Advances in EGFR Inhibitors
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The Role of MET Amplification in EGFR Resistance
Small-molecule tyrosine kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR) have become an important therapeutic option in the treatment of advanced non–small cell lung cancer (NSCLC). In 2004, investigators discovered a class of activating mutations in the tyrosine kinase domain of the EGFR that are associated with sensitivity to EGFR-TKI therapy.7,8,13 In-frame deletions in exon 19 or substitution of arginine for leucine at position 858 (L858R) in exon 21 account for about 85% of mutations in this receptor.18 With the creation and analysis of a database encompassing multiple prospective trials of gefitinib or erlotinib in the first-line setting, improved response rates, median time to progression, and median survival were demonstrated for patients harboring one of these mutations compared to those who are wild-type for EGFR.71 More recently, results were reported for a first-line trial (IPASS Trial) of patients with advanced NSCLC who were randomized to gefitinib or doublet chemotherapy.10 Subset analysis based on EGFR status showed that patients harboring an EGFR mutation had improved progression-free survival with gefitinib compared to chemotherapy, while those patients who were EGFR wild-type showed a progression-free survival benefit in favor of chemotherapy over gefitinib.
Unfortunately, although responses to EGFR-TKIs may extend for months or even years, patients ultimately develop clinical progression. The most common reason for acquired resistance involves a secondary EGFR mutation leading to substitution of methionine for threonine at position 790 (T790M).13 This mutation, which induces resistance to EGFR-TKIs by altering the binding properties for ATP,58 is found in approximately 50% of patients who ultimately progress after initially responding to an EGFR-TKI.13,36,14
The search for secondary mechanisms of resistance has been ongoing, and one has recently been elucidated. There is increasing preclinical and clinical evidence that amplification of MET may promote drug resistance in EGFR-driven NSCLC.
Structure and function of MET
The MET gene encodes a high-affinity transmembrane receptor for hepatocyte growth factor (HGF).73 Binding of HGF to the MET receptor leads to phosphorylation of the tyrosine kinase domain and activation of downstream signaling.74 MET plays a critical role in embryogenesis, liver regeneration, and wound healing.75 In cancer cells, dysregulated MET signaling can pro mote tumor invasion, progression, and metastasis.78 Aberrant MET signaling may result from overexpression of HGF and/or MET and MET gene amplification, mutations, and structural rearrangement. The role of specific MET mutations in human cancer was first demonstrated in patients with germline MET kinase domain mutations who developed papillary renal cell carcinoma.79 In addition, MET amplification and/or over- expression has been detected in a number of cancer cell types.80
MET amplification: Role in EGFR resistance
Recent evidence suggests that aberrant MET signaling may play a key role in the development of secondary resistance to therapy with an EGFR-TKI. Investigators from our institutions created resistant clones of the gefitinib-sensitive EGFR exon 19 deletion NSCLC cell line, HCC827, by exposing it to increasing concentrations of gefitinib over 6 months.14 The resulting resistant cell line did not harbor a T790M mutation. To determine if aberrant activation of another receptor could mediate gefitinib resistance, the effects of gefitinib on 42 phosphorylated RTKs were com pared in both the HCC827 and HCC827 GR (gefitinib-resistant) lines.14 In the parental cell line, EGFR, ERBB3, ERBB2, and MET were all initially phosphorylated, but this was completely or significantly reduced with gefitinib therapy. In contrast, phosphorylation of MET, ERBB3, and EGFR persisted in the HCC827 GR cells, even in the presence of gefitinib. MET was found to be amplified in the resistant cell line but not in the parental line, and no concomitant MET mutation was detected. These data support a role for MET amplification in secondary EGFR resistance as a possible mechanism of persistent ERBB3- mediated AKT signaling.
The clinical relevance of MET amplification has been investigated by examining tumor biopsies from patients who developed secondary resistance to gefitinib or erlotinib. MET copy status was assessed in rebiopsy samples obtained from 18 NSCLC patients at the time of development of secondary resistance following an initial partial response.14 MET amplification was detected in 4 patients (22%). In another study, MET amplification was identified in 9 (21%) of 43 patients who had developed secondary resistance to an EGFR-TKI.74 As a control, only 2 of 62 untreated patients with known sensitizing EGFR mutations were found to have amplification of MET. Of note, in both studies there was a significant overlap between MET amplification and the presence of a T790M mutation.74,14 In samples taken from 61 patients at the time of secondary resistance, the T790M mutation was seen in 30 patients (49%), while 13 (21%) showed evidence of MET amplification. Six patients showed evidence of both the T790M mutation and MET amplification (Table 2).
Table 2
Overcoming resistance
Secondary EGFR resistance through MET amplification appears to involve activation of the PI3K/AKT pathway via aberrant MET signaling through ERBB3. It would be anticipated that an EGFR- TKI, whether reversible or irreversible, would not be able to sup press MET signaling of downstream pathways. Instead, overcoming this resistance would likely require some suppression of MET signaling as well. For example, when Engelman and colleagues treated gefitinib-resistant clones (HCC827 GR) using monotherapy with gefitinib or PHA665752 (Pfizer), a MET inhibitor, there was no downregulation of ERBB3/PI3K/AKT signaling and no suppression of cell growth. However, combined therapy with both gefitinib and PHA665752 resulted in a clear decrease in phosphorylation of ERRB3, AKT, and MET, with growth suppression at a concentration of approximately 50 nM (Figure 3).14
Figure 3
In light of this preclinical data, clinical trials directed at dual MET and EGFR inhibition are underway. Three compounds with activity against MET, XL880 (Exelixis), XL184 (Exelixis), and ARQ197 (Arqule), are each being combined with erlotinib in early-stage clinical trials aimed at circumventing resistance.
Future directions
As the potential role of MET in resistance to EGFR-TKI therapy is explored, several issues must be addressed. First, we need to understand to what extent MET amplification is a secondary change that arises during EGFR-TKI therapy, rather than a pre- existing mutation in a small subpopulation of tumor cells that subsequently thrive in the face of selective pressure on therapy.
In one cohort of 24 patients treated prospectively with gefitinib, pretreatment specimens were analyzed for MET amplification; all patients in this cohort were either never-smokers or had EGFR gene amplification by FISH.81 While 12 patients were found to have sensitizing EGFR mutations (with 9 partial responses to gefitinib), none had MET amplification in their pretreatment specimens. Moreover, there was no correlation between MET copy number and response, time to progression, or survival.
While this small study suggests that MET amplification arises as a secondary mechanism for resistance to EGFR-TKI therapy, other data indicate that MET amplification might be identified prior to EGFR-TKI treatment. In a study of 106 patients who received first- line gefitinib for advanced NSCLC, pretreatment samples from 39 patients were tested for MET copy number.82 One patient had a high MET copy number, along with de novo L858R and T790M mutations. Although no other patients had MET copy numbers >5.5, there was a trend for high MET copy number to predict short time to treatment failure based on logistic regression analysis.
Investigators are also exploring a role for the detection of MET overexpression by immunohistochemistry. In one study of 43 evaluable pretreatment tumor samples from patients treated with an EGFR-TKI, four patients showed strong (3+) immunohistochemical staining for MET. In this cohort, strong MET staining was significantly associated with progressive disease (P= 0.019) and a shorter time to progression (P= 0.041).83
We will need to determine reasonable and appropriate cutoff values for defining overexpression and amplification as we explore the prognostic and predictive potential of these tests. Larger studies involving both pretreatment and posttreatment samples will be required to determine the role of MET in both primary and secondary resistance to EGFR-TKI therapy, and additional clinical trials employing MET inhibitors will have to be conducted.