EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib
Freely available online through the PNAS open access option.
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.
Tyrosine kinases (TKs) regulate signaling pathways that control critical cellular activities (1). When overexpressed or activated by mutations, TKs can contribute to the development of cancers. If tumor cells depend on a mutant TK for survival, as illustrated by certain mouse models of cancer (2, 3), the mutated enzyme can fortuitously serve as an Achilles' heel for cancer therapy (4). Human examples include BCR-ABL-dependent chronic myelogenous and acute lymphoblastic leukemias (5), KIT- and PDGFRA-dependent gastrointestinal stromal tumors (6), and PDGFRA-dependent hypereosinophilic syndrome (7). In each disease, activated oncogenes encode TKs; inhibition by imatinib mesylate (Gleevec) leads to rapid and durable clinical responses.
EGFR is a TK of the ErbB family that is the presumptive target of the TK inhibitor (TKI) gefitinib. This drug is an anilinoquinazoline (Fig. 5, which is published as supporting information on the PNAS web site) that reversibly competes with ATP at a critical ATP-binding site (lysine 745; K745) within the epidermal growth factor (EGF) receptor (EGFR) protein (8, 9). In vitro, gefitinib selectively inhibits the kinase activity of EGFR versus a handful of other kinases (10). In two phase II trials, radiographic regressions of tumors were observed in 28% of patients treated in Japan and 10% of those studied in Europe and the U.S. (11, 12). Dramatic responses occurred within the first two weeks of initiating therapy (e.g., ref. 13), similar to those seen in the murine and human examples noted above. Assuming that the drug did affect a kinase, these kinds of responses suggested that at least some lung tumors depended on a specific genetic lesion for tumor survival. However, when gefitinib was approved as second- or third-line treatment for patients with non-small cell lung cancer (NSCLC), the clinically relevant target(s) of the drug in human tumors were unknown. Analyses of both preclinical xenograft models (14) and specimens from gefitinib-sensitive and -refractory tumors (15) did not reveal any obvious relationship between EGFR expression levels and tumor sensitivity. Retrospective epidemiologic analyses suggested that gefitinib is more likely to be effective in Japanese patients (11), individuals with adenocarcinomas of the bronchioloalveolar carcinoma (BAC) subtype, and “never smokers” (16).
Recently, two groups have shown that mutations in the TK domain of EGFR are associated with sensitivity of NSCLC to gefitinib (17, 18). In total, deletions or amino acid substitutions in exons 18, 19, and 21 of EGFR were found in 13 of 14 tumors sensitive to the drug, but in none of 11 tumors with no response. Lynch and colleagues (17) found mutations in another 2 of 25 primary NSCLCs, and Paez et al. (18) found EGFR mutations in 16 of 119 unselected tumors, with a striking predominance of mutations found in 15 of 58 (28%) specimens from Japan as compared to 1 of 61 from the U.S. (2%).
To confirm and extend data on gefitinib sensitivity, we examined the status of the TK domain of EGFR in tumors that were sensitive and refractory to the drug. To determine whether a related but distinct TKI, erlotinib (Fig. 5), “targets” a similar subset of NSCLCs, we also profiled erlotinib-sensitive and -refractory tumors. The clinically relevant target of erlotinib has not yet been documented. To examine whether smoking history is predictive of the likelihood of EGFR mutations, we determined the incidence of EGFR TK domain mutations in 96 resected NSCLCs from never smokers, as well as former and current smokers who had never received a TKI. Finally, in an effort to explain the selective advantage of cells with mutant EGFR and the drug sensitivity conferred upon mutant-bearing tumors, we began to characterize some biochemical properties of EGFR mutants in vitro.
M, male; F, female. Smoking indicates smoking history; never, smoked <100 cigarettes in a lifetime; former, smoked 100 or more cigarettes and quit > 1 year prior to diagnosis of lung cancer. BWFI, BAC with focal invasion; AWFB, adenocarcinoma with BAC features; ADENO, adenocarcinoma; SQUAM, squamous. Mutation indicates amino acids affected in EGFR; for all tumor specimens, exons 18-24 of EGFR, which encode the TK domain, were examined; asterisks denote homozygous deletions; none, no mutation observed. Duration indicates months of drug-induced response. OS indicates months of overall survival after starting therapy with gefitinib or erlotinib. +, still on drug and/or alive at the time of writing of this manuscript. No mutations were observed in exons 18-24 in 8 and 10 patients refractory to gefitinib and erlotinib, respectively.
Exons 2-28 from EGFR were examined for mutations in 96 surgically resected NSCLCs. Mutations in the TK domain were detected in 7 of 15 never smokers with adenocarcinomas and in 4 of 81 NSCLCs resected from former or current smokers. All abbreviations as per Table 1.Click here to view.
We thank J. Eldred, G. Fewell, T. Miner, J. Reed, C. Jeliti, H. Sun, E. Boatright, H. Bauer, and N. Hershberger from Washington University for technical assistance and data generation/analysis, and M. Watson from the Siteman Cancer Center Tissue Procurement Core for additional preparation of genomic DNA samples. We especially thank R. Bhargava and M. Ladanyi from the Memorial Sloan-Kettering Cancer Center for DNA extraction from paraffin-embedded tumors; D. Tabarini, M. Ilzarbe, and D. Wong from the Memorial Sloan-Kettering Cancer Center Sequencing Facility, and D. K. Pham for management of the surgical tumor bank. Additionally, we acknowledge M. Resh for the wild-type EGFR cDNA; E. Venkatraman for statistical calculations; P. Yurttas and N. Pavletich for discussion about EGFR structure; J. Massague for use of imagequant software; F. Cong for helpful discussions; V. Ty for peripheral blood collection; N. Shah, J. Kunkle, L. LoGuidice, R. Henry, M. Cespon, E. Ro, and J. Jones for clinical research assistance; and AstraZeneca and Genentech for supply of gefitinib and erlotinib, respectively. W.P. was supported by National Cancer Institute Grant CA009512 and the Steps for Breath Foundation. The work was also funded by an anonymous donor.
Abbreviations: TK, tyrosine kinase; TKI, TK inhibitor; EGF, epidermal growth factor; EGFR, EGF receptor; t-EGFR, total EGFR; p-EGFR, phospho-EGFR; NSCLC, non-small cell lung cancer; BAC, bronchioloalveolar carcinoma.
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