRAS mutation was significantly associated with histological type and p53 protein expression (P<0.05). BRAF mutation was significantly associated with right colon cancer (P<0.05). Conclusions: KRAS is closely related to the molecular mechanism of occurrence and development of colorectal cancer. BRAF mutation is only found in right colon cancer, suggesting that the mechanism of right hemicolon cancer may have its particularity. The interaction between p53 protein expression and NRAS mutation is worthy of further study.

Key words: Colorectal cancer, KRAS, NRAS, BRAF, P53

中图分类号:

R735.3⁺5

王子元, 梁婷玉, 陈佳, 韩志宏, 吴丽莉. 结肠直肠癌KRAS、NRAS、BRAF基因突变及其与临床病理特征、p53蛋白表达间相关性研究[J]. 诊断学理论与实践, 2018, 17(06): 687-693.

WANG Ziyuan, LIANG Tingyu, CHEN Jia, HAN Zhihong, WU Lili. Analysis of KRAS, NRAS and BRAF mutations in colorectal cancer and their correlation with clinicopathologic features and p53 protein expression[J]. Journal of Diagnostics Concepts & Practice, 2018, 17(06): 687-693.

参考文献

[1] Hanahan D, Weinberg RA.The hallmarks of cancer[J]. Cell,2000,100(1):57-70.
[2] Rajagopalan H, Nowak MA, Vogelstein B, et al.The significance of unstable chromosomes in colorectal cancer[J]. Nat Rev Cancer, 2003, 3(9):695-701.
[3] Peltomäki P.Mutations and epimutations in the origin of cancer[J]. Exp Cell Res,2012,318(4):299-310.
[4] Esteller M.Epigenetics in cancer[J]. N Engl J Med,2008, 358(11):1148-1159.
[5] 侯慧, 肖明明, 任秋华, 等. 结直肠癌中p53蛋白和Ki67的表达及临床意义[J]. 实用肿瘤学杂志,2008,22(5):424-426.
[6] Li Y, Xu KP, Jiang D, et al.Relationship of Fas, FasL, p53 and bcl-2 expression in human non-small cell lung carcinomas[J]. Int J Clin Exp Pathol,2015,8(11):13978-13986.
[7] Phipps AI, Buchanan DD, Makar KW, et al.KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers[J]. Br J Cancer, 2013,108(8): 1757-1764.
[8] Cappuzzo F, Varella-Garcia M, Finocchiaro G, et al.Primary resistance to cetuximab therapy in EGFR FISH-positive colorectal cancer patients[J]. Br J Cancer,2008, 99(1):83-89.
[9] Baldus SE, Schaefer KL, Engers R, et al.Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases[J]. Clin Cancer Res,2010,16(3):790-799.
[10] Lurkin I, Stoehr R, Hurst CD, et al.Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes[J]. PLoS One,2010,5(1):e8802.
[11] Guedes JG, Veiga I, Rocha P, et al.High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer[J]. BMC Cancer,2013,13:169.
[12] Sidransky D, Tokino T, Hamilton SR, et al.Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors[J]. Science,1992,256(5053):102-105.
[13] McLellan EA, Owen RA, Stepniewska KA, et al. High frequency of K-ras mutations in sporadic colorectal adenomas[J]. Gut,1993,34(3):392-396.
[14] ZhangJ, ZhengJ, Yang Y, et al. Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases[J]. Sci Rep,2015,5:18678.
[15] 吴文辉, 肖隆斌, 汤友珍, 等. K-ras基因突变与结直肠癌生物学行为的关系[J]. 中国病理生理杂志,2009,25(11):2159-2162.
[16] Shen Y, Wang J, Han X, et al.Effectors of epidermal growth factor receptor pathway: the genetic profiling of KRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine[J]. PLoS One,2013,8(12):e81628.
[17] Ye JX, Liu Y, Qin Y, et al.KRAS and BRAF gene mutations and DNA mismatch repair status in Chinese colorectal carcinoma patients[J]. World J Gastroenterol,2015,21(5):1595-1605.
[18] Shimada Y, Kameyama H, Nagahashi M, et al.Comprehensive genomic sequencing detects important genetic differences between right-sided and left-sided colorectal cancer[J]. Oncotarget,2017,8(55):93567-93579.
[19] Gonsalves WI, Mahoney MR, Sargent DJ, et al. Patient and tumor characteristics and BRAF and KRAS mutations in colon cancer, NCCTG/Alliance N0147[J]. J Natl Cancer Inst,2014,106(7):pii: dju106.
[20] Downward J.Targeting RAS signalling pathways in cancer therapy[J]. Nat Rev Cancer,2003,3(1):11-22.
[21] Zenonos K, Kyprianou K.RAS signaling pathways, mutations and their role in colorectal cancer[J]. World J Gastrointest Oncol,2013,5(5):97-101.
[22] Irahara N, Baba Y, Nosho K, et al.NRAS mutations are rare in colorectal cancer[J]. Diagn Mol Pathol,2010,19(3):157-163.
[23] Devitt B, Liu W, Salemi R, et al.Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma[J]. Pigment Cell Melanoma Res,2011,24(4):666-672.
[24] Yoon HH, Shi Q, Alberts SR, et al. Racial Differences in BRAF/KRAS Mutation Rates and Survival in Stage Ⅲ Colon Cancer Patients[J]. J Natl Cancer Inst,2015,107(10):pii: djv186.
[25] Baskin Y, Calibasi G, Amirfallah A, et al.KRAS and BRAF mutation frequencies in a series of Turkish colo-rectal cancer patients[J]. Transl Cancer Res,2014,3(2):160-166.
[26] Tie J, Gibbs P, Lipton L, et al.Optimizing targeted therapeutic development: analysis of a colorectal cancer patient population with the BRAF(V600E) mutation[J]. Int J Cancer,2011,128(9):2075-2084.
[27] Samowitz WS, Albertsen H, Sweeney C, et al.Association of smoking, CpG island methylator phenotype, and V600E BRAF mutations in colon cancer[J]. J Natl Cancer Inst,2006,98(23):1731-1738.
[28] Bhalla A, Zulfiqar M, Bluth MH.Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018[J]. Clin Lab Med,2018,38(2):311-342.
[29] Yokota T, Ura T, Shibata N, et al.BRAF mutation is a powerful prognostic factor in advanced and recurrent colo-rectal cancer[J]. Br J Cancer,2011,104(5):856-862.
[30] Lai E, Pretta A, Impera V, et al.BRAF-mutant colorectal cancer, a different breed evolving[J]. Expert Rev Mol Diagn,2018,18(6):499-512.
[31] Muller PA, Vousden KH.Mutant p53 in cancer: new functions and therapeutic opportunities[J]. Cancer Cell,2014,25(3):304-317.
[32] Wu GS.The functional interactions between the p53 and MAPK signaling pathways[J]. Cancer Biol Ther,2004,3(2):156-161.
[33] Sauer L, Gitenay D, Vo C, et al.Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells[J]. Oncogene,2010,29(18):2628-2637.
[34] Muller PA, Trinidad AG, Timpson P, et al.Mutant p53 enhances MET trafficking and signalling to drive cell scattering and invasion[J]. Oncogene,2013,32(10):1252-1265.
[35] Najem A, Krayem M, Salès F, et al.P53 and MITF/Bcl-2 identified as key pathways in the acquired resistance of NRAS-mutant melanoma to MEK inhibition[J]. Eur J Cancer,2017,83:154-165.