Update on epidermal growth factor receptor mutations in non-small cell lung cancer.

In 2004, several investigators reported that somatic mutations in the epidermal growth factor receptor gene were associated with clinical responses to erlotinib and gefitinib in patients with non-small cell lung cancer. Since then, multiple groups have examined the biological properties that such mutations confer as well as the clinical relevance of these mutations in patients with non-small cell lung cancer. Although a tremendous amount of knowledge has been gained in the past 2 years, there remain a number of important epidemiologic, biological, and clinical questions.     

非小细胞肺癌原发灶与相应转移灶之间EGFR基因突变状况的不一致性研究

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)突变是晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者获益于酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)治疗的预测因子,本研究旨在探讨NSCLC原发灶与相应转移灶之间EGFR基因突变状况的不一致性。方法应用TaqManRT-PCR的方法检测35例病理确诊为NSCLC患者原发灶和相应转移灶的EGFR基因突变状况。结果原发肺癌病灶中有29例为EGFR基因突变型,余下6例为EGFR野生型。35例转移灶中18例为EGFR基因突变型,17例为EGFR野生型。35对配对标本中,11对(31.43%)标本出现原发灶EGFR基因突变,而转移灶为EGFR基因野生型,18对原发灶及转移灶均为EGFR基因突变型,且突变具体位点相同,6对原发灶及转移灶均为EGFR基因野生型。NSCLC原发灶与转移灶的EGFR基因表达不一致率为31.43%(11/35,P=0.008)。结论 NSCLC原发灶与转移灶的EGFR基因表达存在不一致性。     

Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features.

PURPOSE: This study evaluated the mutational profile of epidermal growth factor receptor (EGFR) and KRAS in non-small cell lung cancers in Hong Kong and determined their relation with smoking history and other clinicopathologic features. EXPERIMENTAL DESIGN: Mutational profile of exons 18 to 21 of EGFR and codons 12, 13, and 61 of KRAS were determined in 215 adenocarcinomas, 15 squamous cell (SCC), and 11 EBV-associated lymphoepithelioma-like carcinomas (LELC). RESULTS: EGFR mutations were prevalent in adenocarcinomas (115 of 215), uncommon in LELC (1 of 11), and not found in SCC (P < 0.001). Among adenocarcinomas, mutations were associated with nonsmokers (83 of 111; P < 0.001), female gender (87 of 131; P < 0.001), and well-differentiated (55 of 86) compared with poorly differentiated (11 of 41) tumors (P < 0.001). Decreasing mutation rates with increasing direct tobacco exposure was observed, with 74.8% (83 of 111) in nonsmokers, 61.1% (11 of 18) in passive, 35.7% (10 of 28) in previous, and 19.0% (11 of 58) in current smokers. There were 53% amino acid substitutions, 43% in-frame deletions, and 4% insertions. Complex patterns with 13% double mutations, including five novel substitutions, were observed. For KRAS, mutations occurred in adenocarcinoma only (21 of 215) and were associated with smokers (11 of 58; P = 0.003), men (14 of 84; P = 0.009) and poorly differentiated (7 of 41) compared with well-differentiated (4 of 86) tumors (P = 0.037). EGFR and KRAS mutations occurred in mutually exclusive tumors. Regression analysis showed smoking history was the significant determinant for both mutations, whereas gender was a confounding factor. CONCLUSION: This study shows EGFR mutations are prevalent in lung adenocarcinoma and suggests that it plays an increasing oncogenic role with decreasing direct tobacco damage.     

Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers have generated enormous interest, because they predict for sensitivity to TK inhibitors (TKIs). While mutational status is of great importance in determining response to TKIs, it is not the sole factor, and evidence is accumulating that EGFR gene amplification, other members of the EGFR family (HER2, HER3) and genes downstream of EGFR signaling (KRAS, BRAF), may be involved in cancer pathogenesis and the response of TKIs. EGFR mutations occur in highly selected subpopulations of lung cancer patients: adenocarcinoma histology, never-smoker status, East Asian ethnicity and female gender. The recent finding of "a resistance associated" mutation for TKIs also provides new insights into this complicated mechanism. Thus, molecular-based studies to analyze the biological functions and to assess TKI sensitivity depending on the type of mutations are required. Epidemiological studies to identify possible carcinogenic factor(s) affecting different subpopulations are also of interest. In addition, for optimal therapeutic approach a comprehensive understanding of the genes related to EGFR signaling pathway, including RAS/RAF/MAPK and PI3K-AKT pathways, are required.     

High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.

PURPOSE: Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non-small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non-small cell lung cancer. EXPERIMENTAL DESIGN: Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues. RESULTS: The 101 non-small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non-small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations. CONCLUSIONS: Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.     

Targeting the epidermal growth factor receptor in non-small cell lung cancer

The epidermal growth factor receptor (EGFR) has been implicated in a multiplicity of cancer-related signal transduction pathways like cellular proliferation, adhesion, migration, neoangiogenesis and apoptosis inhibition, all of them important features of cancerogenesis and tumour progression. The inhibition of this receptor has been discovered as a suitable pharmaceutical intervention aimed at interrupting tumour activity. In cancer, both monoclonal antibodies and small molecules with anti-tyrosine kinase activity have been assessed in several trials with significant efficacy in clinical applications. The current review focuses in particular on the clinical data of EGFR inhibition in non-small cell lung cancer with emphasis on tyrosine kinase inhibition.     

表皮生长因子受体与非小细胞肺癌预后之间的关系

目的探讨细胞膜及细胞核内表皮生长因子受体(EGFR)水平与非小细胞肺癌(NSCLC)预后之间的关系。方法选取2010年4月至2013年4月间南京市溧水区人民医院保存的60例非小细胞肺癌患者的病理档案,采用免疫组化的方法分析其病理组织切片中细胞膜及细胞核内EGFR及细胞周期蛋白D1(Cyclin D1)的表达。结果 39例(65.0%)样本细胞膜EGFR呈阳性,7例(11.7%)样本细胞膜EGFR呈强阳性;18例(30.0%)样本细胞核EGFR呈阳性和5例(8.3%)样本细胞核EGFR呈强阳性。Kaplan-Meier生存分析和Log-rank检验发现非小细胞肺癌患者细胞膜EGFR强阳性与总体生存率(OS)无明显相关,差异无统计学意义(P>0.05),细胞核EGFR强阳性组与总体生存率(OS)呈负相关,差异有统计学意义(P<0.01)。细胞核EGFR表达水平与Cyclin D1之间正相关,差异有统计学意义(P<0.05),细胞膜EGFR表达水平与Cyclin D1之间无明显相关,差异无统计学意义(P>0.05)。结论细胞核内EGFR水平与非小细胞肺癌患者的预后相关,核内EGFR强阳性者预后差。     

血清癌胚抗原与非小细胞肺癌患者EGFR突变的关系及其对预后的影响

  目的  探讨血清癌胚抗原（CEA）水平与非小细胞肺癌患者EGFR基因突变的关系及预测价值,分析CEA水平对肺癌患者术后生存期的影响。  方法  选取2009年3月至2011年3月于天津医科大学肿瘤医院肺部肿瘤科行外科手术治疗的患者387例,术前行CEA等肿瘤标志物检测,术后常规行EGFR基因突变检测,分析CEA等肿瘤标志物与EGFR突变关系及其与患者预后的关系。  结果  全组患者中出现EGFR基因突变的患者共168例,突变阳性率43.4%,EGFR突变在女性、非吸烟、腺癌以及年龄 < 60岁的患者中更加常见（P < 0.05）,另发现EGFR突变情况与肿瘤标志物及化疗敏感性指标相关,EGFR野生型患者的Cyfra21-1、SCC升高、ERCC1阳性表达更常见（P < 0.05）,而EGFR基因突变患者的CEA升高则更常见（P=0.015）,并且随着CEA水平的升高,EGFR基因突变阳性率更高（血清CEA水平 < 5 μg/L、5~20 μg/L及>20 μg/L 3组的阳性率分别为40.1%、47.5%和66.6%,P=0.003）。Logistic回归分析发现CEA水平是预测EGFR基因突变的独立因素,并且血清CEA水平也是影响非小细胞肺癌患者预后的独立因素（CEA水平升高组与正常组2年生存率分别为75.9%和88.4%,P < 0.01）。  结论  血清CEA水平高低可以预测手术切除的非小细胞肺癌患者的预后,并且与非小细胞患者EGFR突变相关,可以初步指导EGFR突变情况。      

The relationship of quantitative epidermal growth factor receptor expression in non-small cell lung cancer to long term survival.

Increased expression of epidermal growth factor receptor (EGFr) has been reported in non small cell lung cancers (NSCLC) when compared to normal lung. We have examined post-operative survival in 19 surgically treated patients with NSCLC who had full characterisation of EGFr on primary tumour membrane preparations from resection specimens. There were ten squamous, seven adeno and two large cell carcinomas. The median concentration of high affinity sites was 31 fmol per mg of protein (4-1532) and the median dissociation constant (Kd) of these high affinity sites was 2.3 x 10(-10) per mol (1.2-30 x 10(-10)). Seven patients survived over 5 years. Twelve patients died between 8.5 and 55 months from the time of surgery. When > 5 year survivors were compared to non-survivors there was no difference as regards tumour size or stage, or as regards age or sex. The survivors had a median concentration of high affinity EGFr sites of 16.1 fmol mg-1 protein compared to a median concentration of 68.6 fmol mg-1 protein in the non-survivors (P = 0.01 Wilcoxon test). No long term survivor had > 35 fmol mg-1 protein of receptor. Thus EGFr quantitation may give independent prognostic information in NSCLC and help to select patients for adjuvant therapy after surgery. These results need confirmation in a larger prospective study.     

Amplification of CRKL induces transformation and epidermal growth factor receptor inhibitor resistance in human non-small cell lung cancers

We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage-independent growth and tumorigenicity. Oncogenic CRKL activates the SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in epidermal growth factor receptor (EGFR)-mutant cells induces resistance to gefitinib by activating extracellular signal-regulated kinase and AKT signaling. We identified CRKL amplification in an EGFR inhibitor-treated lung adenocarcinoma that was not present before treatment. These observations demonstrate that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications, and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy. Significance: These studies credential CRKL as an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.     

Erlotinib-induced autophagy in epidermal growth factor receptor mutated non-small cell lung cancer

Purpose

Erlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC.

Methods

Four NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy.

Results

In line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure.

Conclusions

Erlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.     

Relation of epidermal growth factor receptor, phosphorylated-Akt, and hypoxia-inducible factor-1alpha in non-small cell lung cancers

The role of the epidermal growth factor receptor (EGFR)/Posphorylated-Akt (P-Akt) signaling axis in regulating hypoxia-inducible factor-1alpha (HIF-1alpha) expression in vivo is not well understood and is of potential clinical importance since the extent of hypoxia in the tumor environment is thought to be an important determination of resistance to chemotherapy and radiotherapy. We performed the immunohistochemical studies in 80 patients with non-small cell lung cancers to evaluate EGFR, P-Akt, and HIF-1alpha expression. Significant correlation between P-Akt and HIF-1alpha (P=0.0006) and marginal correlation between EGFR and P-Akt (P=0.066) were found. P-Akt was shown to be a poor prognostic factor as to patients with lymph node involvement (P=0.031).     

Epidermal growth factor receptor mutations in patients with non-small cell lung cancer

A year has passed since mutations of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) were discovered in patients with non-small cell lung cancer (NSCLC) who had dramatic clinical responses to treatment with gefitinib. Additional laboratory and clinical studies have provided further insight into the biological impact of EGFR mutations in cell culture experiments and in patients with NSCLC. In vitro characterizations of NSCLC cell lines and host cell lines transfected with these mutant and wild-type EGFR show that most cell lines with mutated EGFR are growth-inhibited by 10- to 100-fold lower concentrations of gefitinib and erlotinib compared with wild-type EGFR. NSCLC lines with mutations of the EGFR treated with concentrations of gefitinib and erlotinib that are achievable in the plasma undergo apoptosis rather than growth arrest. Retrospective studies of patients with NSCLC-treated gefitinib have reported a close association between EGFR mutations, increased chance of clinical response and longer survival. This review will provide information on the impact of EGFR mutations on gefitinib and erlotinib treatment by in vitro experiments, the outcome of NSCLC patients with these mutations when treated with gefitinib and erlotinib, and the subsets of patients with NSCLC in whom these mutations arise.     

扩增耐突变系统对非小细胞肺癌表皮生长因子受体基因突变的检测

目的:探讨扩增耐突变系统检测非小细胞肺癌(NSCLC)患者肿瘤组织EGFR基因突变的应用价值.方法:选取70例NSCLC患者的病理切片,提取基因组DNA,分别使用EGFR外显子19和21突变检测试剂盒检测EGFR外显子19和21的突变情况,并结合临床资料进行分析.结果:70例肺癌组织中检出EGFR基因突变29例,基因突变检出率为41.4％,其中外显子19突变20例(69.0％),外显子21突变9例(31.0％);腺癌基因突变23例(79.3％),检出率为51.1％;鳞癌基因突变6例(20.7％),检出率为24.0％;肺泡癌基因突变4例(13.8％),检出率为66.7％.女性、不吸烟和肺腺癌患者的EGFR 2个外显子基因突变率均较高,P＜0.01.结论:NSCLC患者EGFR基因突变主要表现为外显子19和21的突变,女性、肺腺癌和不吸烟的患者多见.     

非小细胞肺癌患者表皮生长因子受体Kras基因突变检测及与临床病理特征的相关性

目的分析非小细胞肺癌患者表皮生长因子受体(EGFR)、Kras基因突变的情况及其与临床病理学特征的关系。方法收集1110例非小细胞肺癌标本,采用实时荧光PCR法检测标本中EGFR基因第18、19、20和21号外显子及Kras基因第2号外显子12和13密码子的突变情况。结果非小细胞肺癌EGFR基因的突变率为46.1%(512/1 110),Kras基因的突变率为7.1%(79/1110)。EGFR基因突变主要发生在女性、不吸烟和腺癌的患者,突变位点主要集中在第19和21号外显子,第18、19、20和21号外显子突变所占比例分别为3.3%(17/520)、46.0%(239/520)和5.0%(26/520)、45.8%(238/520)。结论非小细胞肺癌患者EGFR基因突变主要发生于女性、不吸烟和腺癌的患者,采用实时荧光PCR法检测分析非小细胞肺癌患者EGFR、Kras基因突变的情况,有助于选择靶向治疗获益人群,为个性化治疗提供依据。     

TaqMan-MGB探针实时荧光聚合酶链反应快速检测非小细胞肺癌表皮生长因子受体基因突变

目的探讨非小细胞肺癌(NSCLC)瘤组织表皮生长因子受体(EGFR)基因突变及TaqMan MGB探针实时荧光PCR快速检测EGFR突变的诊断价值。方法应用聚合酶链(PCR)反应,对80例手术切除NSCLC瘤组织EGFR基因的第18、19和21外显子片段进行扩增和测序, Chromas软件分析基因突变。设计EGFR突变位点的TaqMan MGB探针,采用实时PCR检测瘤组织EGFR突变,并与测序结果比较。实时PCR的敏感性与特异性评价,用不同混合数量的PC-9细胞(19外显子缺失)为阳性参照。结果21例NSCLC瘤组织存在EGFR基因突变,总体突变率为26.3%。其中13例为EGFR第19外显子阅读框内多核苷酸的缺失,8例为第21外显子2573位核苷酸点突变。诊断的特异性与敏感性均为100%。当PC-9突变型细胞仅占10%时或PC-9细胞数低达50只时,PCR仍然检测到EGFR基因突变的存在。女性、不吸烟和肺腺癌患者EGFR基因突变率显著高于男性、吸烟和非腺癌患者(P<0.05)。EGFR基因突变与患者年龄、TNM分期等因素无关。结论NSCLC存在EGFR基因的突变或缺失,其中以女性、腺癌和不吸烟患者突变率较高。TaqMan MGB探针联合实时PCR可有效地检测出EGFR基因突变,操作简便,易于临床推广。