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Keywords:

  • Acquired resistance;
  • EGFR;
  • gene mutation;
  • lung cancer;
  • tyrosine kinase inhibitors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

Epidermal growth factor receptor (EGFR) kinase domain mutations (from exon 18 to 21) alter the efficacy of the EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung cancer patients with drug-sensitive EGFR mutations initially respond, but acquire resistance after approximately one year of tyrosine kinase inhibitor treatment. This review summarizes EGFR gene mutation profiles for East Asian non-small-cell lung cancer patients according to recent publications. Strategies for the treatment of acquired resistance mediated by EGFR T790M are also reviewed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

In 2004, three independent studies reported that most lung cancer patients who responded to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), erlotinib or gefitinib, harbored EGFR mutations.1–3 More recent data has elucidated that EGFR mutations are strong predictors of efficacy for the EGFR TKI. Clinical trials indicated that more than 70% of lung cancer patients harboring EGFR 19 deletions or L858R point mutation are sensitive to the EGFR TKI gefitinib (Iressa) and erlotinib (Tarceva).4,5 In contrast, less than 1% of patients with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations respond to the treatment of EGFR TKI.6–8

Increasing evidence indicates that cancer can be further classified as a group of distinct genetic diseases. Within this group of diseases gene mutations are required for the development, maintenance, and progression of the disease.9–12 Gene mutations capable of establishing and maintaining cancers have been labeled driver mutations.13

The discovery of driver-dependent cancers has signified a new era for cancer treatment. The majority of known driver mutations occur in genes that encode protein kinases. These mutated kinases constitutively activate signaling pathways independent of stimulus, resulting in tumorigenesis. Small molecule inhibitors that specifically target these proteins can inactivate signaling pathways, thus inhibiting continued growth of tumor cells. Currently, there are more than 140 small molecule inhibitors designed to target 20 mutant kinases in clinical trials.14 Fourteen of these have been approved by the Food and Drug Administration (FDA).15

The receptor tyrosine kinase (RTK) EGFR has been defined as a driver of overexpression of the mutant protein. Wild-type EGFR expressed at normal levels is activated by the binding of its cognate ligands, such as epidermal growth factor (EGF). Activated EGFR can signal through the RAS-RAF-MEK pathway, involved in cell proliferation, or the PI3K-AKT- mammalian target of rapamycin (mTOR) pathway, involved in cell survival.16 Mutation of the EGFR can result in constitutive tyrosine kinase activity and signaling in the absence of ligands, leading to the development of tumors. First generation TKI, such as gefitinib and erlotinib, bind to EGFR in the nucleotide-binding pocket, inhibiting tyrosine autophosphorylation and suppressing tumor growth.

The promising results with EGFR TKI use in lung cancer patients have generated tremendous enthusiasm for developing therapeutics that specifically inhibit pathways activated by mutations. Unfortunately, most lung cancer patients who are initially sensitive to TKI such as gefitinib and erlotinib develop resistance after approximately one year of treatment. Roughly 50% of the acquired resistance to EGFR TKI is the result of a second EGFR mutation, T790M.

This review will summarize the EGFR mutation profiles in East Asian non-small-cell lung cancer (NSCLC) patients and update the strategies for overcoming acquired resistance mediated by the EGFR T790M mutant.

EGFR mutations are predictors of TKI treatment response

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

Many retrospective studies have indicated that the response rate to TKI is higher in lung cancer patients with adenocarcinoma histology than in others.17,18 Lung cancer patient characteristics that correlate with a statistically significant higher response to TKI treatment include being of East Asian descent, female, and non-smoking (defined as less than 100 cigarettes in a patient's lifetime). This is in contrast to the lower TKI response reported in Caucasian, male, smoker populations. Examination of the molecular characteristics associated with these patient populations revealed that EGFR mutations are more frequent in patients of East Asian descent than that in Caucasian NSCLC patients (35% versus 10%). Additionally, female non-smoking patients with adenocarcinoma histology also exhibited a higher frequency of EGFR mutations than non-adenocarcinoma, male, smoking patients (Fig. 1a). Thus, EGFR mutations can determine the efficacy of TKI treatment.

image

Figure 1. (a) Correlation of epidermal growth factor receptor (EGFR) mutation frequency in East Asian non-small-cell cancer (NSCLC) patients with histology, gender, and smoking status. The frequencies are calculated based on recent publications.18–31 (b) Mutation frequency in EGFR from Exon 18 to Exon 21 in East Asian NSCLC patients. The frequencies are calculated based on recent publications.18–31 Adeno: Adenocarcinoma; del: deletion; ins: insertion; Non-Adeno: non-adenocarcinoma NSCL.

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Figure 1b summarizes EGFR mutation frequencies found in East Asian NSCLC patients. The majority of EGFR mutations occur in the kinase domain, which is encoded by exons 18 to 21. The exon 19 deletion, which results in the loss of residues 747–750 (Leu Arg Glu Ala), and the exon 21 L858R point mutation account for approximately 90% of primary NSCLC mutations. Patients harboring exon 19 deletion and L858 point mutation are sensitive to TKI treatment (i.e. gefitinib or erlotinib). G719A/C/S substitution in exon 18 and L861Q mutations account for roughly 2.3% of mutations and are also sensitive to the TKI. By contrast, patients with exon 20 insertions, which account for 5.8%, do not respond to TKI treatment.18–31 The T790M mutation accounts for 1.4% and is related to TKI acquired resistance (Fig. 1b).

T790M mutation is associated with acquired resistance to EGFR TKI

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

In 2005, Pao et al. reported that three lung adenocarcinoma patients (two who had never smoked and one smoker) acquired resistance to gefitinib or erlotinib after about 10 months of disease improvement. It was found that these patients acquired a second mutation in EGFR exon 20, T790M.32 To determine if the mechanism of resistance was reliant on the second mutation T790M, Pao and colleagues ectopically expressed the mutant EGFR L858R + T790M or L747_E749del; A759P + T790M in cells with low endogenous levels of EGFR. Both gefitinib and erlotinib treatment significantly decreased the phosphorylation of L858R or L747_E749del; A759P mutant EGFR. However, tyrosine phosphorylation of mutant EGFR containing T790M (L858R + T790M or L747_E749del; A759P + T790M) was not significantly reduced in the presence or absence of TKI.

Concurrently, Kobayashi et al. reported a lung adenocarcinoma patient who relapsed after 24 months of complete remission after gefitinib monotherapy. DNA sequencing identified that this 71-year-old former smoker with a previously documented EGFR exon 19 deletion, acquired a second mutation, T790M.33 After performing structural modeling based on a published crystal structure,34 they suggested that the replacement of threonine (T) with methionine (M) introduces a bulkier amino acid side chain which interferes with the binding of TKI. Another direct binding assay provided a clearer picture for the mechanism of the acquired resistance mediated by T790M. Compared with the gefitinib-sensitive L858R mutant, the introduction of the second mutation T790M (L858R + T790M) decreased the Michaelis–Menten constant (Km) of EGFR for adenosine triphosphate (ATP) greatly (Km 148 versus 8.4). These data suggested that T790M mutation increased ATP affinity, reducing the potency of the ATP-competitive TKI. Based on this data, it was thought that irreversible inhibitors could overcome the resistance mediated by T790M (details below in Second generation TKI).

About 50% of NSCLC patients who acquire resistance to gefitinib or erlotinib harbor the EGFR T790M mutation. Whether the TKI induce the T790M mutation or merely select a small number of clones with pre-existing T790M mutations is unclear. With an enriched polymerase chain reaction assay that can detect one mutant out of 1000 wild-type alleles Inukai et al. were able to detect nine previously unidentified T790M mutants in a cohort of 280 NSCLC samples. Only one case was detected using direct sequencing.35 T790M mutants were also detected in captured circulating tumor cells using a high sensitivity chip prior to the TKI treatment.36 In addition, Bell et al. reported a germline EGFR T790M mutation in a family of European descent in which multiple members developed NSCLC.37

Overcoming resistance to TKI mediated by EGFR T790M mutation

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

Second generation TKI

New inhibitors with a higher affinity for EGFR than that of ATP were designed to overcome the acquired TKI resistance conferred by T790M. Unlike gefitinib or erlotinib, these so called second generation TKI form a covalent bond with a conserved C797 residue in the EGFR, thus binding to EGFR irreversibly. Several irreversible EGFR inhibitors, such as PF00299804, BIBW2992 and HKI-272, showed promising results in preclinical studies of lung cancer models with the T790M mutation.38–40 However, results from a recent phase II clinical trial for HKI-272 (Neratinib) were disappointing. In a large cohort of 167 patients with advanced NSCLC, none of the 12 patients with an EGFR T790M mutation responded to the treatment.41 The gloomy view for this TKI is due to their dose limitation. Because this quinazoline-based TKI has a similar affinity for wild type EGFR and T790M mutant EGFR, the effective concentration for an EGFR T790M mutant also inhibits the wild-type EGFR, leading to a sometimes severe toxicity manifested as a red rash and diarrhea.42Table 1 summarizes some irreversible EGFR TKI in clinical trials.

Table 1.  List of irreversible tyrosine kinase inhibitors currently in clinical trials
Inhibitor nameTargetsStatusCompany
  1. Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany; Pfizer Inc, New York City, New York, USA; Wyeth, Madison, New Jersey, USA.

  2. EGFR, epidermal growth factor receptor.

BIBW2992EGFR/HER2Phase III-NSCLCBoehringer Ingelheim GmbH
CI-1033Pan-ERBBPhase IIPfizer Inc
HKI-272Pan-ERBBPhase IIWyeth
PF 00299804Pan-ERBBPhase IIPfizer Inc

Using a high-throughput screen, Zhou et al. identified three novel EGFR inhibitors selective for EGFR T790M (WZ3146, WZ4002 and WZ8040) with an anilinopyrimidine core, which has an intrinsically better fit to the T790M mutant. These compounds exhibited greater selectivity for the EGFR T790M mutant than EGFR wild type. Mouse lung cancer model studies indicated that WZ4002 reduced the T790M-containing tumor volume significantly without serious toxicity compared to vehicle only treated mice.42

Combination therapy

mRNA expression profiles of lung tumor cells expressing the EGFR L858R/T790M mutant showed that the EGFR ligands amphiregulin and epiregulin were overexpressed compared to normal cells. This implies that interference with ligand-binding to EGFR might, in combination with a TKI, inhibit the tumor more effectively. By combining BIBW2992 (an irreversible EGFR and HER2 TKI) with cetuximab (a FDA approved EGFR antibody for colon cancer therapy), Pao et al. observed a dramatic reduction of the erlotinib- resistant tumor volume in a mouse model.43 A phase I clinical trial of the combination therapy of BIBW-2992 and cetuximab for the treatment of advanced lung cancers with acquired resistance is ongoing (http://www.clinicaltrials.gov).

The PI3K-AKT-mTOR pathway plays a central role in the development and maintenance of lung cancer. mTOR is a serine/threonine kinase activated downstream of the PI3K, which makes mTOR a promising component in the treatment of cancers. By combining HKI-272 and rapamycin (an mTOR inhibitor), Li et al. showed a significant regression of lung tumors generated in a mouse model carrying EGFR L858R/T790M mutations.44 Nakachi et al. also observed a greater inhibition of cell growth by combining the pan-RTK inhibitor AEE788 with an mTOR inhibitor RAD-001, even though the individual use of AEE788 or RAD-001 only had a small effect on cell growth.45

Thymidylate synthase (TS) is a protein involved in generating and repairing DNA. It catalyzes the methylation of deoxyuridine monophosphate to form deoxythymidine monophosphate. Data indicated that high levels of TS may be involved in the development of cancers.46,47 By using cell lines and xenografts containing the T790M mutations, Takezawa et al. showed that a TS-targeting agent, S-1, enhanced the antitumor effect of BIBW2992 in NSCLC with acquired resistance to EGFR-TKI.48

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References

The EGFR T790M mutation accounts for approximately 50% of acquired resistance to EGFR TKI. In addition to T790M, the EGFR T854A mutation was also found to be associated with acquired resistance.49 Recent studies demonstrate that tumor cells can switch on other signaling pathways to escape current therapies.50,51 Engelman et al. found that in an induced gefitinib-resistant cell line (HCC827GR), the copy number of mesenchymal epithelial transition factor (MET) increased (or was amplified). The amplification of MET leads to the activation of the ERBB3-PI3K-AKT signal transduction pathway, which aids tumor cells in escaping from gefitinib treatment. About 20% of acquired EGFR TKI resistance cases are related to MET amplification.50,52 The mechanisms of acquired resistance to EGFR TKI for the remaining 30% of cases are unknown. In the meantime, combination therapy against different targets may represent a more effective way to treat cancers.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. EGFR mutations are predictors of TKI treatment response
  5. T790M mutation is associated with acquired resistance to EGFR TKI
  6. Overcoming resistance to TKI mediated by EGFR T790M mutation
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References