非小细胞肺癌表皮生长因子受体P848L突变及其功能研究

目的:检测一组中国非小细胞肺癌(non-small-cell lung cancer,NSCLC)患者的表皮生长因子受体(epidermal growth factor receptor,EGFR)突变情况,探讨检出的1例EGFR突变类型P848L的功能特征。方法:收集55例手术或活检确诊的NSCLC标本,抽提细胞DNA,对EGFR的1~27号外显子进行筛查以检出突变情况;利用EGFR野生型全长基因载体,通过定点诱变技术构建出P848L突变型,进一步构建出P848L突变与T790M突变的双重突变体,分别转入293T细胞,观察细胞EGFR磷酸化活性及EGFR靶向抑制剂吉非替尼对其磷酸化的抑制作用。结果:共发现8例NSCLC携带EGFR突变,均为腺癌患者,占非吸烟腺癌患者的34.78%(8/23);其中1例NSCLC患者携带P848L突变体的细胞对吉非替尼的抑制反应与野生型相近,低于经典型突变体;当引入T790M形成双重突变后,对吉非替尼的反应性显著下降。结论: EGFR激活突变在非吸烟的肺腺癌患者具有较高的发生率,而在鳞状细胞癌等类型未发现;P848L突变不能增强吉非替尼对EGFR受体酪氨酸磷酸化的抑制效应,在携带P848L突变的EGFR基因上,引进耐药的T790M突变,能完全抑制其对吉非替尼的敏感性。

Epidermal growth factor receptor P848L mutation and its function in non-small-cell lung cancer

Objective：To determine the mutation status of epidermal growth factor receptor（EGFR） in chinese non-small-cell lung cancer（NSCLC） patients,and after detected a rare mutant type of EGFR,P848L,to further study its functional feature.Methods：Fiftyfive NSCLC samples were enrolled in the study.The total DNA was extracted,then the 1-27 exons were sequenced for the detection of mutation.The P848L mutant EGFR construct and then the T790M mutation was generated by introducing a point mutation into the wildtype EGFR vector using a site-directed mutagenesis kit.Results：There were 8 cases of mutations being detected,all the mutationharboring patients were non-smoker with pathologic type of adenocarcinoma.In vitro study demonstrated that the P848L mutant had a similar response to gefitinib treatment,and P848L and T790M double mutant did respond to gefitinib.Conclusions：EGFR mutations occurred in only non-smoker patients with lung adenocarcinoma in this series.P848L mutant EGFR has a similar response as the widetype to gefitinib treatment,while double mutated with T790M diminished the response completely.