Molecular Study of Malignant Gliomas Treated with Epidermal Growth Factor Receptor Inhibitors : Tissue Analysis fromNorth

Purpose:We investigated the molecular effect of the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib in vivo on all available tumors from patients treated on North American BrainTumor Consortium trials 01-03 and 00-01for recurrent or progressive malignant glioma. Experimental Design:EGFRexpressionand signalingduring treatmentwith erlotiniborgefitinib were analyzed byWestern blot and compared with pre ^ erlotinib/gefitinib ^ exposed tissue or unexposed controls.Tumorswere also analyzed forEGFRmutations and for other genomicabnormalities by array-based comparative genomic hybridization. Clinical data were used to associate molecular features with tumor sensitivity to erlotinib or gefitinib. Results: Erlotinib and gefitinib did not markedly affect EGFR activity in vivo. No lung signature mutations of EGFR exons18 to 21were observed.There was no clear association between erlotinib/gefitinib sensitivity and deletion or amplification events on array-based comparative genomic hybridization analysis, althoughnovel genomic changes were identified. Conclusions: As erlotinib and gefitinib were generally ineffective at markedly inhibiting EGFR phosphorylation in these tumors, other assays may be needed to detect molecular effects. Additionally, the mechanism of erlotinib/gefitinib sensitivity likely differs between brain and lung tumors. Finally, novel genomic changes, including deletions of chromosomes 6, 21, and 22, represent new targets for further research. The prognosis for patients with high-grade gliomas is poor, with a median survival of 2 to 5 years for anaplastic astrocytomas and 1 year for glioblastomas (1). Unfortunately, glioblastomas are the most common as well as most aggressive subtype (1). New therapies are needed, and small-molecule inhibitors targeting specific molecular abnormalities important in glioma biology may provide benefit (2). Several types of abnormalities of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, contribute to the growth and proliferation of tumor cells in the majority of glioblastomas, including EGFR gene amplification, protein overexpression, and constitutively activating mutations (3–9). Normally, EGF and other ligands activate the EGFR, causing dimerization/oligomerization and activation of intrinsic tyrosine kinase activity in the cytosolic domain of the receptor (Fig. 1A; ref. 10). When activated, the receptor both autophosphorylates and initiates downstream signaling through the RASMAPK and phosphatidylinositol 3-kinase (PI3K)/AKT signal Cancer Therapy: Clinical Authors’Affiliations: Departments of Neurology, Cancer Biology and Genetics, Medicine, and Surgery (Neurosurgery), Memorial Sloan Kettering Cancer Center, New York, New York ; Roswell Park Cancer Institute, Buffalo, New York ; Department of Neurology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois; University of Pittsburgh Medical Center Cancer Pavilion, Pittsburgh, Pennsylvania; Neuro-Oncology Services, University of California-San Francisco, San Francisco, California; University of Texas Health Science Center, San Antonio,Texas; Genome Sequencing Center,Washington University School of Medicine, St. Louis, Missouri; Department of Neurology, Dana-Farber Cancer Institute, Boston, Massachusetts; and Department of Neuro-Oncology, University of TexasM.D. Anderson Cancer Center, Houston,Texas Received 2/25/05; revised 6/24/05; accepted 8/9/05. Grant support: NIH/National Cancer Institute grants T32 CA009512 (A.B. Lassman andW. Pao), 5-U01CA62399-09 (A.B. Lassman, J.R. Raizer, L.E. Abrey, L.M. DeAngelis, and E.C. Holland), R21CA104504 (M.R. Rossi, N.J. Nowak, and J.K. Cowell), CA62426 (J. Kuhn), U01CA62407-08 (P. Wen), CA62412 and CA62422 (K. Lamborn, S. Chang, and M. Prados), CA62412 (M.R. Gilbert and W.A. Yung), and RO1CA099489, RO1CA100688, and UO1CA894314-1 (A.B. Lassman, C.N. Grefe, A.H. Shih, and E.C. Holland); American Brain Tumor Association (A.B. Lassman); Chest and LUNGevity Foundations of the American College of Chest Physicians and an anonymous donor (W. Pao); and Bressler, Seroussi, and Kirby Foundations (E.C. Holland). 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/). Requests for reprints: Andrew B. Lassman, Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-6037; Fax: 212-717-3519; E-mail: lassmana@mskcc.org. F2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-0421 www.aacrjournals.org Clin Cancer Res 2005;11(21) November1, 2005 7841 Research. on May 2, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from transduction cascades. Activation of EGFR, RAS, and AKT can be detected by analysis of tumor tissue for pEGFR, pERK, and pAKT levels with commercially available antibodies. The EGFR inhibitors erlotinib (OSI774, Tarceva) and gefitinib (ZD1839, Iressa) are currently under evaluation in clinical trials for gliomas. Seeking to determine whether erlotinib or gefitinib therapy affects EGFR signaling in malignant gliomas in vivo and whether observed effects determined clinical response, we analyzed all available surgically resected malignant glioma tissue from patients who were treated with erlotinib or gefitinib through two multi-institution clinical trials. We also analyzed the available tumors for mutations of the EGFR gene and for other genomic alterations beyond EGFR. Although we did not identify consistent inhibition of EGFR signaling by erlotinib or gefitinib, several new genomic abnormalities were identified as common to malignant gliomas and worthy of further study. Materials andMethods Tissue. Patients with recurrent or progressive malignant gliomas were treated with erlotinib through North American Brain Tumor Consortium (NABTC) multicenter phase I/II clinical trial 01-03 or with gefitinib through NABTC trial 00-01. Treatment and correlative analyses were approved by the institutional review board at each center and performed with patient’s informed consent. Patients received daily erlotinib or gefitinib as monotherapy and were followed clinically and radiographically with brain imaging (magnetic resonance imaging or computed tomography scans) every 8 weeks thereafter until tumor progression or death. When surgical resection was medically indicated at the time of enrollment for tumor recurrence or progression (also called the surgical arm of the trials), patients received 150 mg p.o. erlotinib or 500 mg p.o. gefitinib daily for 1 week preoperatively, underwent resection, and then restarted daily erlotinib or gefitinib following recovery from surgery. These specimens were analyzed to determine the effect of erlotinib or gefitinib on EGFR signaling at the molecular level in vivo and to determine if molecular effects were associated with clinical response. Twenty-one malignant gliomas from 18 patients treated with erlotinib or gefitinib were available for molecular analysis (Table 1, tumors 1-21). Criteria for clinical evaluation and final clinical results (overall survival, progression-free survival, etc.) will be reported separately for these and other patients who participated in NABTC trials 01-03 (11) or 00-01 (12). The results reported here are restricted to molecular analysis, and for the purpose of analyzing the biological activity of erlotinib/gefitinib therapy, the following criteria were applied. Ten patients were considered to have erlotinib/gefitinib– insensitive tumors because of radiographically progressive disease (>25% growth; ref. 13) or because of clinical progression within the first 8 weeks of therapy. One patient was considered to have a sensitive tumor because a complete radiographic response (13) was observed, independently confirmed on central review, which was sustained for at least 22 months. Six patients with radiographically stable disease (between 50% reduction and 25% growth; ref. 13) after 8 weeks of treatment were considered to have erlotinib/gefitinib–sensitive tumors because all patients had radiographically enlarging tumors when erlotinib was started; however, it should be noted that the stable responses were not sustained and progressive tumor growth was observed between 8 and 24 weeks after starting erlotinib in all of these cases. Finally, one patient was considered to have a sensitive tumor because of a mixed radiographic response and another because of histologically proven disease control. In sum, there were 11 insensitive tumors and 10 sensitive tumors resected from 18 patients. Among these 21 specimens, there were 12 resected from patients during erlotinib or gefitinib treatment (Table 1, tumors 9-20). In three of these (tumors 9-11), tissue was also available from the same patients from prior excisions before treatment with an EGFR inhibitor (tumors 1-3), and direct comparison of the pretreatment and during-treatment specimens was done. In the other nine specimens (12–20), pretreatment tissue was unavailable for comparison; therefore, we compared these tumors indirectly with 12 controls of matched histology (tumors 22-33) banked from patients who did not receive erlotinib, gefitinib, or other receptor tyrosine kinase inhibitors. Western blots. Tumors that were flash frozen in liquid nitrogen immediately following surgical resection and stored at 80jC were ground into fine powder in liquid nitrogen and dissolved in T-PER buffer (Pierce, Rockford, IL) containing EDTA-free Complete protease inhibitor cocktail (Roche, Indianapolis, IN) and the phosphatase inhibitors NaVO3 (1 mmol/L; pH 10) and NaF (30 mmol/L). Protein concentrations were determined by absorption at