EGFR基因突变L861Q在非小细胞肺癌中的临床意义

发布时间：2018-04-02 09:30

本文选题：非小细胞肺癌　切入点：EGFR突变L861Q　出处：《北京协和医学院》2015年博士论文

【摘要】：研究背景非小细胞肺癌(NSCLC)由于其高发病率和高病死率成为世界性难题,近年来出现的靶向治疗为NSCLC患者提供新的治疗思路。表皮生长因子受体(EGFR)突变是靶向治疗是否有效的关键所在,酪氨酸激酶抑制剂(TKIs)可以延长常见敏感突变患者(del-19, L858R)的非疾病进展生存期(PFS)。然而EGFR罕见突变L861Q对靶向TKIs药物治疗敏感性尚不明确,需要进一步研究。研究目的1 基础实验方面,通过取得大量纯度较高的EGFR野生型和各突变型目的蛋白,体外分析小分子TKIs药物与EGFR突变蛋白的结合情况,探索EGFR突变蛋白的激酶活性以及小分子药物抑制效果。2 临床随访方面,探索L861Q突变患者临床特点和预后情况,并分析该突变患者对TKIs药物靶向治疗的敏感性。研究方法1 基础实验部分：通过NCBI检索获得野生型EGFR蛋白696-1022区域对应的截短体基因,PCR扩增后连接至载体质粒。设计各突变蛋白基因引物,PCR获得含有本研究目的基因的重组质粒。之后利用Bac-to-Bac杆状病毒表达系统将目的基因重组至昆虫真核细胞sf9中,大量传代培养sf9细胞,之后破碎细胞获得目的蛋白,通过镍柱纯化和分子筛层析纯化,最终获得大量纯度高的野生型和各突变性EGFR目的蛋白。下一步,利用取得的目的蛋白进行体外SPR实验初步分析小分子TKIs药物与EGFR目的蛋白的亲和力情况。之后通过蛋白激酶实验分析EGFR目的蛋白的激酶活性及TKIs药物的抑制效果。2 临床随访部分：回顾性收集全国范围内5125例在2009-2012年接受基因突变检测的肺癌患者和北京协和医院489例在2009-2014年接受基因突变检测的肺癌患者,最终确定了6例含有L861Q突变的患者。针对6例患者收集临床资料,包括性别、年龄、吸烟史、病理类型、肿瘤分期、治疗情况等,并随访生存期。最后分析L861Q突变患者的临床特点和靶向治疗敏感性。研究结果1 基础实验部分：经过提纯浓缩获得了500ul浓度2.5mg/ml的野生型EGFR蛋白、500ul浓度200ug/ml的EGFR-L858R突变蛋白、800ul浓度500ug/ml的EGFR-L861Q突变蛋白、500ul浓度315ug/ml的EGFR-L861Q/G719A联合突变蛋白。之后的SPR实验未能拟合计算出亲和力常数。激酶实验部分证实野生型和各突变型EGFR均具有激酶活性,且活性从高到低依次为：L858RL861Q/G719A L861QG719AWT。2 临床随访部分：全国范围的5125例肺癌患者和北京协和医院随访的489例肺癌患者中,EGFR突变率分别为36.2%(1854/5125)和44.2%(216/489),其中联合突变比例分别为7.2%(160/2208)和6.02%(13/216)。本研究的L861Q突变占总突变的比例分别为0.27%(5/1854)和0.46%(1/216),且所有L861Q突变均以联合突变形式出现,L861Q联合突变比例为100%。其中最常见的联合突变为L861Q和G719A联合,占66.7%(4/6)。L861Q患者初期使用吉非替尼治疗有效,肿瘤稳定无进展,4-5个月出现耐药,改用厄洛替尼无效。结论1 成功获得大量纯度高的EGFR野生型及研究的各突变型目的蛋白。2 本研究EGFR-L861Q突变蛋白具有激酶活性,L861Q突变为致癌突变；各目的蛋白激酶活性从高到低为：L858RL861Q/G719AL861QG719AWT。3 EGFR-L861Q突变患者初期吉非替尼靶向治疗有效,但很快出现耐药(4-5个月),之后改用厄洛替尼无效。
[Abstract]:The research background of non-small cell lung cancer (NSCLC) due to its high incidence and high mortality rate has become a worldwide problem, in recent years the emergence of targeted therapy to provide new therapy of NSCLC patients. The epidermal growth factor receptor (EGFR) mutation is targeting key treatment is effective, tyrosine kinase inhibitors (TKIs) can extended common sensitive mutations (del-19, L858R) - progression free survival (PFS). However, EGFR L861Q of rare mutations targeting drug sensitivity of TKIs treatment is not clear, the need for further research. The purpose of the study of 1 basic experimental aspects, made by high purity EGFR wild type and the mutant protein. In vitro analysis combined with drugs and small molecule EGFR TKIs mutant protein, explore the EGFR mutant protein kinase activity and the inhibitory effect of.2 small molecule drugs clinical follow-up, to explore the mutation of L861Q patients The characteristics and prognosis, and to analyze the mutations of TKIs targeted drug sensitivity. Methods 1 basic experimental part: through NCBI retrieve the truncated gene corresponding to wild-type EGFR protein region 696-1022, amplified by PCR and connected to the plasmid. The design of each mutant protein gene primers containing the recombinant plasmid PCR objective to study the gene. After using Bac-to-Bac baculovirus expression system to recombinant eukaryotic cells to insect Sf9, a large number of cultured Sf9 cells were obtained after crushing protein cells, chromatography and molecular sieve purification by nickel column, finally get the wild type with high purity and the mutagenicity of EGFR protein. Step SPR in vitro experiment preliminary affinity analysis of small molecule drugs and EGFR TKIs protein using protein. The protein kinase. Experimental analysis of EGFR protein kinase activity and the inhibitory effect of.2 TKIs drug clinical follow-up part: retrospectively collected 5125 cases nationwide in 2009-2012 years to accept the detection of gene mutation in patients with lung cancer and 489 cases in Peking Union Medical College Hospital in 2009-2014 years for gene mutation assay in patients with lung cancer, and ultimately determine the 6 patients with the L861Q mutation. In 6 cases of patients with clinical data were collected, including gender, age, smoking history, pathological type, tumor staging, treatment, and survival analysis. Finally L861Q mutation of clinical characteristics and target patients to treatment sensitivity. Results of the 1 basic experimental part: after purification and concentration to obtain 500ul concentration of 2.5mg/ml wild type EGFR protein, 500ul concentration of 200ug/ml EGFR-L858R mutant protein, the concentration of 800ul 500ug/ml EGFR-L861Q mutant protein, the concentration of 500ul 315ug/ml EGFR-L861Q/G719A combined process Change of protein. SPR experiments failed to calculate the affinity constant after fitting. The experimental part confirmed that wild-type and kinase of the mutant EGFR have kinase activity, and the activity from high to low is: L858RL861Q/G719A L861QG719AWT.2: clinical follow-up of 489 lung cancer patients nationwide 5125 lung cancer patients and follow-up in Peking Union Medical College Hospital, EGFR mutation rates were 36.2% (1854/5125) and 44.2% (216/489), which combined mutation rates were 7.2% (160/2208) and 6.02% (13/216). The L861Q mutation of total mutation rates were 0.27% (5/1854) and 0.46% (1/216), and all the L861Q mutation was combined with mutation form, L861Q the mutation ratio of 100%. one of the most common mutation for L861Q and G719A, accounting for 66.7% (4/6).L861Q in patients with early use of gefitinib treatment is effective, stable tumor progression occurred in 4-5 months Resistance to erlotinib is invalid. Conclusion 1 successfully obtained a large number of wild type EGFR and study the high purity of the mutant protein the.2 mutant EGFR-L861Q protein with kinase activity, L861Q mutations for oncogenic mutations; the protein kinase activity from high to low: L858RL861Q/G719AL861QG719AWT.3 EGFR-L861Q mutation in patients with early gefitinib target to effective treatment, but soon the emergence of resistant (4-5 months), after the use of erlotinib is invalid.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

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