NovelD761YandCommonSecondary T790MMutationsinEpidermal Growth Factor Receptor^Mutant LungAdenocarcinomaswith AcquiredResistance toKinase Inhibitors

Purpose: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance toTKImonotherapy. Experimental Design: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domainmutations by molecular analyses. Results: Eight of16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations wereT790M and one was a novel D761Y mutation found in a brain metastasis.When combined with a drug-sensitive L858R mutation, the D761Ymutation modestly reduced the sensitivity of mutant EGFR toTKIs in both surrogate kinase and cell viability assays. In an autopsy case, theT790M mutation was found in multiple visceral metastases but not in a brain lesion. Conclusions:TheT790M mutation is common in patients with acquired resistance. The limited spectrum ofTKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds toABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target. Somatic activating mutations in exons encoding the epidermal growth factor receptor (EGFR) tyrosine kinase domain are found in f10% and 25% of non–small-cell lung cancers (NSCLC) from the United States and East Asia, respectively (1). Nearly 90% of these mutations occur as either multinucleotide in-frame deletions in exon 19 or as single missense mutations that result in substitution of arginine for leucine at position 858 (L858R). Both mutations are associated with increased sensitivity to the selective EGFR kinase inhibitors gefitinib and erlotinib (2–4). Our initial analysis suggests that patients whose tumors harbor EGFR exon 19 deletion mutations have a longer median survival than those with EGFR L858R point mutation (5). Patients with drug-sensitive EGFR mutations, whose tumors initially respond to gefitinib or erlotinib, develop acquired resistance after a median of about 12 months (5). We previously reported that in two of five NSCLC patients with such acquired resistance, tumors biopsied after disease progression contained a second-site mutation in the EGFR kinase domain, in addition to a drug-sensitive mutation (6). This C!T mutation at nucleotide 2,369 in exon 20 leads to substitution of methionine for threonine at position 790 (T790M). Whereas the amino acid Cancer Therapy: Clinical Authors’Affiliations: Human Oncology and Pathogenesis Program, Thoracic Oncology Service, Varmus Lab, Program in Cancer Biology and Genetics, Department of Pathology, Organic Synthesis Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center; and Department of Medicine,Weill Medical College of Cornell University, New York, New York Received 6/29/06; accepted 8/22/06. Grant support: Doris Duke Charitable Foundation (W. Pao); Joan’s Legacy: The Joan Scarangello Foundation to Conquer Lung Cancer (W. Pao); NIH grants 5T32CA009207 (G.J. Riely), R21-CA115051 (V.A. Miller), and K08-CA097980 (W. Pao); The Carmel Hill Fund; postdoctoral fellowship from The Canadian Institute of Health Research (R. Somwar); and the Doris Duke Clinical Scientist Development Award (W. Pao). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). The rights to a patent application on the testing of the EGFR T790Mmutation have been licensed to Molecular MD byMSKCC.This applies toW. Pao andV. Miller. Requests for reprints: William Pao, Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, Box 125, 1275 York Avenue, New York, NY 10021. Phone: 212-639-2761; Fax: 212-717-3125; E-mail: paow@ mskcc.org. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-1570 www.aacrjournals.org Clin Cancer Res 2006;12(21) November1, 2006 6494 Research. on April 15, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from change does not impair the catalytic activity of EGFR, it is predicted to block binding of either gefitinib or erlotinib to the EGFR ATP-binding domain. Others have reported similar findings collectively in drug-resistant tumors from five patients (7–10). Certain cases of inherited susceptibility to lung cancer may be associated with the T790M mutation (11), although somatic T790M mutations in patients who have never received gefitinib or erlotinib are very rare (6). In this study, we aimed to elucidate the frequency and nature of secondary EGFR mutations in 16 more patients who progressed on either gefitinib or erlotinib monotherapy. Other exploratory studies were done to uncover additional potential mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Materials andMethods Tissue procurement. Tumor specimens, including paraffin blocks, biopsies, and pleural effusions, were obtained through protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (protocols 92-055, 02-010, and 04-103). All patients provided informed consent. In total, 17 patients with acquired resistance were evaluated, but DNA from one patient was insufficient for analysis. Mutational analyses. Genomic DNA was extracted from tumor specimens and primers for EGFR (exons 18-24) analyses were as published (4). PCR-RFLP assays for exon 19 deletions and L858R and T790M missense mutations were done as published (6, 12). All mutations were confirmed at least twice from independent PCR isolates and sequence tracings were reviewed in the forward and reverse directions by visual inspection. Reverse transcription-PCR. EGFR cDNA was generated and examined as published (6). Functional analyses of EGFR D761Y. Two numbering systems are used for EGFR. The first denotes the initiating methionine in the signal sequence as amino acid 24. The second, used here, denotes the methionine as amino acid +1. Mutations were introduced into the full-length mutant EGFR L858R using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and cloned into the expression vector, pcDNA3.1( ), as described (4). The following primers were used to generate the D761Y mutation: D761Y F, 5¶-GGAAATCCTCTATGAAGCCTA-3¶; D761Y R, 5¶-TAGGCTTCATAGAGGATTTCC-3¶. The generation of the EGFR L858R + T790M cDNA was previously described (6). Immunoblotting. See methods and supplementary methods in ref. 4 for details on transient transfection of 293T cells, cell lysis, immunoblotting, and antibody reagents. At least three independent experiments were done for all analyses. Generation of EGFR mutant-expressing Ba/F3 cells and growth inhibition analyses. Ba/F3 cells (a kind gift of Dr. C. Antonescu, Memorial Sloan-Kettering Cancer Center, New York, NY) were maintained in RPMI supplemented with 10% fetal bovine serum, 10 ng/mL interleukin-3 (IL-3; R&D Systems, Minneapolis, MN), and 10 units/mL penicillin and 10 Ag/mL streptomycin, at 37jC and 5% CO2. To generate polyclonal cell populations, Ba/F3 cells were electroporated (Amaxa, Gaithersburg, MD) with 2 Ag each of the following plasmids: pcDNA3.1( ) empty vector, pcDNA3.1( )-EGFR L858R , pcDNA3.1( )-EGFR L858R + D761Y , and pcDNA3.1( )EGFR L858R + T790M . Following selection in 1.25 mg/mL G418 (Invitrogen, Carlsbad, CA) for 12 days in the presence of 10 ng/mL IL-3, G418-resistant cells were subjected to a second round of selection in the absence of IL-3 for 5 days and then maintained in the presence of 10 ng/mL IL-3 for further study. Ba/F3 growth inhibition assays were done with the CellTiter-Blue cell viability kit (Promega, Madison, WI) as per instructions of the manufacturer. Briefly, Ba/F3 transfectants were washed with PBS twice and resuspended in RPMI medium containing 10% fetal bovine serum and 40 ng/mL EGF (Cell Signaling Technology, Danvers, MA). Cells were then seeded into 96-well plates in triplicate at a density of 100,000 per well and treated with various concentrations of EGFR inhibitors for 48 or 72 hours. Cell viability was calculated according to the CellTiter-Blue–emitted fluorescence at 530 nm (ex)/590 nm (em) using a Fluoroskan Ascent FL plate reader (Thermo Electron Corporation, Waltham, MA). All assays were done at least three