n engl j med 350;21 www.nejm.org may 20, 2004 2129 The new englandjournal of medicine established in 1812 may 20 , 2004 vol. 350 no. 21 Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non–Small-Cell Lung Cancer to Gefitinib Thomas J. Lynch, M.D., Daphne W. Bell, Ph.D., Raffaella Sordella, Ph.D., Sarada Gurubhagavatula, M.D., Ross A. Okimoto, B.S., Brian W. Brannigan, B.A., Patricia L. Harris, M.S., Sara M. Haserlat, B.A., Jeffrey G. Supko, Ph.D., Frank G. Haluska, M.D., Ph.D., David N. Louis, M.D., David C. Christiani, M.D., Jeff Settleman, Ph.D., and Daniel A. Haber, M.D., Ph.D.abstract From the Cancer Center (T.J.L., D.W.B., R.S.,S.G., R.A.O., B.W.B., P.L.H., S.M.H., J.G.S.,F.G.H., D.N.L., D.C.C., J.S., D.A.H.) and theDepartments of Medicine (T.J.L., D.W.B.,J.G.S., F.G.H., D.C.C., J.S., D.A.H.) and Pa-thology (D.N.L.), Massachusetts GeneralHospital and Harvard Medical School; andthe Harvard School of Public Health (S.G.,D.C.C.) — all in Boston. Address reprint re-quests to Dr. Haber at MGH Cancer Center,Bldg. 149, 13th St., Charlestown, MA 02129,or at [email protected]. Lynch, Bell, and Sordella contributedequally to the article.This article was published at www.nejm.orgon April 29, 2004. N Engl J Med 2004;350:2129-39. Copyright © 2004 Massachusetts Medical Society. background Most patients with non–small-cell lung cancer have no response to the tyrosine kinaseinhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). How-ever, about 10 percent of patients have a rapid and often dramatic clinical response. Themolecular mechanisms underlying sensitivity to gefitinib are unknown. methods We searched for mutations in the EGFR gene in primary tumors from patients with non–small-cell lung cancer who had a response to gefitinib, those who did not have a re-sponse, and those who had not been exposed to gefitinib. The functional consequenc-es of identified mutations were evaluated after the mutant proteins were expressed incultured cells. results Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene ineight of nine patients with gefitinib-responsive lung cancer, as compared with none ofthe seven patients with no response (P<0.001). Mutations were either small, in-framedeletions or amino acid substitutions clustered around the ATP-binding pocket of thetyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 pa-tients with primary non–small-cell lung cancer who had not been exposed to gefitinib(8 percent). All mutations were heterozygous, and identical mutations were observedin multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mu-tants demonstrated enhanced tyrosine kinase activity in response to epidermal growthfactor and increased sensitivity to inhibition by gefitinib. conclusions A subgroup of patients with non–small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitorgefitinib. These mutations lead to increased growth factor signaling and confer suscep-tibility to the inhibitor. Screening for such mutations in lung cancers may identify pa-tients who will have a response to gefitinib.Copyright © 2004 Massachusetts Medical Society. All rights reserved. Downloaded from www.nejm.org at Stanford University on October 5, 2005 .n engl j med 350;21 www.nejm.org may 20 , 2004 The new england journal of medicine 2130on–small-cell lung cancer is the leading cause of death from cancer in theUnited States. Chemotherapy slightly pro-longs survival among patients with advanced dis-ease, but at the cost of clinically significant adverseeffects. 1 The success of the ABL tyrosine kinase in-hibitor imatinib in the treatment of chronic myeloidleukemia (CML) has demonstrated the effective-ness of targeting the critical genetic lesion that pro-motes proliferative signals in cancer cells. 2 Gefi-tinib targets the ATP cleft within the tyrosine kinaseepidermal growth factor receptor (EGFR), 3 whichis overexpressed in 40 to 80 percent of non–small-cell lung cancers and many other epithelial can-cers. 4 EGFR signaling is triggered by the bindingof growth factors, such as epidermal growth factor(EGF), resulting in the dimerization of EGFR mol-ecules or heterodimerization with other closely re-lated receptors, such as HER2/neu. Autophospho-rylation and transphosphorylation of the receptorsthrough their tyrosine kinase domains leads to therecruitment of downstream effectors and the acti-vation of proliferative and cell-survival signals. 5 De-spite its ubiquitous expression, inactivation of the EGFR gene in the mouse causes minimal defects, 6,7 suggesting that pharmacologic inhibition of EGFRby gefitinib should have few adverse effects.Gefitinib inhibits the growth of some cancer-derived cell lines and tumor xenografts, althoughthis effect is not well correlated with the level of ex-pression of EGFR or related members of the ErbBfamily of receptors. 3 In initial clinical studies, gefi-tinib had minimal adverse effects, 8-10 but tumorresponses were observed in only 10 to 19 percent ofpatients with chemotherapy-refractory advancednon–small-cell lung cancer. 11,12 The addition ofgefitinib to traditional chemotherapy provided nobenefit. 13,14 Even in gliomas, in which the findingof frequent amplification and rearrangements ofthe EGFR gene suggests that EGFR plays an impor-tant role, gefitinib failed to induce clinically signif-icant responses. 15,16 Despite these discouragingresults, the remarkably rapid and often profoundresponse to gefitinib in a subgroup of patients withnon–small-cell lung cancer led to its approval assingle-drug therapy for refractory lung cancer. 17 Weevaluated tumors from patients with these dramat-ic responses to determine the underlying mecha-nisms. nucleotide-sequence analysis of tumor specimens Tumor specimens were obtained during diagnos-tic or surgical procedures from patients with non–small-cell lung cancer who were subsequently treat-ed with gefitinib according to a protocol approvedby the institutional review board of MassachusettsGeneral Hospital in Boston. Frozen tumor speci-mens, along with matched normal tissue, wereavailable from four patients, and paraffin-embed-ded material was used from the other patients. Inaddition, specimens from 25 patients with primarynon–small-cell lung