發(fā)布時間：2018-06-24 00:14

  本文選題：探討 + sARMS-PCR��； 參考：《安徽醫(yī)科大學》2015年碩士論文

【摘要】：背景每年全世界肺癌新發(fā)病例大約1200000,其中80%為非小細胞肺癌(Non-small cell lung cancer, NSCLC),而且大多數(shù)診斷時已處晚期。近20余年來,針對晚期NSCLC的個體化治療方法不斷發(fā)展,相應針對肺癌的靶向藥物種類也不斷增加,最具代表性的是針對肺癌EGFR、ALK等驅(qū)動基因改變帶來的精準治療增加。肺癌表皮因子受體(Epidermal Growth Factor Receptor,EGFR)突變的發(fā)現(xiàn)及隨后發(fā)展表皮生長因子受體絡氨酸激酶抑制劑(Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors,GRFR-TKIs)在延長EGFR突變?nèi)巳旱纳嫫诘耐瑫r也極大的改善患者生活質(zhì)量,其與臨床的相關(guān)性已經(jīng)得到很好認證。EGFR-TKIs藥物的作用機制是通過EGFR酪氨酸磷酸化來組織絲裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)信號轉(zhuǎn)導通路的啟動,終止腫瘤細胞生長,隨著EGFR基因突變檢測的逐漸普及和EGFR-TKIs藥物如吉非替尼、厄羅替尼在臨床的廣泛使用,臨床觀察發(fā)現(xiàn),雖然有部分患者存在EGFR基因突變,但對EGFR-TKIs的治療效果并不滿意。研究證實,這可能與RAS-RAF-絲裂原激活蛋白激酶的激酶(mitogen-activated protein kinase kinase,MEK)一細胞外信號調(diào)節(jié)激酶(extracel-lular-signal-regulated kinase,ERK)信號傳導系統(tǒng)的下游信號因子KRAS(Kirsten rat sarcoma viral oncogene homolog,鼠類肉瘤病毒癌基因同源基因)或BRAF(V-rafmurine sarcoma viral oncogene homologBl,鼠類肉瘤濾過性毒菌致癌同源體B1)基因突變部分相關(guān)。在NSCLC中,EGFR信號途徑最常檢測到的就是KRAS突變,其在NSCLC患者中的發(fā)生率約為20-30%,主要為吸煙者和腺癌病人。最近,最新的數(shù)據(jù)顯示KRAS突變是NSCLC患者預后不良的指標,而且與NSCLC患者對EGFR-TKIs靶向藥物的原發(fā)性耐藥有關(guān),部分KRAS基因野生型的NSCLC患者也會對EGFR-TKIs產(chǎn)生耐藥性,這主要是由于BRAF基因存在突變而導致,這就意味著是否給予患者行EGFR-TKIs治療僅僅檢測JEGFR基因突變可能是不夠的；因此在行EGFR基因突變檢測同時需行KRAS和BRAF基因檢測。目的建立針對非小細胞肺癌(non-small cell lung cancer, NSCLC)患者組織標本鼠類肉瘤病毒癌基因同源基因(Kirsten rat sarcoma viral oncogene homolog, KRAS)和鼠類肉瘤濾過性毒菌致癌同源體B1驅(qū)動基因(V-rafmurine sarcoma viral oncogene homologBl,BRAF)突變的特異引物雙擴增實時蝎形探針擴增阻滯突變系統(tǒng)(sARMS-PCR)檢測方法。了解NSCLC患者出現(xiàn)KRAS和BRAF突變的臨床、病理特征,為臨床治療藥物選擇提供依據(jù)；探討是否存在多驅(qū)動基因突變,為EGFR靶向藥物選擇提供依據(jù);方法總共收集我院2013年1月～2013年12月經(jīng)胸外科手術(shù)后或穿刺標本89例,病理均確診為非小細胞肺癌,系腫瘤組織甲醛固定石蠟包埋標本(Formalin-fixed paraffin-embedded,FFPE),采用FFPE樣品DNA分離試劑盒(離心柱型,Amoy Diagnostics, Xiamen, China)提取DNA,使用sARMS-PCR檢測方法(廈門艾德生物醫(yī)藥科技有限公司)檢測KRAS和BRAF基因突變。結(jié)果89例非小細胞肺癌患者手術(shù)或穿刺FFPE標本中,檢出KRAS基因突變21例(21/89),突變率23.6%；其中KRAS基因7種熱點突變中,檢出6種熱點突變,常見的突變區(qū)域集中在G12區(qū)：G12A與G12D檢出6例,G12C檢出5例均為高發(fā)區(qū),而G12V只與G12D或G12C各聯(lián)合出現(xiàn)1例,未見單獨存在,G13區(qū)僅有G13D存在。KRAS基因突變好發(fā)于男性,男性檢出率為31.5%(17/54),女性檢出率為12.9%(4/31),兩者比較具有明顯差異(P=0.030)。BRAF基因突變1例(1/89),突變率1.12%,突變位點為V600E,為女性、粘液腺癌；同一患者標本中未見KRAS和BRAF基因同時突變現(xiàn)象。結(jié)論利用sARMS-PCR技術(shù)針對NSCLC患者組織學標本KRAS、BRAF突變進行檢測,該方法有取材方便,檢測穩(wěn)定可靠的優(yōu)點；KRAS基因突變與性別有相關(guān)性(p0.05)；與年齡,臨床分期,吸煙等臨床病例特征無明顯相關(guān)性(p0.05);BRAF基因突變因例數(shù)太少未能觀察與性別,年齡,吸煙及分期的相關(guān)性。以早期為主的標本中(73/89,82.0%)同一患者標本中只發(fā)現(xiàn)KRAS存在復合突變(G12V與G12D或G12C-突變),未見與BRAF同時雙突變現(xiàn)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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R734.2

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相關(guān)期刊論文 前1條

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本文編號：2059086