非小细胞肺癌中EGFR和K_ras基因突变与蛋白表达相关性的研究_栾焕玲

非小细胞肺癌中EGFR和K-ras基因突变

与蛋白表达相关性的研究

　　［摘要］　背景与目的：当前表皮生长因子受体（epidermal growth factor receptor, EGFR）基因靶向治疗已成为晚期非小细胞肺癌（non-small cell lung cancer，NSCLC）治疗的研究的热点之一。本研究旨在探讨NSCLC患者中EGFR基因和K-ras（Kirsten rat sarcoma viral oncogene）基因突变的情况，分析其与蛋白表达的相关性及对吉非替尼（gefitinib）治疗的指导意义。方法：收集200例NSCLC患者新鲜组织，采用基因测序法检测EGFR及K-ras基因突变的状态；同时采用免疫组织化学法检测EGFR和K-ras蛋白的表达。结果：200例患者中，EGFR基因突变率为33%，主要发生在19号和21号外显子；K-ras基因突变率为5.5%，主要位于第12密码子；不存在同时携带有EGFR和K-ras两种基因突变的病例。腺癌尤其是含细支气管肺泡癌（bronchioloalveolar carcinoma，BAC）病理分型的患者、非吸烟患者和女性患者EGFR突变率较高。EGFR蛋白表达阳性率为16%，与总的EGFR突变无关（P>0.05），但与19号外显子突变具有统计学意义（P<0.05）。K-ras蛋白表达阳性率为52.5%，与K-ras基因突变无关（P>0.05）。15例携带有EGFR基因突变的NSCLC患者服用吉非替尼，12例（其中8例为EGFR蛋白表达阴性）有效。结论：EGFR蛋白表达与EGFR基因19号外显子突变具有一定相关性；联合检测EGFR和K-ras基因突变可用来筛选对EGFR酪氨酸激酶抑制剂（tyrosine kinase inhibitor，TKI）治疗敏感的人群，并预测吉非替尼治疗晚期NSCLC的疗效及预后。

　　［关键词］　表皮生长因子受体；　K-ras；　基因突变；　非小细胞肺癌；　蛋白表达；　EGFR酪氨酸激酶抑制剂

　　中图分类号：R734.2　　文献标识码：A　　文章编号：1007-3639(2010)07-0486-05

Study of the correlation of EGFR and K-ras gene mutations with its protein expression in non-small cell lung cancer LUAN Huan-ling, SUN Lei-na, DONG Na, GUO Yan, SUN Bao-cun, ZHAN Zhong-li (Department of Pathology of Tianjin Cancer Hospital, Tianjin Medical University, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin Diagnosis and Therapy Center of Lung Cancer, Tianjin 300060, China)

Correspondence to：ZHAN Zhong-li　E-mail：hyunsungsun@http://

　　［Abstract］ Background and purpose：EGFR (epidermal growth factor receptor) gene targeted therapy has been used for advanced NSCLC (non-small cell lung cancer). This study investigated gene mutations of EGFR and K-ras (Kirsten rat sarcoma viral oncogene) in Chinese patients with NSCLC, analyzed the correlation with its protein expression and its clinical signify cancer after the treatment of gefitinib. Methods：Gene sequencing was used to detect the EGFR and K-ras gene mutations status, immunohistochemistry was used to detect EGFR and K-ras protein expression. Results：The frequency of EGFR mutations, which were mainly located in exon 19 and exon 21, was 33%. The frequency of K-ras mutations was 5.5%, which were mainly located in codon 12. There was no instance of which both had EGFR and K-ras mutations occurring simultaneously. EGFR mutations were more common in adenocarcinomas (particularly those with bronchioloalveolar features), non smokers and females. 16% of patients were detected with EGFR positive expression and had no correlation with EGFR mutation (P>0.05), but were signi cantly correlated with mutation in exon 19 (P<0.05). The frequency of K-ras positive expression was 52.5% and had no correlation with K-ras mutation (P>0.05). 12 (8 cases were protein-negative) out of 15 ge tinib-treated NSCLC patients

通讯作者：战忠利　E-mail:hyunsungsun@http://

under disease control carried EGFR mutations. Conclusion：EGFR protein expression has some correlation with exon 19 mutations. Combined detection of EGFR and K-ras gene mutations can help determined whether the patients may bene t more from EGFR tyrosine kinase inhibitor (EGFR-TKI) and also help predict the response and prognosis of ge tinib.

　［Key words］　epidermal growth factor receptor; Kirsten rat sarcoma viral oncogene; gene mutation; non-small cell lung cancer; protein expression; EGFR tyrosine kinase inhibitor

　　近年来，肺癌的发病率和死亡率迅速上升，已跃居我国甚至世界恶性肿瘤的首位。虽然当前治疗技术有了很大提高，但5年生存率并没有明显改善。研究证实表皮生长因子受体（epidermal growth factor receptor，EGFR）的酪氨酸激酶抑制剂（tyrosine kinase inhibi-tor，TKI）在非小细胞肺癌（non-small cell lung cancer，NSCLC），尤其在肺腺癌的治疗中已取得较好的疗效，但肺癌患者对EGFR-TKI的敏感性与EGFR基因突变密切相关［1］。对于EGFR蛋白表达水平与EGFR-TKI敏感人群的选择，以及与EGFR基因突变的相关性目前尚存在争议［2-3］。K-ras基因是EGFR途径下游的一个重要的信号分子，它的突变可以导致EGFR-TKI原发性耐药，是预后不良的指标。而目前关于K-ras蛋白表达与基因突变相关性研究还很少，两者之间的相关性有待进一步探讨。

琼脂糖凝胶鉴定DNA样本进行质量检查以及浓度估计。

　　采用PCR方法扩增EGFR基因的4个外显子（18、19、20和21）和K-ras基因第2外显子，引物序列见表1。PCR反应体系总体积为20 μL：1’ HotStar-Taq缓冲液，2.0 mmol/L Mg2+，0.2 mmol/LdNTP，上、下游引物各0.2 μmol/L，HotStarTaq聚合酶（Qiagen Inc.）1 u和10 ng模板DNA。PCR反应条件为：94 ℃ 15 s、56 ℃ 30 s、72 ℃ 1 min，共35个循环，最后72 ℃延伸2 min。PCR产物经纯化后，用ABI 3130XL测序仪进行序列分析（基因测序工作由 …… 此处隐藏：9814字，全部文档内容请下载后查看。喜欢就下载吧 ……