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The clinical development of irreversible tyrosine kinase inhibitors that target the human epidermal growth factor receptor family in non-small cell lung cancer is reviewed.

Small-molecule tyrosine kinase inhibitors (TKIs) of the human epidermal growth factor receptor (HER) include the reversible epidermal growth factor receptor (EGFR/HER-1) inhibitors gefitinib and erlotinib. EGFR TKIs have demonstrated activity in the treatment of patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations; however, multiple mechanisms of resistance limit the benefit of these drugs. Although resistance to EGFR TKIs can be intrinsic and correlated with molecular lesions such as in Kirsten rat sarcoma viral oncogene homolog (KRAS; generally observed in a wild-type EGFR background), acquired resistance to EGFR TKIs can evolve in the setting of activating EGFR mutations, such as in the case of EGFR T790M mutations. Several irreversible inhibitors that target multiple members of the HER family simultaneously are currently in clinical development for NSCLC and may have a role in the treatment of TKI-sensitive and TKI-resistant disease. These include PF00299804, an inhibitor of EGFR/HER-1, HER-2, and HER-4, and afatinib (BIBW 2992), an inhibitor of EGFR/HER-1, HER-2, and HER-4. Results of large, randomized trials of these agents may help to determine their potential for the treatment of NSCLC.

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.

The discovery of epidermal growth-factor receptor (EGFR)-activating mutations and the introduction of oral EGFR tyrosine kinase inhibitors (EGFR-TKIs) have expanded the treatment options for patients with non-small cell lung cancer. The first two reversible EGFR-TKIs, erlotinib and gefitinib, are approved for use in the first-line setting in patients with known EGFR-activating mutations and in the second- and third-line settings for all NSCLC patients. These first-generation EGFR-TKIs improve progression-free survival when compared to chemotherapy in patients with EGFR-activating mutations in the first-line setting. However, nearly all patients develop resistance to EGFR-directed agents. There is a need for further therapy options for patients with disease progression after treatment with reversible EGFR-TKIs. Afatinib is an irreversible ErbB family blocker that inhibits EGFR, HER2, and HER4. In vitro and in vivo, afatinib have shown increased inhibition of the common EGFR-activating mutations as well as the T790M resistance mutation when compared to erlotinib and gefitinib. Clinically, afatinib has been evaluated in the LUX-Lung series of trials, with improvement in progression-free survival reported in patients with EGFR-activating mutations in both first- and second-/third-line settings when compared to chemotherapy. Further investigation is needed to determine the precise role that afatinib will play in the treatment of patients with non-small cell lung cancer and EGFR-activating mutations.

Subsets of patients with non-small cell lung cancer respond remarkably well to small molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptor (EGFR) such as gefitinib or erlotinib. In 2004, it was found that EGFR mutations occurring in the kinase domain are strongly associated with EGFR-TKI sensitivity. However, subsequent studies revealed that this relationship was not perfect and various predictive markers have been reported. These include EGFR gene copy numbers, status of ligands for EGFR, changes in other HER family genes or molecules downstream to EGFR including KRAS or AKT. In this review, we would like to review current knowledge of predictive factors for EGFR-TKI. As all but one phase III trials failed to show a survival advantage of the treatment arm involving EGFR-TKIs, it is necessary to select patients by these biomarkers in future clinical trials. Through these efforts, it would be possible to individualise EGFR-TKI treatment for patients suffering from lung cancer.

Gefitinib and erlotinib, small-molecule tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR), were the first molecularly targeted agents to become clinically available for the treatment of non-small cell lung cancer (NSCLC). During the course of their clinical development, it has become clear that the substantial clinical benefit associated with EGFR-TKIs is limited to patients harboring activating mutations of EGFR. Accumulating clinical outcomes in patients with EGFR mutation-positive NSCLC treated with EGFR-TKIs support the notion that this group of individuals constitutes a clinically distinct population. These findings have prompted investigations of the potential role of first-line treatment with EGFR-TKIs in molecularly selected patients, with platinum-based doublet chemotherapy currently being the standard of care for most individuals with advanced NSCLC. This review summarizes the results of recent clinical trials of EGFR-TKIs in selected patients and highlights the efficacy of these drugs in first-line treatment as a form of personalized medicine aimed at improving therapy for advanced NSCLC.

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

Somatic mutations of epidermal growth factor receptor (EGFR) are the strongest predictive markers for the response to EGFR-tyrosine kinase inhibitors (TKIs). Patients with EGFR mutations generally receive EGFR-TKI treatment, and their survival has been significantly improved compared with that before the development of EGFR-TKIs. This study aimed to clarify the impact of EGFR mutational status on the survival of patients with non-small cell lung cancer (NSCLC) receiving cytotoxic agents, but not EGFR-TKIs, as their first-line chemotherapy. In addition, we analyzed patients with EGFR mutations to determine whether the timing of EGFR-TKI administration affects overall survival (OS). A total of 83 NSCLC patients with stage IIIB/IV who received chemotherapy alone and whose EGFR mutational status was known were investigated. Univariate and multivariate analysis for OS was performed using parameters such as age, gender, performance status (PS), histology, disease stage, smoking status, EGFR mutational status and administration of a first-line regimen. Among the 52 patients with EGFR mutations who received EGFR-TKIs, OS between those who received EGFR-TKIs as their first-line treatment and after chemotherapy were similar. Among the 83 patients who received cytotoxic agents as their first-line chemotherapy, the multivariate analysis showed OS to be significantly associated with PS (p<0.001), histology (p=0.039) and EGFR mutational status (p=0.040). OS was almost similar among the 52 patients with EGFR mutations who received EGFR-TKIs in a first- and second-line setting (25.6 vs. 26.8 months, p=0.914). The EGFR mutational status had a significant impact on the survival of NSCLC patients, although these patients did not receive EGFR-TKIs as their first-line chemotherapy. In future randomized trials, even when EGFR-TKIs are not included in experimental regimens, patients may need to be stratified by EGFR mutational status in order that study results be evaluated appropriately.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), gefitinib and erlotinib, are reversible competitive inhibitors of the tyrosine kinase domain of EGFR that bind to its adenosine-5′ triphosphate-binding site. Somatic activating mutations of the EGFR gene, increased gene copy number and certain clinical and pathological features have been associated with dramatic tumor responses and favorable clinical outcomes with these agents in patients with non-small-cell lung cancer (NSCLC). The specific types of activating mutations that confer sensitivity to EGFR TKIs are present in the tyrosine kinase (TK) domain of the EGFR gene. Exon 19 deletion mutations and the single-point substitution mutation L858R in exon 21 are the most frequent in NSCLC and are termed ‘classical’ mutations. The NSCLC tumors insensitive to EGFR TKIs include those driven by the KRAS and MET oncogenes. Most patients who initially respond to gefitinib and erlotinib eventually become resistant and experience progressive disease. The point mutation T790M accounts for about one half of these cases of acquired resistance. Various second-generation EGFR TKIs are currently being evaluated and may have the potential to overcome T790M-mediated resistance by virtue of their irreversible inhibition of the receptor TK domain.

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.

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.

Background

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) have been widely used for the treatment of non-small cell lung cancer (NSCLC). KRAS and EGFR somatic mutations in NSCLC may predict resistance and responsiveness to TKI, respectively. Nevertheless, most research to date has been conducted on samples from primary tumors. For many patients with advanced disease, their samples can only be obtained from metastases for test. The molecular characteristics of metastasized tumors may be different from those of primary tumors.

Materials and methods

Mutation status of KRAS and EGFR between primary tumors and local lymph node metastases of 80 Chinese patients with NSCLC were analyzed by direct sequencing. Five of them were given gefitinib as neoadjunvant treatment after the EGFR-TKI sensitive mutations were detected in their biopsies of mediastinal lymph nodes metastases. McNemar's test was used to compare the EGFR and KRAS mutation status between primary tumors and corresponding local lymph node metastases. Data evaluation was carried out with SPSS_13.0 statistical software.

Results

Among the 160 samples, one primary tumor and seven metastases were identified with KRAS mutations and 21 primary tumors and 26 metastases were found to have EGFR mutations. KRAS and EGFR mutation status was different between primary tumors and corresponding metastases in 6 (7.5%) and 7 (8.75%) patients, respectively. One patient with no TKI sensitive mutations detected in the primary tumor showed disease progression.

Conclusion

Our results suggest that a considerable proportion of NSCLC in Chinese population showed discrepancy in KRAS and EGFR mutation status between primary tumors and corresponding metastases. This observation may have important implication for the use of targeted TKI therapy in the treatment of NSCLC patients.

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.

Purpose

Mutations in the epidermal growth factor receptor (EGFR) have been confirmed as predictors of the efficacy of treatment with EGFR-tyrosine kinase inhibitors (TKIs). We investigated whether polymorphisms of the EGFR gene were associated with clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with EGFR-TKI.

Materials and Methods

A polymorphic dinucleotide repeat in intron 1 [CA simple sequence repeat in intron 1(CA-SSR1)] in intron 1 and single nucleotide polymorphisms (SNP-216) in the promoter region of the EGFR gene were evaluated in 71 NSCLC patients by restriction fragment length polymorphism and DNA sequencing. The relationship between genetic polymorphisms and clinical outcomes of treatment with EGFR-TKIs was evaluated.

Results

SNP-216G/T polymorphisms were associated with the efficacy of EGFR-TKI. The response rate for the SNP-216G/T tended to be higher than that for G/G (62.5% vs. 27.4%, p=0.057). The SNP-216G/T genotype was also associated with longer progression-free survival compared with the GG genotype (16.7 months vs. 5.1 months, p=0.005). However, the length of CA-SSR1 was not associated with the efficacy of EGFR-TKI.

Conclusion

SNP-216G/T polymorphism was a potential predictor of clinical outcomes in NSCLC patients treated with EGFR-TKI.

Introduction

EGFR mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, they develop resistance, often due to acquisition of a second-site mutation (T790M). Current EGFR TKIs select for T790M in preclinical models of acquired resistance. We explored whether all EGFR TKIs similarly select for the T790M mutation using data from early clinical trials and established in vitro models of acquired resistance.

Methods

We analyzed the clinical characteristics of 8 patients with metastatic EGFR mutant lung adenocarcinoma who were treated first-line with XL647 and then progressed. XL647 is an ATP-competitive inhibitor of EGFR, HER2, KDR, and EPHB4. Additional molecular preclinical studies were performed to characterize resistance.

Results

Four patients displayed confirmed partial responses (PRs), three patients had unconfirmed PRs, and one patient displayed stable disease. Only one of five patients’ tumor samples available for analysis after disease progression harbored the T790M mutation. Eight patients subsequently received erlotinib, with (n=3) or without (n=5) chemotherapy. Three of five patients treated with single agent erlotinib derived additional benefit, staying on drug up to 9 months. EGFR mutant PC-9 cells with acquired resistance to XL647 did not harbor the T790M mutation, displayed a distinct mRNA profile from PC-9 cells with T790M-mediated resistance, and were moderately sensitive to erlotinib in growth inhibition assays. Crystal structure analyses of XL647/EGFR T790M did not reveal a different binding mode from that of erlotinib.

Conclusions

The findings of this exploratory study suggest different EGFR TKIs may select for distinct mechanisms of resistance. These results raise the possibility that different EGFR TKIs could be sequentially used to improve outcomes in patients with EGFR mutant lung cancer. Further work investigating this hypothesis is warranted.

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.

Sorafenib is a highly selective multi-targeted agent and has been reported to have potent antitumor effects against various tumors, including human non-small cell lung cancer (NSCLC). In the present study, we explored the antitumor effect and associated molecular mechanisms of sorafenib against human lung cancer cell lines in vitro. We also investigated the efficacy of concurrent and sequential administration of sorafenib and gemcitabine in epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-sensitive and EGFR-TKI-resistant NSCLC cell lines. The PC-9 (EGFR-TKI-sensitive, EGFR-mutated) and A549 (EGFR-TKI-resistant, K-Ras-mutated) NSCLC cell lines were treated with sorafenib and gemcitabine, alone, in combination or with different schedules. Cytotoxicity was assessed by MTT assay, cell cycle distribution was analyzed by flow cytometry and alterations in signaling pathways were analyzed by western blotting. We found that sorafenib exhibited dose-dependent growth inhibition in the EGFR-TKI-sensitive and EGFR-TKI-resistant NSCLC cell lines, and the sequence gemcitabine→sorafenib exhibited the strongest synergism. Sorafenib arrested the cell cycle at G1 phase, whereas gemcitabine caused arrest at S phase. The molecular mechanism of this synergism is that the downstream signaling pathways that were initially activated by gemcitabine exposure were efficiently suppressed by the subsequent exposure to sorafenib. By contrast, the reverse of this sequential administration resulted in antagonism, which may be due to differential effects on cell cycle arrest. The results suggest that sorafenib as a single agent exhibits anti-proliferative effects in vitro in NSCLC cell lines with EGFR and K-Ras mutations and that the sequential administration of gemcitabine followed by sorafenib is superior to sorafenib followed by gemcitabine and concurrent administration.

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.

Background

Lung cancer is the leading cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) - tyrosine kinase inhibitor (TKI) is used for the patients with EGFR-mutant lung cancer. Recently, phase III studies in the patients with EGFR-mutant demonstrated that EGFR-TKI monotherapy improved progression-free survival compared with platinum-doublet chemotherapy. The echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion oncogene represents one of the newest molecular targets in non-small cell lung cancer (NSCLC). Patients who harbor EML4-ALK fusions have been associated with a lack of EGFR or KRAS mutations.

Case presentation

We report a 39-year-old patient diagnosed as adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene. We treated this patient with erlotinib as the third line therapy, but no clinical benefit was obtained.

Conclusion

We experienced a rare case with EGFR mutation and EML4-ALK. Any clinical benefit using EGFR-TKI was not obtained in our case. The therapeutic choice for the patients with more than one driver mutations is unclear. We needs further understanding of the lung cancer molecular biology and the biomarker infomation.

PURPOSE OF REVIEW

Testing for epidermal growth factor receptor (EGFR) mutations has become standard practice in treating patients with advanced nonsmall cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) are being offered as first line therapy in patients with EGFR activating mutations. These drugs offer an increased progression free survival and response rate compared to standard chemotherapy in this setting, however resistance invariably occurs. This review discusses the development of resistance to EGFR TKIs and the progress that is being made to better understand how to overcome this resistance.

RECENT FINDINGS

Results from recently published papers dealing with resistance to EGFR TKIs are allowing for a better understanding of this mechanism. No one treatment allows for overcoming this resistance. Understanding this resistance will likely become an individualized patient/tumor approach. Selecting which drug or drugs that may be suitable can only be determined based on the molecular mechanism of resistance.

SUMMARY

Progress is being made in our understanding of the multiple pathways of resistance. Using a tumors molecular signature at the time of progression can determine the best treatment option.

The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.

In the last 6 years, since the first reports of an association between somatic mutations in epidermal growth factor receptor (EGFR) exons 19 and 21 and response to EGFR tyrosine kinase inhibitors (TKIs), treatment of non-small cell lung cancer (NSCLC) has changed dramatically. Based on laboratory and clinical observations, investigators have anticipated that these mutations could be predictive of response to EGFR TKIs and numerous studies have confirmed that the presence of mutation was associated with longer survival in patients receiving targeted therapy. Prospective trials comparing standard platinum-based chemotherapy with EGFR TKIs in patients with and without activating EGFR mutations validated the predictive value of molecular selection of patients for first-line treatment of advanced NSCLC. Recently, preclinical and first-in-human studies have demonstrated impressive activity of ALK TKI in tumors harboring ALK rearrangement. In this article, we review current data on molecular biology of lung cancer and evidence-based patient selection for targeted therapy.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) were initially established as second- or third-line treatment of advanced non-small-cell lung cancer (NSCLC). Subsequent studies, including IPASS, OPTIMAL, and EURTAC, have demonstrated that these TKIs are effective first-line therapeutic options in patients with tumors harboring activating mutations in the EGFR gene. The TKIs are better tolerated than conventional chemotherapy, with frequent yet mild side effects such as rash and diarrhea, and rarely interstitial lung disease. Because most patients on TKIs develop resistance due to a variety of mechanisms, the use of TKIs in the acquired-resistance setting and in the setting of earlier-staged cancers is being extensively studied. Here we review the major trials leading to the established use of EGFR TKIs in NSCLC, followed by discussion of recently completed and ongoing trials using the next-generation EGFR inhibitor afatinib.

Tyrosine kinase inhibitors (TKIs) that target the epidermal growth factor receptor (EGFR) are effective in most NSCLC patients whose tumors harbor activating EGFR kinase domain mutations. Unfortunately, acquired resistance eventually emerges in these chronically treated cancers. Two of the most common mechanisms of acquired resistance to TKIs seen clinically are the acquisition of a secondary “gatekeeper” T790M EGFR mutation that increases the affinity of mutant EGFR for ATP and activation of MET to offset the loss of EGFR signaling. Although up to one-third of patient tumors resistant to reversible EGFR TKIs harbor concurrent T790M mutation and MET amplification, potential therapies for these tumors have not been modeled in vivo. In this study, we developed a preclinical platform to evaluate potential therapies by generating transgenic mouse lung cancer models expressing EGFR-mutant Del19-T790M or L858R-T790M, each with concurrent MET overexpression. We found that monotherapy targeting EGFR or MET alone did not produce significant tumor regression. In contrast, combination therapies targeting EGFR and MET simultaneously were highly efficacious against EGFR TKI resistant tumors co-driven by Del19-T790M or L858R-T790M and MET. Our findings therefore provide an in vivo model of intrinsic resistance to reversible TKIs and offer preclinical proof of principle that combination targeting of EGFR and MET may benefit patients with NSCLC.

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.

Background

Increased focus surrounds identifying patients with advanced non-small cell lung cancer (NSCLC) who will benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI). EGFR mutation, gene copy number, coexpression of ErbB proteins and ligands, and epithelial to mesenchymal transition markers all correlate with EGFR TKI sensitivity, and while prediction of sensitivity using any one of the markers does identify responders, individual markers do not encompass all potential responders due to high levels of inter-patient and inter-tumor variability. We hypothesized that a multivariate predictor of EGFR TKI sensitivity based on gene expression data would offer a clinically useful method of accounting for the increased variability inherent in predicting response to EGFR TKI and for elucidation of mechanisms of aberrant EGFR signalling. Furthermore, we anticipated that this methodology would result in improved predictions compared to single parameters alone both in vitro and in vivo.

Results

Gene expression data derived from cell lines that demonstrate differential sensitivity to EGFR TKI, such as erlotinib, were used to generate models for a priori prediction of response. The gene expression signature of EGFR TKI sensitivity displays significant biological relevance in lung cancer biology in that pertinent signalling molecules and downstream effector molecules are present in the signature. Diagonal linear discriminant analysis using this gene signature was highly effective in classifying out-of-sample cancer cell lines by sensitivity to EGFR inhibition, and was more accurate than classifying by mutational status alone. Using the same predictor, we classified human lung adenocarcinomas and captured the majority of tumors with high levels of EGFR activation as well as those harbouring activating mutations in the kinase domain. We have demonstrated that predictive models of EGFR TKI sensitivity can classify both out-of-sample cell lines and lung adenocarcinomas.

Conclusion

These data suggest that multivariate predictors of response to EGFR TKI have potential for clinical use and likely provide a robust and accurate predictor of EGFR TKI sensitivity that is not achieved with single biomarkers or clinical characteristics in non-small cell lung cancers.