MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

Abstract

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFR(T790M) mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFR(T790M) mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.

Fig. 1.

Recurrence of chromosomal alterations found in EGFR mutant lung adenocarcinomas from patients with acquired resistance to EGFR tyrosine kinase inhibitors (n = 12) or from untreated patients (n = 38). Shown is the percentage of samples with CNAs after data segmentation (y axis) plotted for each probe evenly aligned along the x axis in chromosome order. The gray areas denote counts of chromosomal gain and loss defined by log₂ ratios ±0.2. Amplifications or deletions having >2-fold change in copy number, defined by log₂ ratios ±1.0, are shown by bright red and bright green lines, respectively. Asterisks denote amplifications that occurred in more than one sample in the acquired resistance cohort.

Fig. 2.

MET status and sensitivity of H820 cells to the MET inhibitor, XL880. (A) H820 cells display, as assessed by dual-color FISH analysis (see Materials and Methods), ≈4–6 copies of chromosome 7 (CEP7), with additional focal amplifications at loci containing MET. The cells shown are representative of the whole population. Scale bar: 10-μm. CEP7 probe, green; MET probe, red; nuclei, blue (DAPI). (B) Lysates from H820 and PC-9 cells were immunoblotted by using anti-phospho-(p)-MET (Y^1234/5), anti-total MET, and anti-actin antibodies. H820 cells harbor an exon 19 deletion (E746-E749), a T790M mutation, and amplified no MET, whereas PC-9 cells harbor an exon 19 deletion (E746-A750), no T790M mutation, and no MET amplification. (C) Growth inhibition curves of H820 and PC-9 cells treated with MET and EGFR inhibitors at various concentrations. Error bars indicate one standard deviation from six replicates.

Fig. 3.

ERBB3 signaling in H820 cells depends on MET status. Lysates from H820 and PC-9 cells, either untreated (DMSO-only control; 0 μM) or treated with 0.3 μM XL880 or 0.3 μM CL-387,785, were immunoblotted by using anti-phospho-(p)-MET (Y^1234/5), anti-total MET, anti-phospho-(p)-ERBB3 (Y¹²⁸⁹), anti-total ERBB3, anti-phospho-(p)-EGFR (Y¹⁰⁹²), anti-total EGFR, and anti-actin antibodies.