【作者】 张新勇；

【导师】 徐丽焱； 汪蕙；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 肿瘤内科， 2007， 硕士

【摘要】 背景: EGFR酪氨酸激酶抑制剂Iressa和Tarceva以表皮生长因子受体(Epithelial Growth Factor Receptor, EGFR)为作用靶点,通过与ATP竞争结合于ATP结合位点而抑制EGFR活性,是目前晚期非小细胞肺癌(Non-small cell lung cancer,NSCLC)治疗研究中的热点。研究表明,Iressa治疗效果显著的患者中EGFR基因突变的发生率明显高于治疗无效者。在有EGFR基因突变的患者中Iressa的肿瘤客观缓解率明显高于有野生型EGFR患者。经治疗的有EGFR基因突变的患者总体生存期较有野生型EGFR患者的总体生存期有增高趋势。EGFR基因突变部位90%左右集中在编码酪氨酸激酶区的19、21外显子,为杂合突变。目前检测EGFR基因突变的方法主要是直接测序,费时,费用较高,不适合用于临床筛查。建立适合临床筛查常见EGFR基因突变的方法,筛选出可能从Iressa或Tarceva治疗中受益的NSCLC患者,从而实现个体化治疗,具有重要的临床意义。目的:本研究对肺泡细胞癌和接受Iressa、Tarceva的晚期NSCLC患者的肿瘤病理组织进行了EGFR基因19、21外显子突变检测,旨在建立EGFR基因常见突变的检测方法,了解EGFR在NSCLC的突变率和该突变与EGFR酪氨酸激酶抑制剂疗效的相关性。方法:本研究回顾性地收集了55例肺泡细胞癌、34例接受Iressa单药治疗和25例接受Tarceva单药治疗的晚期NSCLC患者的病理组织蜡块。用巢式PCR加聚丙烯酰胺凝胶检测EGFR 19外显子突变,用PCR-RFLP检测EGFR 21外显子突变,均用直接测序进行验证。结果: 55例肺泡细胞癌标本中共检测出EGFR基因突变24例,突变率43.6%。59例NSCLC标本中共检测出22例标本中有EGFR基因突变,突变率为37.3%。EGFR基因突变为19外显子的框内缺失突变,和21外显子的错义突变(L858R)。有EGFR基因突变的22例NSCLC患者中7例有19外显子的框内缺失突变,13例有21外显子的L858R,2例同时有该两种突变。EGFR基因突变率在女性、腺癌、不吸烟患者中高。有EGFR基因突变的患者的接受酪氨酸激酶抑制剂治疗的有效率高于无突变患者(41.7%vs15%,p=0.012)。亚组分析Iressa治疗组中EGFR基因突变与治疗有效率相关(p=0.016),但在Tarceva治疗组中突变与治疗有效率没有统计学意义(p=0.063),尚不能认为该突变与Tarceva的疗效相关。有EGFR基因突变的患者的疾病控制率高于无突变患者(86.4% vs 54.1%,p=0.011),在Tarceva治疗组中突变与疾病控制率相关(p=0.027),但Iressa治疗组中突变与疾病控制率间没有统计学意义,尚不能认为该突变与Iressa的疗效相关。结论:有EGFR基因突变的晚期NSCLC接受EGFR酪氨酸激酶抑制剂(Iressa、Tarceva)治疗的有效率和疾病控制率高于无EGFR基因突变者,并有统计学意义。用巢式PCR加聚丙烯酰胺凝胶检测EGFR 19外显子突变,用PCR-RFLP检测EGFR 21外显子突变,经直接测序验证,是较为准确、快速、简便的检验EGFR基因常见突变的方法。更多还原

【Abstract】 Background: Epidermal growth factor receptor(EGFR) is an important molecule target in targeted therapy in non-small-cell lung cancer(NSCLC).EGFR tyrosine kinase inhibitors Iressa (gefitinib)and Tarceva(erlotinib) block the activity of EGFR by competing ATP biding the ATP-bingding pocket on EGFR ,EGFR as their targets,and they are hotspots in the field of treatment of advanced NSCLC .Researches had reported that the mutation rate of EGFR gene was much higher in Iressa responsers than in non-responders.The tumor relief rate was much higher in patients with EGFR mutions than in those without EGFR mutations.The overall survival of NSCLC patients with EGFR mutations had a longer trend than that of patients without mutations, but had no stastical significance. About 90% of EGFR mutations were clusered within exon 19 and exon 21 of EGFR gene, all of which were heterozygous mutations and were activating mutations. Now the most common method to detect EGFR gene mutations was direct sequencing, expensive and time consuming, which didn’t fit clinical screening.Aim : The study was to evaluate the mutations in the exon 19 and exon 21 of EGFR gene in a series of Chinese patients with bronchioloalveolar carcinoma and advanced NSCLC and to evaluate the relationship between EGFR gene mutations and effects of Iressa and Tarceva in advanced NSCLC patients.Method: We collected paraffin-embedded tumor tissue samples from 59 patients with advanced NSCLC ,34 of which were treated with Iressa monotherapy and 25 of which were treated with Tarceva monotherapy, as well as 55 patients with bronchioloalveolar carcinoma. We detected mutations in exon 19 by nested PCR、PAGE electrophoresis and direct sequencing ,and detected mutations in exon 21 by PCR-RFLP method identified by direct sequencing.Results: The activating EGFR tyrosine kinase domain mutations was detected in 22 patients out of the 59 patients treated with Iressa or Tarceva(22/59,37.3%) and in 24 patients out of 55 patients with bronchioloalveolar carcinoma(24/55,43.6%). Mutations in exon 19 were in-frame deletions resulting in amino acid changes ,and mutations in exon 21 were mutation in exon 21(L858R) resulting in amino acid substitutions. From the patients receiving monotherapies, 7 patients had in-frame deletions in exon 19,13 patients had L858R,and 2 patients had both of them. The mutation rates were higher in females、non-smokers and patients with adenocarcinoma rather than in males、smokers and patients with non-adenocarcinoma respectively. The response rate was higher in patients with mutations than those without mutations and there was stastical significance(41.7% vs 15%, p=0.012). Patients with EGFR gene mutations had a higher response rate than those without mutations while receiving Iressa monotherapy and there was statistic significance(p=0.012).When treated with Tarceva the response rate seemed to be higher in patients with EGFR gene mutations than those without mutations, but there was no stastical significance(p=0.063) and we couldn’t conclude that EGFR gene mutations correspond to response of Tarceva. Patients with EGFR gene mutations had a higher disease control rate than those without mutations(86.4% vs 54.1%,p=0.011). Patients with EGFR gene mutations had a higher disease control rate than those without mutations while receiving Tarceva monotherapy and there was statistic significance(p=0.027).When treated with Iressa the disease control rate seemed to be higher in patients with EGFR gene mutations than those without mutations, but there was no stastical significance(p=0.153) and we couldn’t conclude that EGFR gene mutations correspond to disease control rate of Iressa.Conclusion:The activating mutations in exon 19 and exon 21 of EGFR gene was highly associated with the response rate and disease control rate of EGFR tyrosine kinase inhibitors(Iressa and Tarceva).Methods to detect deletion mutations in exon 19 of EGFR gene by nested PCR and PAGE identified by direct sequencing and to detect missense mutations in exon 21 of EGFR gene by PCR-RLFP method followed by direct sequencing were the fast、simple、correct ones to detect the common mutations in EGFR gene.更多还原