Met Activation in Non-Small Cell Lung Cancer Is Associated with de Novo Resistance to EGFR Inhibitors and the Development of Brain Metastasis
2010; Elsevier BV; Volume: 177; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090863
ISSN1525-2191
AutoresElisa Benedettini, Lynette M. Sholl, Michael Peyton, John F. Reilly, Christopher Ware, Lenora J. Davis, Natalie Vena, D. R. Shackleton Bailey, Beow Y. Yeap, Michelangelo Fiorentino, Azra H. Ligon, Bo-Sheng Pan, Victoria M. Richon, John D. Minna, Adi F. Gazdar, Giulio Draetta, Silvano Bòsari, Lucian R. Chirieac, Bart Lutterbach, Massimo Loda,
Tópico(s)HER2/EGFR in Cancer Research
ResumoMost non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors. Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors. Activating mutations in EGFR (primarily del19 EGFR and L858R) are associated with sensitivity to epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitor therapy, but patients uniformly develop disease recurrence.1Asahina H Yamazaki K Kinoshita I Sukoh N Harada M Yokouchi H Ishida T Ogura S Kojima T Okamoto Y Fujita Y Dosaka-Akita H Isobe H Nishimura M A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.Br J Cancer. 2006; 95: 998-1004Crossref PubMed Scopus (267) Google Scholar, 2Chang AY The role of gefitinib in the management of Asian patients with non-small cell lung cancer.Expert Opin Investig Drugs. 2008; 17: 401-411Crossref PubMed Scopus (11) Google Scholar, 3Inoue A Suzuki T Fukuhara T Maemondo M Kimura Y Morikawa N Watanabe H Saijo Y Nukiwa T Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations.J Clin Oncol. 2006; 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2: 22-28Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar In addition, about 30% of patients with EGFR sensitizing mutations show primary resistance to EGFR inhibitor therapy.1Asahina H Yamazaki K Kinoshita I Sukoh N Harada M Yokouchi H Ishida T Ogura S Kojima T Okamoto Y Fujita Y Dosaka-Akita H Isobe H Nishimura M A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.Br J Cancer. 2006; 95: 998-1004Crossref PubMed Scopus (267) Google Scholar, 2Chang AY The role of gefitinib in the management of Asian patients with non-small cell lung cancer.Expert Opin Investig Drugs. 2008; 17: 401-411Crossref PubMed Scopus (11) Google Scholar, 3Inoue A Suzuki T Fukuhara T Maemondo M Kimura Y Morikawa N Watanabe H Saijo Y Nukiwa T Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations.J Clin Oncol. 2006; 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2: 22-28Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 7Jackman D, Pao W, Riely GJ, Engelman JA, Kris MG, Janne PA, Lynch T, Johnson BE, Miller VA: Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer, J Clin Oncol 28:357–360Google Scholar While disease recurrence in formerly responsive patients has been associated with an EGFRT790M mutation,8Pao W Miller VA Politi KA Riely GJ Somwar R Zakowski MF Kris MG Varmus H Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.PLoS Med. 2005; 2: e73Crossref PubMed Scopus (3089) Google Scholar MET amplification,9Bean J Brennan C Shih JY Riely G Viale A Wang L Chitale D Motoi N Szoke J Broderick S Balak M Chang WC Yu CJ Gazdar A Pass H Rusch V Gerald W Huang SF Yang PC Miller V Ladanyi M Yang CH Pao W MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.Proc Natl Acad Sci U S A. 2007; 104: 20932-20937Crossref PubMed Scopus (1456) Google Scholar, 10Engelman JA Zejnullahu K Mitsudomi T Song Y Hyland C Park JO Lindeman N Gale CM Zhao X Christensen J Kosaka T Holmes AJ Rogers AM Cappuzzo F Mok T Lee C Johnson BE Cantley LC Janne PA MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.Science. 2007; 316: 1039-1043Crossref PubMed Scopus (3902) Google Scholar or hepatocyte growth factor (HGF) expression,11Yano S Wang W Li Q Matsumoto K Sakurama H Nakamura T Ogino H Kakiuchi S Hanibuchi M Nishioka Y Uehara H Mitsudomi T Yatabe Y Nakamura T Sone S Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations.Cancer Res. 2008; 68: 9479-9487Crossref PubMed Scopus (545) Google Scholar the factors involved in de novo resistance remain unidentified. Sequist et al reported primary resistance to EGFR tyrosine kinase inhibitor (TKI) in a patient harboring Met copy gain, but analysis of larger cohorts has not been reported and Met protein expression and activation were not tested.12Sequist LV Martins RG Spigel D Grunberg SM Spira A Janne PA Joshi VA McCollum D Evans TL Muzikansky A Kuhlmann GL Han M Goldberg JS Settleman J Iafrate AJ Engelman JA Haber DA Johnson BE Lynch TJ First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.J Clin Oncol. 2008; 26: 2442-2449Crossref PubMed Scopus (760) Google Scholar We examined a cohort of EGFR mutant non-small cell lung cancer (NSCLC) patients before treatment with EGFR TKI (designated cohort 1), and we report here that Met protein expression and phosphorylation were found in a subset of tumors before EGFR TKI therapy. Importantly, the subset harboring Met expression and phosphorylation was associated with poor response to subsequent EGFR TKI therapy despite the presence of EGFR inhibitor sensitizing mutations. In addition to primary resistance, poor outcome to EGFR targeted therapy in NSCLC can result from development of metastases, especially to the central nervous system. Since HGF/Met signaling is uniquely positioned to be a key factor in cell migration and tumor dissemination,13Birchmeier C Birchmeier W Gherardi E Vande Woude GF Met, metastasis, motility and more.Nat Rev Mol Cell Biol. 2003; 4: 915-925Crossref PubMed Scopus (2225) Google Scholar, 14Boccaccio C Comoglio PM Invasive growth: a MET-driven genetic programme for cancer and stem cells.Nat Rev Cancer. 2006; 6: 637-645Crossref PubMed Scopus (466) Google Scholar we compared Met status in a separate cohort of NSCLC patients (designated cohort 2) with paired brain metastases. Met expression has been correlated with both development of metastases and poor prognosis in some tumor types,13Birchmeier C Birchmeier W Gherardi E Vande Woude GF Met, metastasis, motility and more.Nat Rev Mol Cell Biol. 2003; 4: 915-925Crossref PubMed Scopus (2225) Google Scholar but no studies have demonstrated Met activation in metastatic lung cancer. In this study we found that Met expression and phosphorylation in primary NSCLC tumors were strongly associated with subsequent development of brain metastases. Furthermore, we showed an enrichment of cells positive for Met expression and phosphorylation in brain lesions compared with matched primary lung tumors. Cell lines were from American Type Culture Collection (ATCC) except: EBC-1 (Health Science Research Resources Bank, Japan Health Sciences Foundation) and H1993 and H2073 (from J. Minna and A. Gazdar, Hamon Cancer Center, University of Texas Southwestern). Cells were maintained in RPMI plus 10% fetal calf serum and 100 μg/ml Pennicillin/Streptavidin (Sigma, St. Louis, MO). SU1127415Sattler M Pride YB Ma P Gramlich JL Chu SC Quinnan LA Shirazian S Liang C Podar K Christensen JG Salgia R A novel small molecule met inhibitor induces apoptosis in cells transformed by the oncogenic TPR-MET tyrosine kinase.Cancer Res. 2003; 63: 5462-5469PubMed Google Scholar was from Sigma. Sixty-nine patients with a diagnosis of lung adenocarcinoma were identified who had undergone EGFR mutation analysis between 2003 and 2007 and who had archived formalin-fixed paraffin-embedded (FFPE) samples at Brigham and Women’s Hospital Department of Pathology. Of these, 46 were excluded because they either did not receive TKI therapy or did not have available follow-up data. Of the remaining 23, all were women, 11 were nonsmokers, and 10 were smokers; smoking status was not available for two patients. Patient response was scored according to Response Evaluation Criteria in Solid Tumors (RECIST).16Therasse P Arbuck SG Eisenhauer EA Wanders J Kaplan RS Rubinstein L Verweij J Van Glabbeke M van Oosterom AT Christian MC Gwyther SG New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada.J Natl Cancer Inst. 2000; 92: 205-216Crossref PubMed Scopus (14471) Google Scholar FFPE tumor samples were from a subset of a previously described collection of primary NSCLC diagnosed between 1989 and 2003.17Saad AG Yeap BY Thunnissen FB Pinkus GS Pinkus JL Loda M Sugarbaker DJ Johnson BE Chirieac LR Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma.Cancer. 2008; 113: 2129-2138Crossref PubMed Scopus (52) Google Scholar Patients with adenocarcinoma or squamous cell carcinoma with metastases to the brain were matched for clinicopathological features with a control group of NSCLC patients who did not develop brain metastasis during six years of follow-up or until death. We excluded from the Control Group patients who developed metastases in a distant site different from brain, to have a homogeneous control group that did not develop metastases. We also excluded neuroendocrine tumors. We analyzed 40 patients and a total of 58 specimens, including 18 primary tumors (10 adenocarcinoma and 8 squamous cell carcinoma), with paired 18 brain metastases and 22 nonmetastatic tumors (14 adenocarcinoma and 8 squamous cell carcinoma). The MET probe (BAC clone RP11-95i20,CHORI; http://bacpac.chori.org) was labeled with SpectrumOrange dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to full tissue sections along with the centromere probe (CEP7/D7Z1, SpectrumGreen, Abbott Molecular Inc., Des Plaines, IL), using standard conditions.18Varella-Garcia M Stratification of non-small cell lung cancer patients for therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence in situ hybridization assay.Diagn Pathol. 2006; 1: 19Crossref PubMed Scopus (106) Google Scholar A minimum of 100 cells were analyzed. MET amplification was defined as MET:CEP7 ratio greater than 2.2.19Wolff AC Hammond ME Schwartz JN Hagerty KL Allred DC Cote RJ Dowsett M Fitzgibbons PL Hanna WM Langer A McShane LM Paik S Pegram MD Perez EA Press MF Rhodes A Sturgeon C Taube SE Tubbs R Vance GH van de Vijver M Wheeler TM Hayes DF American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.Arch Pathol Lab Med. 2007; 131: 18-43Crossref PubMed Google Scholar Samples having MET:CEP7 ratio between 1 and 2.2 were further defined as either low copy gain (MET copy number from 3 to 5) or high copy gain (MET copy number >5).20Cappuzzo F Marchetti A Skokan M Rossi E Gajapathy S Felicioni L Del Grammastro M Sciarrotta MG Buttitta F Incarbone M Toschi L Finocchiaro G Destro A Terracciano L Roncalli M Alloisio M Santoro A Varella-Garcia M Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients.J Clin Oncol. 2009; 10: 1667-1674Crossref Scopus (467) Google Scholar, 21Cappuzzo F Varella-Garcia M Finocchiaro G Skokan M Gajapathy S Carnaghi C Rimassa L Rossi E Ligorio C Di Tommaso L Holmes AJ Toschi L Tallini G Destro A Roncalli M Santoro A Janne PA Primary resistance to cetuximab therapy in EGFR FISH-positive colorectal cancer patients.Br J Cancer. 2008; 99: 83-89Crossref PubMed Scopus (159) Google Scholar A score analogous to the H score for immunohistochemistry (IHC) was derived by multiplying the percentage of cells harboring MET abnormalities by the level of copy gain (low copy gain multiplied by 1, high copy gain multiplied by 2). The following antibodies were used: anti-c-Met (Zymed-Invitrogen, clone3D4, diluted 1:100); anti-Y1234/35 Met (3077, Cell Signaling Technology, 1:50); anti-EGFR (Novocastra, clone EGFR.25, 1:50). Negative controls for the Y1234/35 Met antibody included multiple FFPE embedded cell lines with various well-described kinase amplification or activation as indicated in Supplemental Table S2. Negative control for Met was LNCAP cells.22Humphrey PA Zhu X Zarnegar R Swanson PE Ratliff TL Vollmer RT Day ML Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma.Am J Pathol. 1995; 147: 386-396PubMed Google Scholar Positive controls for Met and Y1234/35 Met were Met-amplified GTL16 and nonamplified A549 cells ± HGF. The negative control for EGFR staining was MDAMB453 cells (negative for EGFR23Moasser MM Basso A Averbuch SD Rosen N The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells.Cancer Res. 2001; 61: 7184-7188PubMed Google Scholar), while HCC827 cells were a positive control. Epitope retrieval used boiling 0.01 M citrate buffer, pH 6.0 for 15 minutes in microwave (anti-c-Met,) or in pressure cooker (anti-EGFR). For Y1234/35 Met, Ventana Benchmark autostainer and Rabbit Ultra-HRP were used. Detection was with ChromoMap kit (Ventana Molecular Discovery Systems, Tucson, AZ). Results of the IHC were reviewed independently by two pathologists (M.F. and L.R.C.), who were blinded as to the outcome of the tumors. Membrane stain intensity was scored from 0 to 3+ for total Met and EGFR: 0, absent or faint staining in less than 5% of cells; 1+, ≥5% tumor cells faint stain; 2+, tumor cells moderate stain; 3+, tumor cells strong staining. For Y1234/35 Met phosphospecific antibody membrane stain intensity was scored from 0 to 2+ as follows: 0, absent or faint staining in less than 5% of cells; 1+, tumor cells with weak staining; 2+, tumor cells with moderate-strong staining. The results of the two reviewers were compared, and any tumors for which there was not agreement were reviewed together by both pathologists and a consensus was reached. The H score for each sample was calculated by multiplying the stain intensity value by the percentage of positive cells. Images were acquired using a Leitz Diaplan microscope bright field and a CRI Nuance spectral analyzer (CRI Inc., Woburn, MA) and collected at 5-nm wavelength intervals from 450 to 700 nm. DNA was extracted from five 0.8-mm cores of each FFPE specimen with Qiagen BioRobot (Qiagen, Hilden, Germany). Whole genome amplification (WGA) was performed with GenomePlex (Sigma) as described.24Thomas RK Baker AC Debiasi RM Winckler W Laframboise T Lin WM Wang M Feng W Zander T MacConaill L Lee JC Nicoletti R Hatton C Goyette M Girard L Majmudar K Ziaugra L Wong KK Gabriel S Beroukhim R Peyton M Barretina J Dutt A Emery C Greulich H Shah K Sasaki H Gazdar A Minna J Armstrong SA Mellinghoff IK Hodi FS Dranoff G Mischel PS Cloughesy TF Nelson SF Liau LM Mertz K Rubin MA Moch H Loda M Catalona W Fletcher J Signoretti S Kaye F Anderson KC Demetri GD Dummer R Wagner S Herlyn M Sellers WR Meyerson M Garraway LA High-throughput oncogene mutation profiling in human cancer.Nat Genet. 2007; 39: 347-351Crossref PubMed Scopus (875) Google Scholar The set of somatic EGFR kinase domain activating mutations along with 15 MET mutations (E168D, L229F, R988C, T1010I, H1112Y, H1112R, N1118Y, L1213V, Y1248H, Y1248C, Y1253D, M1268T, splicing site IVS13del22, exon 14 splice site mutation, splicing site ex14del28)25Kong-Beltran M Seshagiri S Zha J Zhu W Bhawe K Mendoza N Holcomb T Pujara K Stinson J Fu L Severin C Rangell L Schwall R Amler L Wickramasinghe D Yauch R Somatic mutations lead to an oncogenic deletion of met in lung cancer.Cancer Res. 2006; 66: 283-289Crossref PubMed Scopus (378) Google Scholar were tested as described.24Thomas RK Baker AC Debiasi RM Winckler W Laframboise T Lin WM Wang M Feng W Zander T MacConaill L Lee JC Nicoletti R Hatton C Goyette M Girard L Majmudar K Ziaugra L Wong KK Gabriel S Beroukhim R Peyton M Barretina J Dutt A Emery C Greulich H Shah K Sasaki H Gazdar A Minna J Armstrong SA Mellinghoff IK Hodi FS Dranoff G Mischel PS Cloughesy TF Nelson SF Liau LM Mertz K Rubin MA Moch H Loda M Catalona W Fletcher J Signoretti S Kaye F Anderson KC Demetri GD Dummer R Wagner S Herlyn M Sellers WR Meyerson M Garraway LA High-throughput oncogene mutation profiling in human cancer.Nat Genet. 2007; 39: 347-351Crossref PubMed Scopus (875) Google Scholar DNA typing was done with Gene Print Multiple PowerPlex 2.1 (Promega). Cell identity typing (Wellcome Trust Sanger Institute, Cambridge, UK) is listed at http://www.sanger.ac.uk/genetics/CGP/Genotyping/synlinestable.shtml. Wounding migration assays used 1-mm-thick scotch tape (Bristol) placed in the center of 24-well plates. Cells were plated at confluency, and tape was removed after to create a clear zone. Cells that migrated into the cleared region were photographed for counting at 72 hours later. A separate migration assay format used 96-well Fluoroblock insert wells (BD Biosciences). H1993 or H2073 cells (n = 10,000) were plated and 24 hours later treated with compounds for 12 hours before HGF addition to both the top and bottom wells. After 12 hours, calcein AM (Invitrogen) at 2.5 μg/ml was added to the bottom chamber for 1 hour. Signal was measured on a Wallace 1420 Multilabel Victor V counter (Perkin Elmer) with bottom reading capacity. Met and control shRNA were as described.26Lutterbach B Zeng Q Davis LJ Hatch H Hang G Kohl NE Gibbs JB Pan BS Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival.Cancer Res. 2007; 67: 2081-2088Crossref PubMed Scopus (265) Google Scholar Growth factor–mediated rescue of gefitinib growth inhibition in HCC827 cells used 4000 cells per well in 96-well plates. Gefitinib (LC labs, 1 μmol/L) was added, followed by addition of growth factors (R&D Systems) at 50 ng/ml. Cell growth was measured by Vialight 4 days later. Lysis buffer containing 30 mmol/L Tris-HCL, pH 7.5, 50 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 30 mmol/L NaPPi, 1% Triton, 0.5% IGEPAL, 10% Glycerol, 1 mmol/L Sodium Vanadate, 1 mmol/L bpPhen (Calbiochem), and protease inhibitor cocktail (Roche) was added with shaking for 10 minutes at 4°C. Lysates were clarified by centrifugation at 20,000g for 5 minutes at 4°C. SDS-PAGE on 40 μg of cell lysates and Western blotting followed standard procedures. Antibody to Met (AF276) was from R&D Systems, GAPDH was from Fitzgerald industries, and antibodies from Cell Signaling Technology included Y1234/35 Met (3077), Met (3127), Ser473 Akt (4058), Akt (4691), T202/Y204 Erk (4370), S235/236 S6 (4857), cleaved PARP (9544), Y1173 EGFR (4407), and EGFR (4267). Wilcoxon rank-sum test was used to compare the distribution of phosphorylation H scores between independent tumor groups defined by Met expression and MET copy number. The Signed rank test was applied to the analysis of paired difference in H scores between primary lung tumors and brain metastases. When H scores were dichotomized as positive versus negative, the Fisher exact test was used to compare the frequencies of expression and phosphorylation between independent patient groups, while the McNemar test was used in the analysis of paired binary data. Exact P values were computed for the log rank test to compare the time of progression between patient groups, stratifying by stage to control for the underlying prognosis. Analysis was performed using SAS 9.1 (SAS Institute, Cary, NC) and StatXact 6.1 (Cytel Software Corp, Cambridge, MA). All P values are based on a two-sided hypothesis test. To investigate the role of Met in de novo resistance to EGFR TKI therapy, we correlated Met expression, phosphorylation, and gene copy gain with patient response. We analyzed 23 NSCLC samples, including ten with activating EGFR mutations (L858R, L861Q, or exon19 deletion) and 13 with wild-type EGFR. Importantly, our analysis was conducted on patient tumors resected before EGFR inhibitor therapy (cohort 1, see supplemental Table S1, available at http://ajp.amjpathol.org). Met phosphorylation was tested using a recently developed antibody specific for Y1234/35 activation loop phosphorylation (Ab 3077, Cell Signaling Technology). Importantly, this antibody did not cross-react by Western blot or IHC with cell lines harboring other amplified or activated receptor tyrosine kinases (including EGFR family kinases, PDGFR, FGFR, insulin receptor, and Ron kinases), providing a specificity not available with prior reagents (see supplemental Table S2, available at http://ajp.amjpathol.org). We determined whether progression free survival (PFS) was different in tumors that expressed Y1234/35 Met compared with tumors that did not express Y1234/35 Met. In a stage-matched analysis, Met expression and phosphorylation were more common in tumors that progressed during EGFR TKI therapy, but this trend was not significant (P = 0.221 and P = 0.148, respectively; Figure 1A). However, when considering only patients harboring EGFR mutation, both Met phosphorylation and expression were strongly associated with shorter time to progression (P = 0.024 and P = 0.005, respectively; Figure 1B and C). Furthermore, two patients with EGFR– activating mutations exhibited primary resistance to EGFR TKI therapy, displaying progressive disease (PD) three months after treatment initiation (Table 1, patients 1 and 8). Importantly, these resistant tumors exhibited extensive Met expression and activation (in 70% of tumor, P = 0.022).Table 1NSCLC Patient Outcome on EGFR TKI Therapy in Relation to Met Expression, Phosphorylation, and Gene Copy Number GainMetY1234/35 MetEGFR mutationPtRECISTPFS (months)% Pos cellsIntensityH scoreFISH% Pos cellsIntensityH scoreType% Pos cellsSiteEGFR TKI1PD3703+210H702+140ex19del90local LNEr2PR15101+10H302+60ex19del100PWREr3PR1540, 101+, 2+60L101+10ex19del90PWREr4SD500000000ex19del20local LNGe5SD4031+3L000ex19del70PWRGe6PR420, 51+, 2+30H302+60ex19del90PWREr7PR38000na000ex19delnaPWREr8PD3702+140L702+140L861QnaPWREr9PR1540, 101+, 2+60H000ex19del40local LNEr10SD12000L000L858R90lobeErAll tumors harbor EGFR-activating mutations and were resected before EGFR TKI therapy. RECIST indicates patient response according to RECIST criteria; PFS, progression-free survival during EGFR inhibitors treatment. IHC: when multiple intensity values are present in the same sample, the respective percentages of positive cells are separated by a comma. FISH for Met is scored as described in Materials and Methods: D indicates disomic; L, low copy gain; H, high copy gain; na, not assessable; Local LN, loco-regional lymph node; PWR, pulmonary wedge resection; Er, erlotinib; Ge, gefitinib. Open table in a new tab All tumors harbor EGFR-activating mutations and were resected before EGFR TKI therapy. RECIST indicates patient response according to RECIST criteria; PFS, progression-free survival during EGFR inhibitors treatment. IHC: when multiple intensity values are present in the same sample, the respective percentages of positive cells are separated by a comma. FISH for Met is scored as described in Materials and Methods: D indicates disomic; L, low copy gain; H, high copy gain; na, not assessable; Local LN, loco-regional lymph node; PWR, pulmonary wedge resection; Er, erlotinib; Ge, gefitinib. We also conducted fluorescence in situ hybridization (FISH) analysis and found MET copy gain resulting from chromosome 7 polysomy but not MET focal amplification (MET/CEP7 ratio >2.2). However MET copy gain was not associated with time to progression (P = 0.198, data not shown), similar to a previous report.27Cappuzzo F Janne PA Skokan M Finocchiaro G Rossi E Ligorio C Zucali PA Terracciano L Toschi L Roncalli M Destro A Incarbone M Alloisio M Santoro A Varella-Garcia M MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients.Ann Oncol. 2009; 20: 298-304Crossref PubMed Scopus (271) Google Scholar We conclude that Met activation (in the absence of gene amplification) was correlated with de novo resistance to EGFR tyrosine kinase inhibitors. We next used the EGFR mutation containing HCC827 cell line to define a potential mechanism for the poor outcome of EGFR-mutated tumors harboring Met activation. We found that Met activation by HGF in HCC827 cells caused a striking resistance to Gefitinib (Figure 2A). In fact, HGF was the only ligand in a panel of 12 growth factors (HGF, EGF, Amphiregulin, Epiregulin, neuregulin, TGFα, FGF1, PDGFBB, Kit ligand, insulin, IGF1, IGF2) able to maintain HCC827 cell growth in the presence of Gefitinib (data not shown). HGF-mediated rescue also occurred in 0.1% serum, revealing that Met activation did not require cooperation with serum factors (Figure 2A). Importantly, the HGF-mediated rescue was blocked by an anti-HGF antibody or the Met-specific inhibitor SU11274 (Figure 2A). HGF also rescued Gefitinib growth inhibition in the EGFR mutation-containing cell lines HCC4006 and H2935 (see supplemental Figure S1, available at http://ajp.amjpathol.org). These in vitro results provide a mechanism for our observation that Met activation, independent of MET amplification, is associated with de novo resistance and poor disease control with EGFR targeted therapy. We next identified the signal transduction pathways necessary for Met t
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