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1.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987
2.  BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations  
PLoS Medicine  2007;4(10):e315.
Background
Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.
Methods and Findings
To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.
Conclusions
Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.
Susumu Kobayashi and colleagues provide evidence that the polypeptide BIM is involved in tyrosine kinase inhibitor (TKI)-induced apoptosis in sensitiveEGFR-mutant cells and suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC). Many patients with NSCLC die within a year of their diagnosis, but recently, “targeted” therapies have increased the life expectancy of some of them. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genes. Targeted therapies specifically attack these changes and, unlike standard chemotherapy drugs, kill cancer cells without damaging normal cells. The targeted drugs used to treat NSCLC are gefitinib and erlotinib, two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In normal cells, messenger proteins bind to EGFR and activate its tyrosine kinase, an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These “phosphorylated” proteins then tell the cell to divide. In some NSCLCs, EGFR drives uncontrolled cell division because its tyrosine kinase is mutated and the cancer becomes dependent on or “addicted” to EGFR signaling for its survival. TKI treatment can dramatically shrink this subset of NSCLCs, most of which lack a specific part of EGFR (the gene that encodes EGFR) or have the amino acid leucine instead of arginine at position 858 (an L858R mutation) of EGFR.
Why Was This Study Done?
TKI-sensitive NSCLCs eventually become resistant to TKIs because they acquire additional (secondary) mutations. In half of these TKI-resistant tumors, the additional mutation is replacement of threonine by methionine at position 790 (T790M) in EGFR. However, the mutations responsible for the remaining cases of TKI resistance are not known. In addition, little is known about how TKIs induce cell death other than that they induce a type of cell death called apoptosis. A better understanding of how TKIs kill tumor cells and how secondary mutations block their effects could reveal ways to enhance their action and improve the outcome for patients with NSCLC. In this study, the researchers have studied the mechanism of TKI-induced cell death and of resistance to TKIs.
What Did the Researchers Do and Find?
The researchers first measured the ability of gefitinib to cause apoptosis (genetically programmed cell death) in NSCLC cell lines (tumor cells adapted to grow indefinitely in dishes) that had the EGFR deletion, the L858R mutation, or normal EGFR. Gefitinib caused apoptosis only in cell lines with altered EGFR. Then they asked whether a proapoptotic protein called BIM (a member of the BCL2 family of pro- and antiapoptotic proteins) is involved in TKI-induced cell death—BIM is known to be involved in this process in leukemia (blood cancer) cells. Gefitinib treatment increased the expression of BIM in TKI-sensitive NSCLC cell lines and reduced the phosphorylation of BIM (which makes BIM more active). By contrast, blocking BIM expression using a technique called RNA interference reduced TKI-induced apoptosis in TKI-sensitive NSCLC cells. Furthermore, introduction of the T790M resistance mutation into these cells blocked gefitinib-induced up-regulation of BIM and apoptosis. Finally, the researchers identified a new TKI resistance mutation (L747S, substitution of serine for leucine at position 747) in a patient whose TKI-sensitive NSCLC had become resistant to gefitinib, and showed that this resistance mutation also reduced TKI-induced apoptosis in cells growing in dishes by interfering with BIM up-regulation.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Cragg et al.) show that BIM is required for TKI-induced apoptosis in EGFR mutant NSCLC cells. They also show that mutations that make TKI-sensitive cells resistant to these drugs reduce TKI-induced apoptosis by preventing the upregulation of BIM. These results were obtained by examining the behavior of established cell lines growing in dishes and need to be confirmed in cells freshly isolated from tumors and in tumors themselves. However, they suggest that the efficacy of TKIs could be increased by finding ways to increase BIM expression or to activate other proteins involved in apoptosis Such approaches might be particularly beneficial for patients with NSCLC whose initially TKI-sensitive tumors have acquired mutations that make them resistant to TKIs.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040315.
Ingo Mellinghoff discusses this paper and two related ones in a perspective article
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer, including information on treatment with TKIs
CancerQuest information on all aspects of cancer from Emory University (in several languages)
Wikipedia pages on apoptosis, epidermal growth factor receptor, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040315
PMCID: PMC2043012  PMID: 17973572
3.  Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1α in non-small cell lung cancer cells 
Oncogene  2010;29(18):2616-2627.
Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1α pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1α. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1α and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.
doi:10.1038/onc.2010.16
PMCID: PMC3378055  PMID: 20154724
EGFR; MET; non-small cell lung cancer; HIF-1α; invasiveness
4.  Molecular predictors of response to tyrosine kinase inhibitors in patients with Non-Small-Cell Lung Cancer 
Introduction
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have become a treatment option in non-small-cell lung cancer (NSCLC) patients. However, despite their use in this disease, a significant number of patients will eventually develop resistance and relapse. In this study, we aimed to characterize several molecular events involved in potential resistance mechanisms to anti-EGFR treatment and correlate our findings with clinical outcome.
Material and methods
The medical records of patients with NSCLC who received anti-EGFR TKIs in any line within the participating centers were reviewed and available paraffin embedded tissue was retrieved. Mutational analysis for EGFR, KRAS, BRAF and intron-exon 14 deletions of MET; FISH analysis for chromosomal gain or amplification for EGFR, MET and the deletion marker D7S486 were performed. Furthermore, the expression of EGFR and MET were analysed by immunohistochemistry. All results were correlated with treatment outcomes.
Results
Between 10/2001 and 12/2009 from an initial cohort of 72 treated patients, 59 cases (28 gefitinib/ 31 erlotinib) were included in the analysis. The majority had adenocarcinoma histology (68%), and received treatment in the second line setting (56%). Disease control rate (DCR) was 25.4% for all patients. EGFR and RAS mutational rates were 33% and 10% respectively, no other mutations were identified. High EGFR expressing tumors were found in 7 of 45 cases and pEGFR positivity (IHC) was found in 56% of the cases; MET expression was found in 48% of tumors. EGFR gene amplification was found in 4 cases, two cases showed high polysomy; overall, 13% cases were FISH positive for EGFR. High polysomy of MET gene was detected in 1/43 cases tested. D7S486 locus deletion was detected in 15/37 (40%) of cases. EGFR mutational status and gene gain were both associated with more favorable DCR. No other associations between examined biomarkers and DCR or survival were noted.
Conclusions
EGFR mutational status is a predictor for disease control in patients with NSCLC treated with anti-EGFR TKIs. The predictive role of several other molecules involved in potential resistance to anti-EGFR TKIs is worthy of additional investigation.
doi:10.1186/1756-9966-31-77
PMCID: PMC3533816  PMID: 22992338
Predictive; Somatic mutation; EGFR; Gefitinib; Erlotinib; Response
5.  Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: http://www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.html
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis
Objective
The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa®) or erlotinib (Tarceva®) in patients with advanced non-small cell lung cancer (NSCLC).
Clinical Need: Target Population and Condition
With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib).
Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined.
The Technology: EGFR mutation testing
The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%.
Research Questions
In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?
What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?
What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?
What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting?
Methods
A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms:
Non-Small-Cell Lung Carcinoma
Epidermal Growth Factor Receptor
An automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
The inclusion criteria were as follows:
Population: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)
Procedure: EGFR mutation testing before treatment with gefitinib or erlotinib
Language: publication in English
Published health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract)
Outcomes: progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).
The exclusion criteria were as follows:
Studies lacking outcomes specific to those of interest
Studies focused on erlotinib maintenance therapy
Studies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drug
Grey literature, where relevant, was also reviewed.
Outcomes of Interest
PFS
ORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)
OS
QoL
Quality of Evidence
The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria.
Summary of Findings
Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel.
In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). However, erlotinib did not confer a significant survival benefit compared with placebo in patients with EGFR mutations (HR, 0.55; 95%CI, 0.25-1.19) versus wild-type (HR, 0.74; 95%CI, 0.52-1.05). The interaction between EGFR mutation status and erlotinib use was not significant (P=0.47). The lack of significance could be attributable to a type II error since there was a low sample size that was available for subgroup analysis.
A series of phase II studies have examined the clinical effectiveness of erlotinib in patients known to have EGFR mutations. Evidence from these studies has consistently shown that erlotinib yields a very high ORR (typically 70% vs. 4%) and a prolonged PFS (9 months vs. 2 months) in patients with EGFR mutations compared with patients with wild-type EGFR. Although having a prolonged PFS and higher respond in EGFR mutated patients might be due to a better prognostic profile regardless of the treatment received. In the absence of a comparative treatment or placebo control group, it is difficult to determine if the observed differences in survival benefit in patients with EGFR mutation is attributed to prognostic or predictive value of EGFR mutation status.
Conclusions
Based on moderate quality of evidence, patients with locally advanced or metastatic NSCLC with adenocarcinoma histology being treated with gefitinib in the first-line setting are highly likely to benefit from gefitinib if they have EGFR mutations compared to those with wild-type EGFR. This advantage is reflected in improved PFS, ORR and QoL in patients with EGFR mutation who are being treated with gefitinib relative to patients treated with chemotherapy.
Based on low quality of evidence, in patients with locally advanced or metastatic NSCLC who are being treated with erlotinib, the identification of EGFR mutation status selects those who are most likely to benefit from erlotinib relative to patients treated with placebo in the second or third-line setting.
PMCID: PMC3377519  PMID: 23074402
6.  MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients 
Annals of Oncology  2008;20(2):298-304.
Background: MET amplification has been detected in ∼20% of non-small-cell lung cancer patients (NSCLC) with epidermal growth factor receptor (EGFR) mutations progressing after an initial response to tyrosine kinase inhibitor (TKI) therapy.
Patients and methods: We analyzed MET gene copy number using FISH in two related NSCLC cell lines, one sensitive (HCC827) and one resistant (HCC827 GR6) to gefitinib therapy and in two different NSCLC patient populations: 24 never smokers or EGFR FISH-positive patients treated with gefitinib (ONCOBELL cohort) and 182 surgically resected NSCLC not exposed to anti-EGFR agents.
Results: HCC827 GR6-resistant cell line displayed MET amplification, with a mean MET copy number >12, while sensitive HCC827 cell line had a mean MET copy number of 4. In the ONCOBELL cohort, no patient had gene amplification and MET gene copy number was not associated with outcome to gefitinib therapy. Among the surgically resected patients, MET was amplified in 12 cases (7.3%) and only four (2.4%) had a higher MET copy number than the resistant HCC827 GR6 cell line.
Conclusions: MET gene amplification is a rare event in patients with advanced NSCLC. The development of anti-MET therapeutic strategies should be focused on patients with acquired EGFR-TKI resistance.
doi:10.1093/annonc/mdn635
PMCID: PMC2733067  PMID: 18836087
EGFR; gefitinib; MET; non-small cell lung cancer; tyrosine kinase inhibitor
7.  Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with tyrosine kinase inhibitors or cetuximab 
BMC Medicine  2012;10:28.
Background
The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab.
Methods
NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index.
Results
EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA.
Conclusions
EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream "resistance" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.
doi:10.1186/1741-7015-10-28
PMCID: PMC3334713  PMID: 22436374
EGFR; RNA interference; tyrosine kinase inhibitors (TKIs); anti-EGFR monoclonal antibodies (mAbs); proliferation; apoptosis; lung cancer
8.  Synergistic Effect of Afatinib with Su11274 in Non-Small Cell Lung Cancer Cells Resistant to Gefitinib or Erlotinib 
PLoS ONE  2013;8(3):e59708.
Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase−3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.
doi:10.1371/journal.pone.0059708
PMCID: PMC3601073  PMID: 23527257
9.  HGF-independent Potentiation of EGFR Action by c-Met 
Oncogene  2011;30(33):3625-3635.
The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant Epidermal Growth Factor Receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors is minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met which begins at 8 h continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of MAPK or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of HGF or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pre-treatment with pan-SFK inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared to control, further, confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft anti-tumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.
doi:10.1038/onc.2011.84
PMCID: PMC3126872  PMID: 21423210
c-Met; c-Src; EGFR; cross-talk
10.  Primary concomitant EGFR T790M mutation predicted worse prognosis in non-small cell lung cancer patients 
OncoTargets and therapy  2014;7:513-524.
Purpose
We performed this analysis to improve the understanding of the clinicopathological characteristics and clinical outcome of non-small cell lung cancer (NSCLC) patients harboring the primary epidermal growth factor receptor (EGFR) T790M mutation along with activating EGFR mutation.
Methods
Resected tumors from 1903 NSCLC patients were analyzed for mutation in EGFR, as well as KRAS (Kirsten rat sarcoma viral oncogene homolog), BRAF (v-raf murine sarcoma viral oncogene homolog B), HER2 (human epidermal growth factor 2), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and EML4 (echinoderm microtubule associated protein like 4)–ALK (anaplastic lymphoma receptor tyrosine kinase) fusion. Fluorescence in situ hybridization was performed to define EGFR and c-MET (met proto-oncogene gene amplification. Expression of PIK3CA and p-Akt (phosphorylated protein kinase B) were tested using immunohistochemistry. Clinical and pathological data, including sex, age at diagnosis, stage, tumor differentiation, smoking history, histological subtype, relapse-free and overall survival, were further analyzed.
Results
In all, 16 NSCLC patients were found to harbor primary EGFR T790M mutation, including 14 adenocarcinomas and two adenosquamous carcinomas, accounting for 2.04% of all the EGFR mutant cases and 0.84% of the total. No c-MET amplification was found to coexist with primary EGFR T790M. Fewer EGFR copy-number variations were found in samples harboring EGFR T790M mutations compared with those in patients with exon 19 deletions and L858R. Overall survival was significantly shorter for patients harboring EGFR T790M mutation than it was for patients with exon 19 deletions (logrank P=0.008). When taking patients harboring EGFR L858R or exon 19 deletions as one group, the overall survival was also significantly longer than that in patients with T790M mutation (logrank P=0.012). There was no significant difference in relapse-free survival among three subgroups of patients.
Conclusion
Our study described the clinicopathological and molecular characteristics of NSCLC patients harboring primary EGFR T790M mutations. Its value of being a predictor for worse prognosis was established. Primary EGFR T790M mutation is a rare event in NSCLC cases, but the therapeutic strategies for this subtype of patients should be precisely considered.
doi:10.2147/OTT.S60122
PMCID: PMC3979794  PMID: 24729716
driver mutation; survival; clinicopathological profile; EGFR tyrosine kinase inhibitor; acquired resistance
11.  Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met 
Breast Cancer Research : BCR  2012;14(4):R104.
Introduction
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC).
Methods
The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis.
Results
Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI.
Conclusions
Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.
doi:10.1186/bcr3224
PMCID: PMC3680928  PMID: 22788954
12.  Acquired resistance to MET kinase inhibition in MET-dependent non-small cell lung cancer cells mediated by a switch to EGFR dependency 
Cancer research  2010;70(4):1625-1634.
The MET receptor tyrosine kinase has been implicated in several solid tumor settings, with MET gene amplification present in a subset of patients with epithelial tumors, including non-small cell lung cancer (NSCLC). MET gene amplification also appears to underlie acquired resistance to EGFR kinase inhibitor therapy in ~20% of NSCLC patients that initially respond to such treatment. In vitro studies have demonstrated that cancer cells harboring MET amplification demonstrate striking sensitivity to small molecule selective MET kinase inhibitors, prompting efforts to evaluate such inhibitors in clinical trials. Previous experience with kinase-targeted therapeutics such as the selective tyrosine kinase inhibitors (TKIs) imatinib and erlotinib, in chronic myeloid leukemia and NSCLC, respectively, has revealed that most treatment-responsive patients eventually relapse with drug-resistant disease. Here, we have modeled acquired resistance to the investigational MET kinase inhibitor PF2341066 in MET-amplified NSCLC cell lines to identify specific drug resistance mechanisms that are anticipated in the course of clinical development. Through this analysis, we identified EGFR (epidermal growth factor receptor) pathway engagement as a PF2341066 resistance mechanism in MET-amplified cells following prolonged drug exposure. While combined inhibition of MET and EGFR kinases in MET-dependent NSCLC cells did not detectably enhance their acute PF2341066 sensitivity, this combination dramatically suppressed the eventual emergence of drug-resistant clones following prolonged exposure. Conversely, activating the EGFR pathway with the receptor ligand EGF increased the yield of PF2341066-resistant clones, confirming the significance of this pathway in conferring resistance to MET TKIs. These findings further support an intimate relationship between EGFR and MET signaling pathways in NSCLC, and suggest that a combination treatment strategy with MET and EGFR kinase inhibitors may be beneficial in MET-amplified NSCLC.
doi:10.1158/0008-5472.CAN-09-3620
PMCID: PMC3057521  PMID: 20124471
MET; EGFR; TKI resistance; cross-talk; acquired drug resistance
13.  Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics 
PLoS Medicine  2007;4(10):e316.
Background
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.
Methods and Findings
We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.
Conclusions
Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation.
Editors' Summary
Background.
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC.
Why Was This Study Done?
Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR.
What Did the Researchers Do and Find?
The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040316.
A perspective by Ingo Mellinghoff discusses this article and two related research articles
Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
CancerQuest provides information on all aspects of cancer from Emory University (in several languages)
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040316
PMCID: PMC2043013  PMID: 17973573
14.  Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung Adenocarcinomas 
PLoS Medicine  2007;4(10):e294.
Background
Mutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy.
Methods and Findings
Using a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro.
Conclusions
In drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.
Using a panel of human drug-sensitive EGFR mutant lung cancer cells, William Pao and colleagues show that induction of BIM, a member of the BCL2 family, is essential for apoptosis triggered by EGFR kinase inhibitors.
Editors' Summary
Background.
Lung cancer, a common type of cancer, has a very low cure rate. Like all cancers, it occurs when cells begin to divide uncontrollably because of changes (mutations) in their genes. Chemotherapy drugs kill these rapidly dividing cells but, because some normal tissues are sensitive to these agents, it is hard to destroy the cancer without causing serious side effects. Recently, “targeted” therapies have brought new hope to some patients with cancer. These therapies attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One of the first molecules for which a targeted therapy was developed was the epidermal growth factor receptor (EGFR). In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive uncontrolled cell division in some cancers so blocking the EGFR signaling pathway should stop these cancers growing. Indeed, some lung cancers with mutations in the tyrosine kinase of EGFR shrink dramatically when treated with gefitinib or erlotinib, two tyrosine kinase inhibitors (TKIs).
Why Was This Study Done?
TKI-sensitive lung cancers shrink when treated with TKIs because of drug-induced cell death, but what are the molecular mechanisms underlying this death? A better understanding of how TKIs kill cancer cells might provide new insights into why not all cancer cells with mutations in EGFR (the gene from which EGFR is made) are sensitive to TKIs. It might also uncover new targets for therapy. TKIs do not completely kill lung cancers, but if the mechanism of TKI-induced cell death were understood, it might be possible to enhance their effects. In this study, the researchers have investigated how cell death occurs after kinase inhibition in a panel of human lung cancer cell lines (cells isolated from human tumors that grow indefinitely in dishes) that carry EGFR mutations.
What Did the Researchers Do and Find?
The researchers show, first, that erlotinib induces a type of cell death called apoptosis in erlotinib-sensitive cell lines but not in resistant cell lines. Apoptosis can be activated by two major pathways. In this instance, the researchers report, the so-called “intrinsic” pathway activates apoptosis. This pathway is stimulated by proapoptotic members of the BCL2 family of proteins and is blocked by antiapoptotic members, so the researchers examined the effect of erlotinib treatment on the expression of BCL2 family members in the EGFR mutant cell lines. Erlotinib treatment increased the expression of the proapoptotic protein BIM in sensitive but not in resistant cell lines. It also removed phosphate groups from BIM—dephosphorylated BIM is a more potent proapoptotic protein. Conversely, blocking BIM expression using a technique called RNA interference virtually eliminated the ability of erlotinib to kill EGFR mutant cell lines. The researchers also report that erlotinib treatment increased BIM expression in erlotinib-sensitive lung tumors growing in mice and that an inhibitor of the anti-apoptotic protein BCL2 enhanced erlotinib-induced death in drug-sensitive cells growing in dishes.
What Do These Findings Mean?
These findings indicate that BIM activity is essential for the apoptosis triggered by TKIs in drug-sensitive lung cancer cells that carry EGFR mutations, and that treatment of these cells with TKIs induces both the expression and dephosphorylation of BIM. The finding that the intrinsic pathway of apoptosis activation is involved in TKI-induced cell death suggests that changes in this pathway (possibly mutations in some of its components) might influence the sensitivity of EGFR mutant lung cancers to TKIs. Finally, these findings suggest that giving drugs that affect the intrinsic pathway of apoptosis activation at the same time as TKIs might further improve the clinical outcome for patients with EGFR mutant tumors. Such combinations will have to be tested in clinical trials before being used routinely.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040294.
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Wikipedia pages on apoptosis, epidermal growth factor receptor, and BCL-2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040294
PMCID: PMC2001209  PMID: 17927446
15.  TAK-701, a humanized monoclonal antibody to HGF, reverses gefitinib resistance induced by tumor-derived HGF in non–small cell lung cancer with an EGFR mutation 
Molecular cancer therapeutics  2010;9(10):2785-2792.
Purpose
Most non–small cell lung cancer (NSCLC) tumors with an activating mutation of the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib but ultimately develop resistance to these drugs. Hepatocyte growth factor (HGF) induces EGFR-TKI resistance in NSCLC cells with such a mutation. We investigated strategies to overcome gefitinib resistance induced by HGF.
Experimental Design
Human NSCLC cells with an activating EGFR mutation (HCC827 cells) were engineered to stably express HGF (HCC827-HGF cells). The effects of TAK-701, a humanized monoclonal antibody to HGF, in combination with gefitinib were examined on signal transduction and cell growth in vitro or in vivo.
Results
HCC827-HGF cells secreted large amounts of HGF and exhibited resistance to gefitinib in vitro to an extent similar to that of HCC827 GR cells, in which the gene for the HGF receptor MET is amplified. A MET-TKI reversed gefitinib resistance in HCC827-HGF cells as well as in HCC827 GR cells, suggesting that MET activation induces gefitinib resistance in both cell lines. TAK-701 in combination with gefitinib inhibited the phosphorylation of MET, EGFR, ERK, and AKT in HCC827-HGF cells, resulting in suppression of cell growth and indicating that autocrine HGF-MET signaling contributes to gefitinib resistance in these cells. Combination therapy with TAK-701 and gefitinib also markedly inhibited the growth of HCC827-HGF tumors in vivo.
Conclusions
The addition of TAK-701 to gefitinib is a promising strategy to overcome EGFR-TKI resistance induced by HGF in NSCLC with an activating EGFR mutation.
doi:10.1158/1535-7163.MCT-10-0481
PMCID: PMC3208321  PMID: 20716641
TAK-701; hepatocyte growth factor; gefitinib; resistance; non–small cell lung cancer
16.  EGFR Exon-Level Biomarkers of the Response to Bevacizumab/Erlotinib in Non-Small Cell Lung Cancer 
PLoS ONE  2013;8(9):e72966.
Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.
doi:10.1371/journal.pone.0072966
PMCID: PMC3769372  PMID: 24039832
17.  EGFR/Met association regulates EGFR TKI resistance in breast cancer 
Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.
doi:10.1186/1750-2187-5-8
PMCID: PMC2911419  PMID: 20624308
18.  Dual MET–EGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer 
British Journal of Cancer  2008;99(6):911-922.
Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV=1.1). At 2 μM, SU11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P=0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET–EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.
doi:10.1038/sj.bjc.6604559
PMCID: PMC2538758  PMID: 19238632
MET; EGFR; inhibitor; erlotinib; resistance; lung cancer
19.  EGF Receptor activates MET through MAP kinases to enhance non-small cell lung carcinoma invasion and brain metastasis 
Cancer research  2013;73(16):5053-5065.
MET amplification as a mechanism of acquired resistance to EGFR targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by co-expression of ERBB3. EGFR-MET crosstalk was not direct but occurred by a combined regulation of MET levels and intermediary signaling through MAP kinases. In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAP kinases reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Lastly, EGFR-MET signaling was enhanced in a highly metastatic EGFR mutant cell subpopulation, compared to the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.
doi:10.1158/0008-5472.CAN-12-3775
PMCID: PMC3745527  PMID: 23794705
EGFR; MET; NSCLC; metastasis; MAP kinases
20.  Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain 
PLoS Medicine  2006;3(12):e485.
Background
Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.
Methods and Findings
Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.
Conclusions
Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.
Ingo Mellinghoff and colleagues sequenced theEGFR gene in glioblastoma samples and cell lines and identified missense mutations in the extracellular domain that suggest a new mechanism for EGFR activation.
Editors' Summary
Background.
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the tissues and organs of the human body in working order. But sometimes, cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. Cancer treatments often involve drugs that kill rapidly dividing cells but, although these hit cancer cells hardest, they also damage some normal tissues. Now, though, some of the specific changes that allow cancer cells to divide uncontrollably have been identified and drugs that attack only these abnormal cells are being developed. One of these—erlotinib—inhibits the activity of epidermal growth factor receptor (EGFR), a “receptor tyrosine kinase” that sits in the cell membrane. The interaction of epidermal growth factor (EGF)—a messenger protein—with the extracellular portion (or domain) of EGFR activates its intracellular part (a kinase enzyme). This adds phosphate groups to tyrosine (an amino acid) in proteins that form part of a signaling cascade that tells cells to divide. Cancer cells often have alterations in EGFR signaling. Some have extra copies of the EGFR gene (EGFR amplification); others make a short version of EGFR that is always active because it lacks the extracellular domain that binds EGF; yet others contain EGFR that is permanently active because of mutations in its kinase domain.
Why Was This Study Done?
Erlotinib can help only patients whose tumor growth is dependent on EGFR signaling. To identify these patients it is necessary to have a detailed catalog of the mutations that occur in EGFR in tumors and to know which mutations drive uncontrolled cell growth. In this study, the researchers have catalogued and characterized the mutations in EGFR that occur in glioblastoma, a deadly type of brain tumor. The researchers chose this tumor type for their study because EGFR amplification and loss of the extracellular domain of EGFR are both common in glioblastomas and because about one in five patients with glioblastoma responds well to EGFR kinase inhibitors.
What Did the Researchers Do and Find?
The researchers sequenced the whole coding sequence of the EGFR gene in more than 100 glioblastomas. Nearly 15% of the tumors contained missense mutations—changes that alter the amino acid sequence of EGFR. Only one tumor had a mutation in the EGFR kinase domain; the rest had mutations in its extracellular domain. To test whether these newly identified mutations might contribute to cancer development (oncogenesis), the researchers introduced mutated or normal EGFR genes into nontumorigenic mouse cells. Only the cells that contained the mutated EGFR genes formed tumors when injected into mice, indicating that the nontumorigenic cells had been “transformed” into cancer cells by the mutated EGFR genes. Finally, the researchers showed that EGFR containing the extracellular missense mutations had kinase activity in the absence of EGF when expressed in human and mouse cells, and that the growth of cells transformed by expression of the mutated genes was sensitive to erlotinib.
What Do These Findings Mean?
These findings identify missense mutations in the extracellular domain of EGFR as a new way to oncogenically activate this protein. Until now researchers have concentrated on the kinase domain of this and other receptor tyrosine kinases in their search for oncogenic mutations, but the results of this study suggest that future searches should be much broader. The distribution of EGFR missense mutations in glioblastoma contrasts with that in lung cancer, in which alterations in EGFR signaling are also implicated in cancer development but all the oncogenic mutations are in the kinase domain. Fortunately, EGFR kinase inhibitors like erlotinib have broad activity: They inhibit the growth of cells transformed by the expression of EGFR containing extracellular domain mutations or kinase mutations, or by the expression of the short EGFR variant. This bodes well for the use of these drugs in patients with glioblastoma. However, before these inhibitors become a standard part of cancer treatments, sensitive techniques need to be developed to analyze tumors for these mutations so that the patients who will benefit from these targeted therapies can be identified.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030485.
MedlinePlus encyclopedia entries on cancer and on brain tumors
US National Cancer Institute information for patients and professionals on brain tumors
Wikipedia pages on protein kinases, epidermal growth factor receptor, and erlotinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030485
PMCID: PMC1702556  PMID: 17177598
21.  Curcumin Induces EGFR Degradation in Lung Adenocarcinoma and Modulates p38 Activation in Intestine: The Versatile Adjuvant for Gefitinib Therapy 
PLoS ONE  2011;6(8):e23756.
Background
Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.
Methods/Principal Findings
After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.
Conclusions/Significance
Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.
doi:10.1371/journal.pone.0023756
PMCID: PMC3157465  PMID: 21858220
22.  Increased MET Gene Copy Number Negatively Affects Survival of Surgically Resected Non–Small-Cell Lung Cancer Patients 
Journal of Clinical Oncology  2009;27(10):1667-1674.
Purpose
To investigate the prognostic role of genomic gain for MET and epidermal growth factor receptor (EGFR) genes in surgically resected non–small-cell lung cancer (NSCLC).
Patients and Methods
This retrospective study included 447 NSCLC patients with available tumor tissue from primary lung tumor and survival data. EGFR and MET status was evaluated by fluorescent in situ hybridization (FISH) in tissue microarray sections.
Results
EGFR FISH results were obtained in 376 cases. EGFR gene amplification and high polysomy (EGFR FISH+) were observed in 10.4% and 32.4% of cases, respectively. EGFR FISH-positive patients had a nonsignificant shorter survival than EGFR FISH-negative patients (P = .4). Activating EGFR mutations were detected in 9.7% of 144 stage I-II disease with no impact on survival. MET FISH analysis was performed in 435 cases. High MET gene copy number (mean ≥ 5 copies/cell) was observed in 48 cases (MET+, 11.1%), including 18 cases with true gene amplification (4.1%). MET+ status was associated with advanced stage (P = .01), with grade 3 (P = .016) and with EGFR FISH+ result (P < .0001). No patient with activating EGFR mutation resulted MET+. In the whole population, MET-positive patients had shorter survival than MET-negative patients (P = .005). Multivariable model confirmed that MET-negative patients had a significant reduction in the risk of death than MET-positive patients (hazard ratio, 0.66; P = .04).
Conclusion
MET increased gene copy number is an independent negative prognostic factor in surgically resected NSCLC. EGFR gene gain does not impact survival after resection.
doi:10.1200/JCO.2008.19.1635
PMCID: PMC3341799  PMID: 19255323
23.  EGFR and HER2 Genomic Gain in Recurrent Non-small Cell Lung Cancer After Surgery 
Journal of Thoracic Oncology  2009;4(3):318-325.
Background
Sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and frequency of activation mutations in EGFR is lower in Caucasian than Asian non small-cell lung cancer (NSCLC) patients. Increased EGFR gene copy numbers evaluated by fluorescence in situ hybridization (FISH) has been reported as predictor of clinical benefit from EGFR-TKIs in Caucasian NSCLC patients. This study was carried out to verify whether EGFR FISH had similar performance in Japanese patients.
Methods
A cohort of 44 Japanese patients with recurrent NSCLC after surgery was treated with gefitinib 250 mg daily. The cohort included 48% females and 52% never-smokers; 73% had prior chemotherapy and 57% had stage III-IV at the time of surgery. Adenocarcinoma was the most common histology (86%). FISH was performed using the EGFR/Chromosome Enumeration Probe 7 and PathVysion DNA probes (Abbott Molecular). Specimens were classified as FISH positive when showing gene amplification or high polysomy (≥4 copies of the gene in ≥40% of tumor cells). Tumor response to gefitinib was assessed by RECIST for 33 patients with measurable diseases.
Results
Twenty-nine tumors (66%) were EGFR FISH+ and 23 (53%) were HER2 FISH+. Overall response rate was 52%, representing 65% of EGFR FISH+ patients and 29% of EGFR FISH+ patients (p = 0.0777). Survival was not impacted by the EGFR FISH (p = 0.9395) or the HER2 FISH (p = 0.0671) status. EGFR FISH= was significantly associated with HER2 FISH+ (p = 0.015) and presence of EGFR mutation (p = 0.0060). EGFR mutation significantly correlated with response (p < 0.0001) and survival after gefitinib (p = 0.0204). EGFR and HER2 FISH status were not associated with KRAS mutation.
Conclusion
Frequency of EGFR FISH+ status was higher and its predictive power for TKI sensitivity was lower in this Japanese cohort than in Western NSCLC cohorts. These findings support differences in the mechanisms of EGFR pathway activation in NSCLC between Asian and Caucasian populations. Confirmation of these results in larger cohorts is warranted.
doi:10.1097/JTO.0b013e31819667a3
PMCID: PMC3379811  PMID: 19247083
FISH; EGFR; HER2; KRAS; Biomarkers; NSCLC; Tyrosine inhibitors
24.  The Fibroblast Growth Factor Receptor Signaling Pathway as a Mediator of Intrinsic Resistance to EGFR-specific Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer 
The EGFR has been targeted through the development of selective tyrosine kinase inhibitors (TKIs) that have proven effective in a subset of non-small cell lung cancer (NSCLC) patients, many bearing gain-of-function EGFR mutations or egfr gene amplification. However, the majority (~80–90%) of NSCLC patients do not respond to EGFR-specific TKIs and a high rate of acquired resistance to these therapeutics is observed in those that do respond. Thus, EGFR-specific TKIs will not, as single agents, make a high impact on overall lung cancer survival. A number of studies support the activities of other receptor tyrosine kinase pathways including cMet, IGF-1R and FGFRs as mechanisms for both intrinsic and acquired resistance to EGFR TKIs. While the role of cMet and IGF-1R signaling systems as mechanisms of resistance to EGFR TKIs has been widely reviewed in recent years, the potential role of FGFR-dependent signaling as a mechanism for EGFR TKI resistance has more recently emerged and will be highlighted herein. Due to the high degree of homology of FGFRs with VEGFRs and PDGFRs, FGFR-active TKIs already exist via development of VEGFR-targeted TKIs as angiogenesis inhibitors. Thus, these agents could be rapidly advanced into clinical investigations as FGFR inhibitors, either alone or in combination with TKIs selective for EGFR, cMet or IGF-1R as a means to expand the spectrum of NSCLC patients that can be effectively targeted with TKI-directed therapies.
doi:10.1016/j.drup.2009.05.001
PMCID: PMC2763047  PMID: 19501013
FGF; FGFR; NSCLC; intrinsic resistance; receptor tyrosine kinase; tyrosine kinase inhibitors
25.  USP8 is a novel target for overcoming gefitinib-resistance in lung cancer 
PURPOSE
Common treatment modalities for non-small cell lung cancer (NSCLC) involve the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due in large part to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non-small cell lung cancer (NSCLC).
EXPERIMENTAL DESIGN
Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model.
RESULTS
Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs, while having little toxicity toward normal cells. Genetic silencing of USP8 led to the down-regulation of several receptor tyrosine kinases (RTKs), including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression, while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells.
CONCLUSIONS
Our results demonstrate for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells.
doi:10.1158/1078-0432.CCR-12-3696
PMCID: PMC3891300  PMID: 23748694
USP8; selective killing; gefitinib-resistance; lung cancer; receptor tyrosine kinases

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