Optimal management of patients with non-small cell lung cancer and epidermal growth factor receptor mutations

In recent years, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, which have promising activity and a favourable toxicity profile, have been used in the management of advanced non-small cell lung cancer (NSCLC). The knowledge that EGFR-activating mutations confer sensitivity to EGFR TKIs has led to the design and analysis of phase II and III studies of gefitinib or erlotinib treatment in various clinical scenarios. We review the important NSCLC clinical trials of the efficacy of EGFR TKIs in the context of EGFR-activating mutations. In all phase II single-arm studies or phase III randomized comparative studies, EGFR TKIs as monotherapy were superior to combination chemotherapy in terms of response rate and progression-free survival in patients with activating EGFR mutations. EGFR TKIs have contributed to the superior overall survival time in NSCLC patients with EGFR mutations compared with those patients without EGFR mutations. The results of these studies have led to a paradigm shift in the treatment of patients with advanced NSCLC. NSCLC with EGFR mutations constitutes a new entity requiring different personalized treatment strategies.     

Correlation between serum CEA levels and EGFR mutations in Chinese nonsmokers with lung adenocarcinoma

Aim： To evaluate the relationship between epidermal growth factor receptor （EGFR） mutations and serum carcinoembryonic antigen （CEA） levels in Chinese nonsmokers with pulmonary adenocarcinoma. Methods： We sequenced exons 18-21 of the EGFR gene in 98 cases. The patients were divided into two groups based on their pre- treatment serum CEA levels （below or above 5 n~/mL） for analyzing the correlations with EGFR mutations. Results： Sixty-seven cases harbored EGFR mutations. The rates of EGFR mutations and exon 19 mutations in the high-CEA group （78.2% and 49.1%, respectively） were significantly higher than those in the Iow-CEA group （55.8% and 20.9%, respectively）. Serum CEA levels were found to be the only independent predictor of EGFR mutation （OR 2.837; 95% CI： 1.178-6.829） and exon 19 mutation （OR 3.618; 95% CI： 1.319-9.918）. Furthermore, a higher serum CEA level was associated with a higher EGFR mutation rate and a higher exon 19 mutation rate： patients with serum CEA levels 〈5 ng/mL, 〉5 and 〈20 ng/mL, 〉20 ng/mL showed the EGFR mutation rate of 55.8%, 74.1%, 82.1%, respectively, and the exon ：19 mutation rate of 20.9%, 40.7%, 57.1%, respectively. Patients with EGFR mutations displayed a significantly higher incidence of abnormal serum CEA levels （〉5 ng/mL） than patients without EGFR mutations （64.2% vs 38.7%）. Conclusion： Elevated serum CEA levels predict the presence of EGFR gene mutations in Chinese nonsmokers with pulmonary adenocarcinoma.     

23例非小细胞肺癌患者肺癌组织和血浆中EGFR外显子21的基因突变检测

目的证明可以从非小细胞肺癌患者血浆中检测到表皮生长因子受体(epidermal growth factorreceptor,EGFR)突变,且与肿瘤组织的突变情况相一致,建立一种从外周血液中快速、经济、准确检测EGFR外显子21基因突变的方法。方法同时取得23例非小细胞肺癌患者的肺癌组织和血浆,采用突变富集PCR方法检测外周血液中EGFR外显子21的基因突变,并将检测结果与肺癌组织的检测结果进行对比。结果 23例非小细胞肺癌患者的肺癌组织中共检测到8例EGFR外显子21突变,其中7例突变可以从患者的外周血液中检测到。结论可以从非小细胞肺癌患者外周血液中检测到EGFR突变,通过本方法,可以从外周血液中检测EGFR外显子21基因突变。     

突变体富集PCR法检测非小细胞肺癌病理组织EGFR基因突变

目的：建立突变体富集PCR法检测非小细胞肺癌病理组织中的EGFR基因突变。方法：选取55例细支气管肺泡癌和59例非小细胞肺癌病理组织蜡块，提取基因组DNA，采用不同的突变体富集PCR法（PCR-PAGE和PCR-RLFP）检测EGFR基因常见的19和21外显予突变，并经过直接测序验证。结果：在59例非小细胞肺癌中共检测出EGFR基因突变22例,突变率为37．3％（22／59）。55例细支气管肺泡癌中共检测出24例基因突变，突变率为43．6％（24／55）。经直接测序验证，EGFR19外显予有3种类型缺失突变。EGFR21外显予的错义突变为L858R。结论：突变体富集PCR法准确、快速、经济，便于临床筛查非小细胞肺癌病理组织中的EGFR基因突变。     

EGFR-TKI类药物治疗晚期非小细胞肺癌的研究进展

表皮生长因子受体酪氨酸激酶抑制剂（epithelial growth factor receptor tyrosine kinase inhibitors,EGFR-TKI）引领EGFR突变的晚期非小细胞肺癌精准治疗十多年,EGFR-TKI对EGFR常见突变的晚期非小细胞肺癌的疗效已得到明确的证实。然而对于EGFR罕见突变,EGFR-TKI的疗效在临床上存在众多争议。本文列举了目前已经进入临床阶段的EGFR-TKI类药物在EGFR突变阳性晚期非小细胞肺癌中的中位无进展及总生存期并进行比较,增加了EGFR罕见突变对EGFR-TKI疗效的相关研究,以期为临床EGFR-TKI的合理使用提供参考。     

Mechanisms of resistance to epidermal growth factor receptor tyrosine kinase inhibitors in patients with advanced non-small-cell lung cancer: clinical and molecular considerations

Non-Small-Cell Lung Cancer (NSCLC) with somatic mutations of the epidermal growth factor receptor (EGFR) is anticipated to respond to small-molecule tyrosine kinase inhibitors (TKIs) of the EGFR tyrosine kinase. There are, however, patients with EGFR mutated tumors who do not demonstrate tumor response. The most widely accepted mechanism of 'de novo' (inherent) resistance to these TKIs involves mutations of the KRAS gene. KRAS is a downstream mediator of EGFR-induced cell signaling, such mutations appear to be mutually exclusive from EGFR mutations in lung cancer. The first molecular modifier of resistance identified in patients who developed resistance (termed 'acquired resistance') to TK inhibition was a new acquired somatic EGFR mutation (T790M). Today there is an ever-growing series of molecular events that have recently come to the forefront to explain other instances of TKI resistance not attributable to T790M or KRAS. These include a number of molecules that interact with EGFR or form part of its downstream signaling pathway such as HER-2, IGFR-1, MET and B-RAF. Considering that the majority of studies carried out to date with respect to the identification of resistant clones have not used highly sensitive techniques (e.g. allelic discrimination to identify somatic mutations), coupled with the relatively low number of studies examining multiple molecular markers and the accepted molecular heterogeneity of NSCLC raise question as to the existence of 'acquired' versus 'de-novo' resistance. By examining the current knowledge base with respect to mechanisms of resistance to EGFR TKIs in NSCLC, we explore whether 'acquired' resistance is 'de-novo' resistance in disguise, and discuss the promises and limitations of molecular stratification with respect to strategies incorporating TKIs in the treatment of NSCLC.     

Lack of somatic mutations in EGFR tyrosine kinase domain in hepatocellular and nasopharyngeal carcinoma

Activating EGFR somatic mutations have been shown to predict treatment response to small molecules targeting the EGFR intracellular tyrosine kinase domain. Recent work on cell-lines and animal models had demonstrated an inhibitory effect of EGFR tyrosine kinase inhibitors in hepatocellular and nasopharyngeal carcinoma, and clinical trials in these tumour types are ongoing. There are few data on the presence or prevalence of EGFR mutations in hepatocellular and nasopharyngeal carcinomas. We studied exons 18-21 of the EGFR gene from 100 hepatocellular and 102 nasopharyngeal carcinomas, and found no exonic mutations of potential significance. Alternative mechanisms may be important for the observed activity of small molecule EGFR tyrosine kinase inhibitors in hepatocellular and nasopharyngeal carcinomas.     

Sanger法检测分析非小细胞肺癌患者组织EGFR

目的探讨Sanger法检测非小细胞肺癌（non-small cell lung cancer,NSCLC）患者组织表皮生长因子受体（epidermal growth factor receptor,EGFR）基因突变情况,为肺癌病人“个体化靶向分子治疗”提供依据。方法收集63例NSCLC患者组织标本（石蜡切片）,提取组织DNA,用EGFR外显子18、19、20和21四位点的分子扩增（PCR）试剂盒进行外显子扩增,扩增产物先后进行电泳鉴定、酶解,酶解产物PCR扩增、纯化,纯化产物用ABI-3130型基因分析仪检测得出EGFR外显子18、19、20、21的野生或突变状态结果,并分析EGFR基因突变和患者临床病理生理特征、临床靶向用药疗效的关系。结果测试显示肺腺癌患者EGFR基因19、21外显子的突变率为33．3％（21／63）,而EGFR基因的突变与患者的病理组织学类型、吸烟状态有关（P〈0．05）。同时EGFR突变患者能从临床靶向用药获益。结论Sanger法检测NSCLC突变情况可为临床靶向用药提供技术支持。     

Recent understanding of the molecular mechanisms for the efficacy and resistance of EGF receptor-specific tyrosine kinase inhibitors in non-small cell lung cancer

INTRODUCTION: The epidermal growth factor receptor (EGFR) and its family members are involved in many aspects of tumor biological processes. Aberrant activation of the EGFR tyrosine kinase by mutations or protein overexpression is observed in various types of human cancer, including lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, are highly effective in lung cancer patients who harbor active mutations in the EGFR gene. However, patients who are initially sensitive to EGFR-TKIs eventually relapse within few years. AREAS COVERED: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with a high frequency of EGFR mutations. This review describes the EGFR mutations that determine the sensitivity to EGFR-TKIs and the current understanding of the molecular mechanisms of acquired resistance to EGFR-TKIs in NSCLC. Furthermore, the authors describe recent strategies developed to overcome acquired resistance using second-generation EGFR-TKIs and combination therapies with several molecular-targeting drugs. EXPERT OPINION: Although recent findings have contributed to our understanding of the mechanism of acquired resistance and helped the development of novel strategies to overcome such resistance, the underlying mechanisms are complex and additional research is necessary to develop effective therapeutic strategies for individual patients with lung cancer.     

The epidermal growth factor receptor gene family as a target for therapeutic intervention in numerous cancers: what's genetics got to do with it?

Over the past 30 years, a relatively simple growth factor and its cognate receptor have provided seminal insights into the understanding of the genetic basis of cancer, as well as growth factor signalling. The epidermal growth factor (EGF), its cognate receptor (EGFR) and related family members have been shown to be important in normal, as well as the malignant growth of many cell types including: glioblastomata, astrocytomas, medulloblastomata, non-small cell lung carcinoma (NSCLC) and breast cancer. This review summarises the history of the EGFR gene and the v-ErbB oncogene, as well as diverse approaches developed to inhibit EGFR activity. The two most advanced therapies use either small-molecule cell membrane permeable kinase inhibitors or antibodies which prevent receptor activation. Recent clinical trials indicate that certain NSCLC patients have mutations in the EGFR gene which makes them more responsive to kinase inhibitors. These mutations appear to enhance the ability of the ligand to activate EGFR activity and also prolong the binding of the EGFR inhibitor to the kinase domain. Evidence to date suggests that these EGFR mutations in NSCLC occur more frequently in Japan than in the western hemisphere. Although these mutations are correlated with enhanced efficacy to the inhibitors in NSCLC, they can not explain or predict the sensitivity of many other cancer patients to the beneficial effects of the EGFR kinase inhibitors or antibody mediated therapy. As with as other small-molecule kinase inhibitors and susceptible diseases (e.g., imatinib and chronic myeloid leukaemia), resistance to EGFR inhibitors has been reported recently, documenting the requirement for development of multi-pronged therapeutic approaches. EGFR kinase inhibitors are also being evaluated as adjuvants in hormonal therapy of breast cancer - especially those which overexpress EGFR. Genetically engineered antibodies specific for the EGFR family member ErbB2 have been developed which show efficacy in the treatment of primary, and prevent the relapse of, breast cancer. Clearly, the EGF/EGFR signalling cascade has, and continues to play, an important role in the development of novel anticancer targeted therapies.     

Afatinib for the first-line treatment of patients with metastatic EGFR-positive NSCLC: a look at the data

Introduction: Epidermal growth factor receptor (EGFR) mutations are detected in about 10–15% of Caucasian and 30–40% of Asian patients with advanced or metastatic non-small-cell lung cancer (NSCLC). In patients harbouring EGFR mutations, the treatment with different available EGFR tyrosine kinase inhibitors (TKIs) showed to be more effective and safe than platinum-based chemotherapy regimens.

Areas covered: The current evidences about the role of afatinib for patients with EGFR-positive NSCLC are reviewed and discussed. We report a review based on a MEDLINE/PubMed, searched for randomized phase II or III trials evaluating afatinib in EGFR-positive NSCLC.

Expert commentary: Afatinib is the third EGFR TKI approved for the treatment of NSCLC harbouring EGFR mutations, showing high efficacy in this setting of patients.     

EGFR基因突变直接测序检测方法的建立

目的 建立一种适用于临床表皮生长因子受体（EGFR）基因直接测序突变检测方法.方法 以EGFR基因突变热点19、21外显子为突变检测靶位点设计特异性扩增、测序引物,以已知19、21外显子野生型、突变型样品分别做为阴、阳性对照品,建立EGFR基因突变直接测序检测方法,进行方法学评估.结果 成功建立了EGFR基因突变直接测序检测方法,该方法检测灵敏度为4.7 ng/μl,重复性良好.检测30例结非小细胞肺癌样品,突变率为26.7％.结论 本研究建立了可用于临床样品检测的EGFR基因突变直接测序检测方法.     

Detection of EGFR Somatic Mutations in Non-Small Cell Lung Cancer (NSCLC) Using a Novel Mutant-Enriched Liquidchip (MEL) Technology

We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.     

Rociletinib,a third generation EGFR tyrosine kinase inhibitor: current data and future directions

Introduction: Major advances have been made since the discovery of driver mutations and their targeted therapies, especially in the treatment of patients with epidermal growth factor receptor (EGFR) mutations. Despite their initial efficacy in the majority of the patients with such driver mutations, all targeted therapies are limited by the eventual development of resistance mechanisms.

Areas Covered: EGFR T790M mutation is a common resistance mechanism after treatment with first or second generation EGFR tyrosine kinase inhibitors (TKI). Rociletinib is one of the third generation EGFR TKIs with activity against T790M and activating EGFR mutations while sparing the wild-type EGFR. In this review, we discuss the current understanding and available data on rociletinib, including the side effects associated with the medication. We will also review the BEAMing plasma test to detect T790M mutation without the need for repeat biopsy. Lastly, we review the potential resistance mechanisms after progression on rociletinib, and future directions.

Expert Opinion: It is important to note that there are other 3^rd generation EGFR TKIs with activity against T790M already approved by the US FDA (osimertinib) and many others in development. Future research will focus on figuring out which patients can benefit the most from a particular medication with minimal side effects, and further resistance mechanisms after rociletinib.     

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.     

非小细胞肺癌患者外周血与组织中EGFR基因突变的研究

目的 检测非小细胞肺癌患者外周血与肿瘤组织EGFR基因突变率及两者的一致性,探讨外周血EGFR基因突变的临床应用价值.方法 应用实时荧光定量PCR方法检测453例非小细胞肺癌组织,229例非小细胞肺癌外周血,其中132例外周血标本和组织标本配对,检测其中EGFR基因突变,比较外周血和组织标本突变一致性,分析EGFR基因突变和患者临床特征相关性.结果 外周血标本中检测出66例突变(28.82％),组织中185例突变(40.84％),两种标本一致性为84.09％,差异有统计学意义(Kappa=0.652,P=0.000).结论 非小细胞肺癌患者外周血与组织EGFR基因突变一致性较高,在无法获得组织学标本时可以检测外周血EGFR基因状态,为非小细胞肺癌的临床诊疗提供帮助.     

The epidermal growth factor receptor gene family as a target for therapeutic intervention in numerous cancers: what’s genetics got to do with it?

Over the past 30 years, a relatively simple growth factor and its cognate receptor have provided seminal insights into the understanding of the genetic basis of cancer, as well as growth factor signalling. The epidermal growth factor (EGF), its cognate receptor (EGFR) and related family members have been shown to be important in normal, as well as the malignant growth of many cell types including: glioblastomata, astrocytomas, medulloblastomata, non-small cell lung carcinoma (NSCLC) and breast cancer. This review summarises the history of the EGFR gene and the v-ErbB oncogene, as well as diverse approaches developed to inhibit EGFR activity. The two most advanced therapies use either small-molecule cell membrane permeable kinase inhibitors or antibodies which prevent receptor activation. Recent clinical trials indicate that certain NSCLC patients have mutations in the EGFR gene which makes them more responsive to kinase inhibitors. These mutations appear to enhance the ability of the ligand to activate EGFR activity and also prolong the binding of the EGFR inhibitor to the kinase domain. Evidence to date suggests that these EGFR mutations in NSCLC occur more frequently in Japan than in the western hemisphere. Although these mutations are correlated with enhanced efficacy to the inhibitors in NSCLC, they can not explain or predict the sensitivity of many other cancer patients to the beneficial effects of the EGFR kinase inhibitors or antibody mediated therapy. As with as other small-molecule kinase inhibitors and susceptible diseases (e.g., imatinib and chronic myeloid leukaemia), resistance to EGFR inhibitors has been reported recently, documenting the requirement for development of multi-pronged therapeutic approaches. EGFR kinase inhibitors are also being evaluated as adjuvants in hormonal therapy of breast cancer – especially those which overexpress EGFR. Genetically engineered antibodies specific for the EGFR family member ErbB2 have been developed which show efficacy in the treatment of primary, and prevent the relapse of, breast cancer. Clearly, the EGF/EGFR signalling cascade has, and continues to play, an important role in the development of novel anticancer targeted therapies.     

EGFR mutational status in penile cancer

Objective: No substantial improvement in overall survival has been obtained over the past two decades in penile cancer (PC). Clinical data are available on the role of epidermal growth factor receptors (EGFR) inhibitors in PC but no EGFR mutational analysis has been conducted.

Research design and methods: We reviewed formalin-fixed, paraffin-embedded blocks of PC at the Pathology Department of the National Cancer Institute since 2000 through 2012 to evaluate activating mutations in the tyrosine kinase domain of EGFR: EGFR E746 ? A750 specific deletion in exon 19 and EGFR L858R specific point mutation in exon 21.

Results: Thirty tumor samples were available for our analysis. EGFR was expressed in all samples at immunohistochemistry. Tested mutations were not identified in any of the samples analyzed.

Conclusions: The most frequent activating EGFR mutations detected in non-small setting lung cancer are absent in penile cancer (PC). Sequencing of the entire EGFR gene in patients with PC may provide useful insights, as its mechanism of overexpression and activation in PC remains unknown.     

非小细胞肺癌表皮生长因子受体基因突变的结果分析

目的回顾性分析301例非小细胞肺癌(NSCLC)组织表皮生长因子受体(EGFR)基因突变检测结果,比较三种实验方法检查EGFR突变的差异。探讨肺癌临床靶向个体化治疗进行EGFR分子病理检查的最佳方法。方法应用聚合酶链反应(PCR)结合直接测序法检测171例肺癌DNA样本;TaqMan探针荧光定量PCR法检测88例肺癌DNA样本;扩增阻碍突变系统(ARMS)法检测42例肺癌DNA样本。结果 PCR直接测序法、TaqMan探针荧光定量PCR法、ARMS法的阳性总检出率分别是31.58%、27.27%、42.86%。结论 PCR直接测序法和TaqMan探针荧光定量PCR法阳性总检出率差异无统计学意义(P>0.05),ARMS法阳性总检出率高于PCR直接测序法和TaqMan探针荧光定量PCR法(P均<0.01)。对于不同来源的肺癌组织标本,不同方法检测EGFR突变具有各自的优点和不足。     

Role of erlotinib in the treatment of non-small cell lung cancer: clinical outcomes in wild-type epidermal growth factor receptor patients

Erlotinib is an orally administered small molecule inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. Erlotinib at the standard oral daily dose of 150 mg is approved for the treatment of unselected chemorefractory advanced non-small cell lung cancer patients as well as maintenance therapy after first-line chemotherapy. The European Medicines Agency has recently also approved erlotinib as the first-line therapy in patients with EGFR mutations. Although recent studies have identified higher response rates and improved survival with erlotinib in a subset of patients with EGFR mutations, the survival benefit from single agent erlotinib in chemorefractory patients and in the maintenance setting is well observed in EGFR wild-type patients. The role of single agent erlotinib in the first-line setting in special subsets of EGFR wild-type patients (elderly, poor performance status, non-smokers) needs to be further determined. The combination of erlotinib with other targeted therapies has shown promising results and warrants further studies in EGFR wild-type patients.