结直肠癌中KRAS基因突变及其检测方法概述

结直肠癌（Colorectal Cancer,CRC）是世界上发病率最高的恶性肿瘤之一.CRC的发生与细胞的跨膜信号转导系统功能异常有关,目前已经明确Ras/Raf/MAPK级联反应通路在其中起到很重要的作用.在CRC中,KRAS基因突变十分常见,KRAS基因的突变使信号转导通路下游各种基因的激活不再依赖于表皮生长因子受体（Epidermal Growth Factor Receptor,EGFR）的启动,这对CRC的发生、发展、治疗与预后起着非常重要的作用.因此,在临床工作中,检测KRAS基因突变情况显得尤为重要,这也对检测技术及其准确性提出了更高的要求.本文对KRAS在CRC中的作用、机制及其突变的概率、检测方法做一简要综述.     

结直肠癌中KRAS基因突变与SETDB1表达的相关性及临床意义

  目的  探讨KRAS基因突变与SETDB1（set domain bifurcated 1）蛋白表达在结直肠癌中的临床意义及两者间的关系。  方法  收集2017年1月至2017年12月于西南医科大学附属医院住院接受诊治的122例结直肠癌患者的组织标本及病例资料,采用二代测序技术（NGS）检测KRAS基因突变情况,免疫组织化学染色检测SETDB1蛋白的表达情况。  结果  122例结直肠癌组织中KRAS基因突变率为41.8%,KRAS基因突变状态与肿瘤部位、术前血清CEA水平相关（P < 0.05）,与年龄、性别、分化程度、肿瘤大小、pT?NM分期、淋巴结转移等无关（P>0.05）。KRAS基因突变状态与SETDB1蛋白表达呈正相关（r=0.232,P=0.008）。SETDB1蛋白水平与肿瘤分化程度、大小及术前血清CEA水平相关（P < 0.05）,与年龄、性别、肿瘤部位、pTNM分期、淋巴结转移等无关（P>0.05）。  结论  SETDB1蛋白在KRAS突变型结直肠癌组织中呈现高表达,KRAS基因突变可能影响SETDB1蛋白表达。      

KRAS基因突变检测与结直肠癌分子靶向治疗的研究进展

结直肠癌是常见的恶性肿瘤之一,其治疗一直是国内外学者研究的重要方向。随着靶向治疗药物表皮生长因子受体（EGFR）抑制剂西妥昔单抗的问世,结直肠癌的治疗进入了靶向治疗时代。近年来,多项临床研究表明：KRAS基因突变状态与转移性结直肠癌抗EGFR单抗治疗的疗效密切相关。许多国家已经将KRAS基因突变作为是否选择EGFR抑制剂治疗的必检指标。如何快速准确地检测KRAS基因突变,对个体化治疗显得尤为重要。     

COLD-PCR-HRM 检测结直肠癌患者外周血KRAS基因突变

目的  评价COLD-PCR-HRM 检测结直肠癌患者外周血KRAS 基因突变的临床应用价值。方法  将已知突变型 KRAS 组织DNA 与野生型DNA 做系列稀释,突变DNA 所占比例分别为50%、25%、10%、5%、3%、2% 和 1%。分别应 用常规PCR-HRM 法和COLD-PCR-HRM 法对不同比例KRAS 突变DNA 样本进行检测,验证两种检测方法的灵敏度;同 时应用 COLD-PCR-HRM 法对 62 例外周血和肿瘤组织配对样本进行一致性验证。结果  PCR-HRM 法最低检测浓度为 3%; COLD-PCR-HRM 法最低检测浓度为1%,两种方法的检测灵敏度均明显高于直接测序法（P ＜ 0.05）。应用COLD-PCRHRM 法检测两种样本中KRAS 基因突变状态,其中血清样本中检测出13 例突变（突变率21.0%,13/62）,组织中检测出 12 例突变（突变率 19.4%,12/62）,两种标本同时存在突变的有 9 例,以组织中检测的 KRAS 状态为准,两者突变一致性 为 75.0%（9/12）。KRAS 基因突变在组织与外周血中存在一致性（κ=0.649;P ＜ 0.001）。结论  COLD-PCR 结合 HRM 明 显提高了 KRAS 突变检测灵敏度,为其应用于低丰度突变样本检测提供了有利条件。     

KRAS基因突变检测与结直肠癌分子靶向治疗的研究进展

结直肠癌是常见的恶性肿瘤之一,其治疗一直是国内外学者研究的重要方向。随着靶向治疗药物表皮生长因子受体(EGFR)抑制剂西妥昔单抗的问世,结直肠癌的治疗进入了靶向治疗时代。近年来,多项临床研究表明:KRAS基因突变状态与转移性结直肠癌抗EGFR单抗治疗的疗效密切相关。许多国家已经将KRAS基因突变作为是否选择EGFR抑制剂治疗的必检指标。如何快速准确地检测KRAS基因突变,对个体化治疗显得尤为重要。     

高分辨率熔解曲线技术检测结直肠癌患者KRAS基因突变

目的探讨高分辨率熔解曲线技术(HRM)检测结直肠癌患者KRAS基因突变的可行性及其临床意义。方法采用HRM技术检测60例结直肠癌患者石蜡包埋组织标本KRAS基因2号外显子12和13位密码子突变,并与Sanger测序法检测结果进行对比分析。结果 HRM法检测结直肠癌患者KRAS基因2号外显子12和13位密码子突变检出率为36.67%(22/60),Sanger测序法检测突变检出率为33.33%(20/60)。HRM法检测突变检出率高于Sanger测序法,HRM法检测敏感性为100%,特异性为95.24%。结论 HRM法检测KRAS基因突变,灵敏度高,敏感性和特异性好,具有操作简便、节约时间、成本低的特点,方法可行。     

结直肠癌KRAS和BRAF基因突变分子病理流行病学研究

目的 探讨结直肠癌患者KRAS和BRAF基因突变、临床病理特征以及流行病因素的分子病理流行病学.方法 应用多重等位基因特异性PCR(ARMS-PCR)技术检测1997-12-01-2014-09-26中国医学科学院北京协和医学院肿瘤医院病理科确诊的945例结直肠癌患者KRAS和BRAF基因突变情况,分析性别、体质量指数...     

结直肠癌患者KRAS和BRAF基因突变检测及其临床意义

目的探讨结直肠癌患者组织中KRAS和BRAF基因突变情况,分析突变与临床病理特征的关系。方法应用荧光PCR-优化寡核苷酸探针法检测304例结直肠癌石蜡包埋标本中KRAS基因2号外显子的12和13密码子、BRAF基因的15号外显子的突变情况,分析KRAS和BRAF基因突变与临床病理特征的关系。结果 KRAS和BRAF基因在结直肠癌的突变率分别为38.8%(118/304)和4.3%(13/304)。KRAS基因突变阳性标本中12密码子的突变率为78.0%,其中p.G12D发生率最高,占总突变的45.3%;13密码子的突变率为22.0%。高年龄组(≥60岁)患者的KRAS基因突变率为47.2%(58/123),高于低年龄组(<60岁)的33.1%(60/181),差异有统计学意义(P<0.05)。转移性结直肠癌患者19例,其KRAS基因突变率为36.8%(7/19),与285例原发性结直肠癌患者的突变率(38.9%,111/285)差异无统计学意义(P>0.05)。BRAF基因在结肠、低分化、黏液腺癌患者中的突变率明显高于直肠、中或高分化和管状腺癌的患者。结论结直肠癌患者中KRAS基因突变的发生率较高,与年龄相关,而与性别、部位、病理类型和分化程度不相关。BRAF基因突变与肿瘤部位、病理类型和分化程度有关。原发性与转移性结直肠癌患者KRAS基因突变率无明显差异。     

结直肠癌KRA S基因突变阳性与P53及MCC基因的相关性研究

目的 探究结直肠癌KRAS基因突变阳性与P53基因及MCC基因的相关性研究.方法 回顾性研究2017年3月至2020年7月于桂林医学院第二附属医院确诊为结直肠癌患者89例,将其中26例KRAS突变阳性患者纳入突变组,63例KRAS突变阴性且无其他突变患者纳入未突变组.采用荧光定量PCR Taqman-ARMS探针法测定...     

结直肠癌组织中KRAS和BRAF基因突变与临床病理特征及预后的关系

目的:探讨结直肠癌组织中KRAS和BRAF基因突变与临床病理和预后的关系.方法:采用扩增阻滞突变系统(ARMS)方法对134例结直肠癌组织进行回顾性分析.结果:134例CRC组织中KRAS基因突变率49.3％(66/134),第2外显子突变率42.5％(57/134),其中12及13密码子突变率分别为35.1％(47/...     

Association between KRAS mutation and lung metastasis in advanced colorectal cancer

Background:

KRAS mutations have been associated with lung metastases at diagnosis of metastatic colorectal cancer (mCRC), but the impact of this mutation on subsequent development of lung metastasis is unknown. We investigated KRAS mutation as a predictor of lung metastasis development.

Methods:

We retrospectively evaluated data from patients with mCRC whose tumour was tested for KRAS mutation from 2008 to 2010. The relationships of KRAS mutational status with time-to-lung metastasis (TTLM) and overall survival (OS) were analysed.

Results:

Of the 494 patients identified, 202 (41%) had tumours with KRAS mutation. KRAS mutations were associated with a shorter TTLM (median 15.2 vs 22.4 months; hazard ratio=1.40; P=0.002) and a two-fold greater odds of developing lung metastases during the disease course in patients with liver-limited mCRC at diagnosis (72 vs 56%, P=0.007). Overall survival did not differ by KRAS status.

Conclusions:

Lung metastasis was more likely to develop during the disease course in patients whose tumour had a KRAS mutation than in those whose tumour did not have a KRAS mutation. This finding may have an impact on decision making for surgical resection of metastatic disease.     

Influence of KRAS mutation status in metachronous and synchronous metastatic colorectal adenocarcinoma

BACKGROUND:

Mutations in the v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) are present in approximately 30% to 40% of colorectal adenocarcinomas. Wild‐type (WT) KRAS mutation status is predictive of tumor response with epidermal growth factor receptor‐directed therapies, but the results from studies evaluating the prognostic value of KRAS status in localized disease have been contradictory. The prognostic value of KRAS in metastatic disease, specifically according to whether patients have synchronous or metachronous disease at presentation, is less understood.

METHODS:

One‐hundred ten consecutive patients with metastatic colorectal adenocarcinoma underwent testing for KRAS exon 2 mutations by polymerase chain reaction amplification and direct nucleotide sequencing. The clinical characteristics, treatments, and outcomes of these patients were then analyzed retrospectively, stratified according to whether patients presented with synchronous or metachronous metastasis and according to KRAS mutation status (WT or mutated).

RESULTS:

For the entire cohort, the median overall survival from the date of diagnosis of metastatic disease was 34.3 months (95% confidence interval, 28.3‐49.4 months) for patients with WT KRAS (n = 70). The median overall survival for patients with mutated KRAS (n = 40) was 40.3 months (95% confidence interval, 27.9‐51.1 months; log‐rank P = .91). Kaplan‐Meier survival analysis indicated that 3‐year overall survival and 5‐year overall survival were not statistically different. Within the subgroups of patients with synchronous and metachronous metastatic disease, no significant differences were observed in median overall survival, 3‐year overall survival, or 5‐year overall survival between the WT KRAS and mutated KRAS groups.

CONCLUSIONS:

In this study, KRAS mutation status did not influence overall survival in either synchronous or metachronous metastatic colorectal adenocarcinoma and, as such, had no prognostic role in this disease setting. Cancer 2012. © 2012 American Cancer Society.     

KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis

Background:

The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.

Methods:

After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.

Results:

HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan–Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.

Conclusions:

HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients.     

Relationship between expression of CEA,E-cadherin and liver metastasis in colorectal cancer

OBJECTIVE To investigate the expression of E-cadherin and CEA in serum in colorectal carcinoma and their relationship with liver metastasis.
METHODS CEA level was measured post-operatively by radioimmunoassay of 60 patients with colorectal cancer. Immunohistochemical analysis was used to evaluate the expression of E-cadherin.
RESULTS In liver metastasis group, 24 patients （24/26, 92.3%） were high level of CEA, but only 9 patients in non-liver metastasis group. The difference is significant （P = 0.004）. Expression of E-cadherin significantly correlated with differentiation, but was not associated with T stage or N stage. Liver metastatic rate in negative expression was higher than that in positive expression. And the survival analysis showed that time of liver metastasis was significant different in two groups （P 〈 0.05）.
CONCLUSION The expression of CEA in serum can be used to predict liver mestatasis of colorectal cancer after operation. E-cadherin, associated with tumor differentiation, is also a hopeful indicator for the prediction of liver metastasis in patients with colorectal cancer.