Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain

Abstract

Background: Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of "acquired" resistance.

Methods and findings: We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).

Conclusion: In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.

Conflict of interest statement

Competing Interests: See Acknowledgments.

Figures

Figure 1. Re-Biopsy Studies

(A.) Patient 1. CT-guided biopsy of progressing lung lesions after 10 mo on gefitinib (left panel). Two months later, fluid from a right-sided pleural effusion (right panel) was collected for molecular analysis. (B) Patient 2. CT-guided biopsy of a progressing thoracic spine lesion (left panel) and fluoroscopic-guided biopsy of a progressing lung lesion (right panel). The biopsy needles are indicated by white arrows.

Figure 2. Sequencing Chromatograms with the T790M EGFR Exon 20 Mutation in Various Clinical Specimens and the NSCLC Cell Line H1975

(A–C) In all three patients—patient 1 (A), patient 2 (B), and patient 3 (C)—the secondary T790M mutation was observed only in lesions obtained after progression on either gefitinib or erlotinib. (D) Cell line H1975 contains both an exon 21 L858R mutation (upper panel) and the exon 20 T790M mutation (lower panel). The asterisks indicate a common SNP (A or G) at nucleotide 2361; the arrows indicate the mutation at nucleotide 2369 (C→T), which leads to substitution of methonine (ATG) for threonine (ACG) at position 790. In the forward direction, the mutant T peak is blue. In the reverse direction, the mutant peak is green, while the underlying blue peak represents an “echo” from the adjacent nucleotide.

Figure 3. A Novel PCR-RFLP Assay Independently Confirms Presence of the T790M Mutation in Exon 20 of the EGFR Kinase Domain

(A) Design of the assay (see text for details). “F” designates the fluorescent label, FAM. At the bottom of this panel, the assay demonstrates with the 97-bp NlaIII cleavage product the presence of the T790M mutation in the H1975 cell line; this product is absent in H2030 DNA. The 106-bp NlaIII cleavage product is generated by digestion of wild-type EGFR. (B) The PCR-RFLP assay demonstrates that pre-drug tumor samples from the three patients lack detectable levels of the mutant 97-bp product, while specimens obtained after disease progression contain the T790M mutation. Pt, patient.

Figure 4. EGFR Mutants Containing the T790M Mutation Are Resistant to Inhibition by Gefitinib or Erlotinib

293T cells were transiently transfected with plasmids encoding wild-type (WT) EGFR or EGFR mutants with the following changes: T790M, L858R, L858R + T790M, del L747–E749;A750P, or del L747–E749;A750P + T790M. After 36 h, cells were serum-starved for 24 h, treated with gefitinib or erlotinib for 1 h, and then harvested for immunoblot analysis using anti-p-EGFR (Y1092), anti-t-EGFR, anti-phosphotyrosine (p-Tyr), and anti-actin antibodies as described in Methods. The EGFR T790M mutation, in conjunction with either wild-type EGFR or the drug-sensitive L858R EGFR mutant, prevents inhibition of tyrosine phosphorylation (A) or p-EGFR (B) by gefitinib. Analogously, the T790M mutation, in conjunction with the drug-responsive del L747–E749;A750P EGFR mutant, prevents inhibition of p-EGFR by erlotinib (C).

Figure 5. Sensitivity to Gefitinib Differs Among NSCLC Cell Lines Containing Various Mutations in EGFR or KRAS

The three indicated NSCLC cell lines, H3255 (L858R mutation), H1975 (both T790M and L858R mutations), and H2030 (wild-type EGFR, mutant KRAS) (see Table 2), were grown in increasing concentrations of gefitinib, and the density of live cells after 48 h of treatment was measured using a Calcein AM fluorescence assay. Fluorescence in vehicle-treated cells is expressed as 100%. Results are the mean ± standard error of three independent experiments in which there were four to eight replicates of each condition. Similar results were obtained with erlotinib (see Figure S3).