探讨sARMS-PCR检测肺腺癌肿瘤组织KRAS、BRAF基因突变

发布时间：2018-06-24 00:14

本文选题：探讨 + sARMS-PCR　；参考：《安徽医科大学》2015年硕士论文

【摘要】：背景每年全世界肺癌新发病例大约1200000,其中80%为非小细胞肺癌(Non-small cell lung cancer, NSCLC),而且大多数诊断时已处晚期。近20余年来,针对晚期NSCLC的个体化治疗方法不断发展,相应针对肺癌的靶向药物种类也不断增加,最具代表性的是针对肺癌EGFR、ALK等驱动基因改变带来的精准治疗增加。肺癌表皮因子受体(Epidermal Growth Factor Receptor,EGFR)突变的发现及随后发展表皮生长因子受体络氨酸激酶抑制剂(Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors,GRFR-TKIs)在延长EGFR突变人群的生存期的同时也极大的改善患者生活质量,其与临床的相关性已经得到很好认证。EGFR-TKIs药物的作用机制是通过EGFR酪氨酸磷酸化来组织丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)信号转导通路的启动,终止肿瘤细胞生长,随着EGFR基因突变检测的逐渐普及和EGFR-TKIs药物如吉非替尼、厄罗替尼在临床的广泛使用,临床观察发现,虽然有部分患者存在EGFR基因突变,但对EGFR-TKIs的治疗效果并不满意。研究证实,这可能与RAS-RAF-丝裂原激活蛋白激酶的激酶(mitogen-activated protein kinase kinase,MEK)一细胞外信号调节激酶(extracel-lular-signal-regulated kinase,ERK)信号传导系统的下游信号因子KRAS(Kirsten rat sarcoma viral oncogene homolog,鼠类肉瘤病毒癌基因同源基因)或BRAF(V-rafmurine sarcoma viral oncogene homologBl,鼠类肉瘤滤过性毒菌致癌同源体B1)基因突变部分相关。在NSCLC中,EGFR信号途径最常检测到的就是KRAS突变,其在NSCLC患者中的发生率约为20-30%,主要为吸烟者和腺癌病人。最近,最新的数据显示KRAS突变是NSCLC患者预后不良的指标,而且与NSCLC患者对EGFR-TKIs靶向药物的原发性耐药有关,部分KRAS基因野生型的NSCLC患者也会对EGFR-TKIs产生耐药性,这主要是由于BRAF基因存在突变而导致,这就意味着是否给予患者行EGFR-TKIs治疗仅仅检测JEGFR基因突变可能是不够的；因此在行EGFR基因突变检测同时需行KRAS和BRAF基因检测。目的建立针对非小细胞肺癌(non-small cell lung cancer, NSCLC)患者组织标本鼠类肉瘤病毒癌基因同源基因(Kirsten rat sarcoma viral oncogene homolog, KRAS)和鼠类肉瘤滤过性毒菌致癌同源体B1驱动基因(V-rafmurine sarcoma viral oncogene homologBl,BRAF)突变的特异引物双扩增实时蝎形探针扩增阻滞突变系统(sARMS-PCR)检测方法。了解NSCLC患者出现KRAS和BRAF突变的临床、病理特征,为临床治疗药物选择提供依据；探讨是否存在多驱动基因突变,为EGFR靶向药物选择提供依据;方法总共收集我院2013年1月～2013年12月经胸外科手术后或穿刺标本89例,病理均确诊为非小细胞肺癌,系肿瘤组织甲醛固定石蜡包埋标本(Formalin-fixed paraffin-embedded,FFPE),采用FFPE样品DNA分离试剂盒(离心柱型,Amoy Diagnostics, Xiamen, China)提取DNA,使用sARMS-PCR检测方法(厦门艾德生物医药科技有限公司)检测KRAS和BRAF基因突变。结果89例非小细胞肺癌患者手术或穿刺FFPE标本中,检出KRAS基因突变21例(21/89),突变率23.6%；其中KRAS基因7种热点突变中,检出6种热点突变,常见的突变区域集中在G12区：G12A与G12D检出6例,G12C检出5例均为高发区,而G12V只与G12D或G12C各联合出现1例,未见单独存在,G13区仅有G13D存在。KRAS基因突变好发于男性,男性检出率为31.5%(17/54),女性检出率为12.9%(4/31),两者比较具有明显差异(P=0.030)。BRAF基因突变1例(1/89),突变率1.12%,突变位点为V600E,为女性、粘液腺癌；同一患者标本中未见KRAS和BRAF基因同时突变现象。结论利用sARMS-PCR技术针对NSCLC患者组织学标本KRAS、BRAF突变进行检测,该方法有取材方便,检测稳定可靠的优点；KRAS基因突变与性别有相关性(p0.05)；与年龄,临床分期,吸烟等临床病例特征无明显相关性(p0.05);BRAF基因突变因例数太少未能观察与性别,年龄,吸烟及分期的相关性。以早期为主的标本中(73/89,82.0%)同一患者标本中只发现KRAS存在复合突变(G12V与G12D或G12C-突变),未见与BRAF同时双突变现象。
[Abstract]:Background there are about 1200000 new cases of lung cancer in the world every year, of which 80% are Non-small cell lung cancer (NSCLC), and most of the diagnosis is in the late period. In the last 20 years, the individualized treatment method for advanced NSCLC has been developing continuously, and the corresponding target drugs for lung cancer are increasing, most representative Accurate treatment for lung cancer EGFR, ALK and other driven gene changes. The discovery of lung cancer epidermal factor receptor (Epidermal Growth Factor Receptor, EGFR) mutation and the subsequent development of the epidermal growth factor receptor (Epidermal Growth Factor Receptor-Tyrosine Kinase) (Epidermal Growth Factor Receptor-Tyrosine Kinase) The survival time of the mutant population also greatly improves the quality of life of the patient, and its clinical relevance has been well certified that the mechanism of the.EGFR-TKIs drug is through EGFR tyrosine phosphorylation to organize the initiation of the mitogen activated protein kinase (mitogen-activated protein kinases, MAPKs) signal transduction pathway and to terminate the tumor. Cell growth, with the gradual popularization of EGFR gene mutation detection and the extensive use of EGFR-TKIs drugs such as gefitinib and errotinib, clinical observations have found that although some of the patients have EGFR mutations, they are not satisfied with the therapeutic effect of EGFR-TKIs. This study has proved that this may be associated with the excitation of the RAS-RAF- mitogen activated protein kinase. Mitogen-activated protein kinase kinase (MEK) a downstream signal regulated kinase (extracel-lular-signal-regulated kinase, ERK) signal transduction system, the downstream signal factor KRAS (Kirsten rat sarcoma) Bl, the gene mutation is partly related to the oncogenic homologous B1 of mouse sarcomas. In NSCLC, the most frequently detected EGFR signal pathway is the KRAS mutation, and its incidence in NSCLC patients is about 20-30%, mainly smokers and adenocarcinoma. Recently, the latest data show that KRAS mutation is a poor prognostic indicator in NSCLC patients, and with N, and N. SCLC patients are related to the primary drug resistance of EGFR-TKIs targeted drugs, and some of the NSCLC patients in the wild type of KRAS gene may also have resistance to EGFR-TKIs, which is mainly due to the mutation of the BRAF gene, which means that it is not enough to give patients with EGFR-TKIs therapy only to detect the mutation of the JEGFR gene; therefore, EGFR is performed in EGFR. KRAS and BRAF genes need to be detected at the same time. Objective to establish the oncogene homologous gene of mouse sarcoma virus (Kirsten rat sarcoma viral oncogene) and mouse sarcomas carcinogenic carcinogenic homologous gene for non small cell lung cancer (non-small cell lung cancer, NSCLC). Rine sarcoma viral oncogene homologBl, BRAF) mutation specific primer double amplification in real-time scorpion probe amplification block mutation system (sARMS-PCR) detection method. To understand the clinical and pathological features of KRAS and BRAF mutations in NSCLC patients and to provide the basis for the selection of clinical drugs, and to explore whether there is a multi drive gene mutation for EGFR targeting. A total of 89 cases in the Department of thoracic surgery from January 2013 to 2013 in 12 menstruation or puncture specimens were collected in our hospital. All the cases were confirmed to be non small cell lung cancer (Formalin-fixed paraffin-embedded, FFPE), and the DNA separation kit of FFPE samples (centrifuge column, Amoy Diagnos) was used. Tics, Xiamen, China) extracted DNA and detected the mutation of KRAS and BRAF gene by sARMS-PCR detection method (Xiamen Eide medical science and Technology Co., Ltd.). Results in 89 cases of non small cell lung cancer, 21 cases of KRAS gene mutation (21/89) were detected, and the sudden change rate was 23.6%. Among them, 6 kinds of hot spots were detected among 7 hot spots of KRAS gene. Mutation, common mutation area concentrated in the G12 region: G12A and G12D detected 6 cases, G12C detection in 5 cases are high incidence area, and G12V only with G12D or G12C in the combination of 1 cases, no single existence, G13 region only G13D exist.KRAS gene mutation good hair in men, male detection rate is 31.5% (17/54), the female detection rate is 12.9% (4/31), both of the more clear 1 cases (P=0.030).BRAF gene mutation (1/89), the mutation rate was 1.12%, the mutation site was V600E, which was a female, mucous adenocarcinoma. There was no simultaneous mutation of KRAS and BRAF in the same specimen. Conclusion the sARMS-PCR technique was used to detect KRAS and BRAF mutations in the histologic specimens of NSCLC patients. The method was convenient and stable. The KRAS gene mutation was associated with sex (P0.05); there was no significant correlation with age, clinical staging, smoking and other clinical features (P0.05); the BRAF gene mutation was too few to observe the correlation with sex, age, smoking and staging. In the early specimens (73/89,82.0%), only KRA was found in the same patient. There was a compound mutation in S (G12V and G12D or G12C- mutation), and there was no simultaneous double mutation with BRAF.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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