EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib

Abstract

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene are reportedly associated with sensitivity of lung cancers to gefitinib (Iressa), kinase inhibitor. In-frame deletions occur in exon 19, whereas point mutations occur frequently in codon 858 (exon 21). We found from sequencing the EGFR TK domain that 7 of 10 gefitinib-sensitive tumors had similar types of alterations; no mutations were found in eight gefitinib-refractory tumors (P = 0.004). Five of seven tumors sensitive to erlotinib (Tarceva), a related kinase inhibitor for which the clinically relevant target is undocumented, had analogous somatic mutations, as opposed to none of 10 erlotinib-refractory tumors (P = 0.003). Because most mutation-positive tumors were adenocarcinomas from patients who smoked <100 cigarettes in a lifetime ("never smokers"), we screened EGFR exons 2-28 in 15 adenocarcinomas resected from untreated never smokers. Seven tumors had TK domain mutations, in contrast to 4 of 81 non-small cell lung cancers resected from untreated former or current smokers (P = 0.0001). Immunoblotting of lysates from cells transiently transfected with various EGFR constructs demonstrated that, compared to wild-type protein, an exon 19 deletion mutant induced diminished levels of phosphotyrosine, whereas the phosphorylation at tyrosine 1092 of an exon 21 point mutant was inhibited at 10-fold lower concentrations of drug. Collectively, these data show that adenocarcinomas from never smokers comprise a distinct subset of lung cancers, frequently containing mutations within the TK domain of EGFR that are associated with gefitinib and erlotinib sensitivity.

Figures

Fig. 1.

Deletion mutations in exon 19 of EGFR from NSCLCs sensitive to TKIs, gefitinib (G) or erlotinib (E); all lack four amino acids, LREA, which are conserved among vertebrate species. (A) Nucleotide alignments. (B) Amino acid alignments. (C) Amino acid alignments of EGFR from various species. All alignments were generated by using Vector nti software. See Table 1 for characteristics of patients.

Fig. 2.

The del L747-S752 mutant EGFR appears to have reduced kinase activity. 293T cells were transiently transfected with vector alone (V) or vector containing wild-type (WT) EGFR, del L747-S752, or L858R. Thirty-six hours later, cells were serum-starved for 24 h and then harvested for immunoblot analyses using anti-phosphotyrosine (p-Tyr), anti-phospho-EGFR (p-EGFR Y1092), anti-total EGFR (t-EGFR), and anti-actin antibodies as described in Methods.(A and B) Time course of ligand-induced activation of del L747-S752 mutant EGFR. Cells were treated with 100 ng/ml EGF for 0-180 min. (B Left) A 5-min exposure of an immunoblot assayed with an anti-phosphotyrosine antibody. (B Right) A 15-min exposure. (C) Comparison of WT EGFR with two EGFR mutants, as assessed by detection of p-Tyr and p-EGFR (Y1092).

Fig. 3.

Compared to wild-type EGFR, the del L747-S752 mutant has similar sensitivity to TKIs, whereas the L858R mutant is inhibited at ≈10-fold lower concentrations of drug. (A) Dose-dependent inhibition by gefitinib of del L747-S752 mutant EGFR as compared to wild type. Cells were treated with gefitinib at various concentrations for 1 h before lysis. Results with erlotinib and in the presence of EGF were similar (data not shown). (B) Dose-dependent inhibition by erlotinib of L858R mutant as compared to wild-type EGFR. Dilution points at 0.0001 micromolar are not shown. Results with gefitinib were similar (data not shown). V, vector alone.

Fig. 4.

Summary of mutations reported here and previously detected in the TK domain of EGFR in NSCLCs. Schematic view of EGFR and key domains, with an expanded view of the TK domain encoded by exons 18-24 (amino acids 718-964). Yellow, sensitive to gefitinib; red, sensitive to erlotinib; black, never treated with TKI. Data are from this paper and refs. and . All exon 19 deletions lack amino acids LREA except for one reported by Paez et al. (18) (del S752-I759). In our series, one tumor with an L858R mutation also had an R776C mutation (see Table 1). Functional landmarks include the GXGXXG motif at position 719, the K745 critical for ATP-binding, the DFG motif at position 855, and a tyrosine at 869. The sites of described mutations are G719, L747-A750, S752, R776, H835, L858, and L861.