2244 Vol. 11, 2244–2251, March 15, 2005 Clinical Cancer Research Predictors of the Response to Gefitinib in Refractory Non–Small Cell Lung Cancer Kyu-Sik Kim,1,2 Ju-Yeon Jeong,1,2 Young-Chul those with non-adenocarcinoma showed a mutation of the Kim,1,2 Kook-Joo Na,1,3 Yun-Hyeon Kim,1,4 EGFR gene, the genetic profile may replace those variables as an independent predictor of a response. Sung-Ja Ahn,1,5 Sun-Mi Baek,1 Chang-Soo Park,6 Chang-Min Park,2 Yu-Il Kim,2 Sung-Chul Lim,2 INTRODUCTION and Kyung-Ok Park7 Lung cancer has been the leading cause of cancer death in 1Lung and Esophageal Cancer Clinic, Chonnam National University Hwasun Hospital, Jeollanam-do, South Korea; Departments of South Korea, as in many other parts of the world, since the year 2Pulmonology and Critical Care Medicine, 3Thoracic and Cardiovascular 2000 (1). As the global burden of lung cancer continues to increase, Surgery, 4Diagnostic Radiology, 5Radiation Oncology, and 6Pathology, new agents are being developed for more effective treatment and Chonnam National University Medical School, Gwangju, South Korea; palliation of symptoms. Lung cancer has been treated with a wide 7 and Department of Internal Medicine, Seonam University Medical range of modalities, including surgery, radiotherapy, and chemo- School, Jeonbuk, South Korea therapy, as first and second lines of treatment. However, many patients experience relapse after cytotoxic chemotherapy, whereas ABSTRACT they are still in a competent physical status. Gefitinib, an epidermal growth factor receptor (EGFR) Gefitinib (ZD1839, Iressa) is an epidermal growth factor tyrosine kinase inhibitor, has a response rate of 10% to receptor (EGFR) tyrosine kinase inhibitor. Although the Iressa 20% in refractory non–small cell lung carcinoma. Although Non–Small Cell Lung Carcinoma (NSCLC) Trial Assessing female gender, adenocarcinoma, and never having smoked Combination Therapy study (2, 3) showed no benefit of adding are possible markers of a favorable response, mutations of gefitinib to cytotoxic chemotherapy, two phase II monotherapy the EGFR gene have also been reported to be highly trials (Iressa Dose Evaluation in Advanced Lung Cancer; refs. 4, 5) significant predictors of response. Seventy patients with showed response rates of 10% to 20% with single-agent gefitinib relapsed non–small cell lung carcinoma were enrolled in in relapsed refractory NSCLC. Considering the increasing the Expanded Access Program. After the drug became demand for third or fourth lines of treatment and the results of available commercially, 28 more patients were treated with previous trials, a recent American Society of Clinical Oncology gefitinib. Response evaluations were feasible in 80 patients. guideline (6) adopted gefitinib as a third line of treatment. Twenty-seven tumor specimens (8 responders and 19 non- EGFR mediates cancer cell growth, proliferation, invasion, responders) were available for the sequence analysis of the and metastasis, and inhibits apoptosis (7). Studies showed that EGFR gene. The response rate was 25% (20/80) and the gefitinib targets the ATP cleft within the tyrosine kinase domain disease control rate (remission + stable disease) was 47.5% of EGFR that is triggered by the binding of EGF. The (38/80). The response rate was significantly higher for phosphorylation of EGFR through tyrosine kinase domains adenocarcinoma (41.0%) versus non-adenocarcinoma inhibits the growth of human tumors (8). These results indicate (9.8%, P = 0.001), in those who never smoked (58.8%) that gefitinib has potential for treating lung cancers that are versus smokers (15.9%, P < 0.001), and in females (42.1%) dependent on the activation of the EGFR pathway. versus males (19.7%, P = 0.049). A deletion or mutation of Despite a low response rate, the very rapid and often the EGFR gene was found in six of eight responders. complete response to gefitinib in a subgroup of patients with Remission was noted in all patients with a mutation, NSCLC led us to search for predictors of the response. Although whereas the response rate was 9.5% (2/21) in patients female gender, the presence of adenocarcinoma, and no smoking without a mutation (P < 0.001). The predictors of response history might be markers of a favorable response, mutations of showed significant correlations with survival and time to the EGFR gene are reported to show high positive predictive progression. In a multivariate logistic analysis, the inde- value for a response (9, 10). pendent predictors of response were smoking history and In South Korea, gefitinib was used to treat 961 patients with adenocarcinoma. Given that 9.5% of smokers and 6.7% of NSCLC in the Expanded Access Program between December 2002 and July 2003. At our institution, we enrolled 70 patients in this program; after gefitinib became available commercially, 28 more patients were treated with this drug. This is a retrospective Received 10/11/04; revised 12/9/04; accepted 12/16/04. review of 98 NSCLC patients who were treated with gefitinib in The costs of publication of this article were defrayed in part by the a single Korean institution. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PATIENTS AND METHODS Requests for reprints: Young-Chul Kim, Lung and Esophageal Cancer Patients. Seventy patients were enrolled in the Expand- Clinic, Chonnam National University Hwasun Hospital, 160 Ilsim-ri, Hwasun, Jeollanam-do, 519-809 South Korea. Phone: 82-61-379-7614; ed Access Program after they showed evidence of relapse af- Fax: 82-61-379-7010; E-mail: [email protected]. ter at least one course of cytotoxic chemotherapy. After it D2005 American Association for Cancer Research. was approved by the Korean Food and Drug Administration, Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Clinical Cancer Research 2245 a commercial form of gefitinib was given to 28 more patients. Immunohistochemical Staining for Epidermal Growth Therefore, 98 patients were included in this analysis. The Factor Receptor Protein. The cancerous tissues from characteristics of the subjects are summarized in Table 1. 30 patients were stained immunohistochemically for the EGFR. Eleven patients were labeled as having non–small cell lung All the steps in the staining procedure were done using cancer because their diagnoses were made by cytologic the Microprobe System (Fisher Scientific, Pittsburgh, PA), taking examination and the histologic type could not be differen- advantage of capillary gap action (12). The tissue section was tiated further. exposed to primary antibody for EGFR (Zymed, 31G7, For most of the patients, gefitinib was the second (n = 44), San Francisco, CA) for 20 minutes after blocking endogenous third (n = 45), or fourth (n = 5) line of treatment for refractory peroxidase activity with Autoblocker (Research Genetics, Hunts- relapse after previous cytotoxic chemotherapy, whereas gefitinib ville, AL) for 5 minutes. Antigen/antibody complexes were was given as the first-line treatment for four patients. detected with a goat anti-mouse antibody (Sigma, St. Louis, MO) The cytotoxic chemotherapy consisted of platinum-based for 10 minutes followed by streptavidin-horseradish peroxidase combination regimens with paclitaxel, docetaxel, gemcitabine, (DAKO, Glostrup, Denmark) for 10 minutes. The chromogen etoposide, or vinorelbine. This study was carried out in reaction consisted of liquid 3,3V-diaminobenzidine (DAKO) for accordance with the institutional review board of our institution, 10 minutes followed by a 30-second application of hematoxylin. and written informed consent was obtained from all of the Then, the sections were mounted in Faramount (DAKO). patients for use of gefitinib and potential studies with their tumor Negative controls consisted of staining with primary or blood specimen. antibody diluent (Research Genetics) only. All the slides were Treatment and Response Evaluation. Gefitinib was examined using standard light microscopy and were scored given at a dose of 250 mg once a day for between 5 and 952 independently by two observers in quartiles as staining intensity days. To evaluate the therapeutic response and adverse reactions, from grades 1 to 4. chest radiographs, computed tomography (CT) scans, complete Sequence Analysis of the EGFR Gene. Twenty-seven blood cell counts, and blood chemistries were monitored. tumor specimens (8 responders and 19 nonresponders) were We classified the therapeutic results as complete or partial available for the sequence analysis of exons 18, 19, and 21 of the remission and stable or progressive disease according to the EGFR gene. Tumor DNA and matched normal DNA were response evaluation criteria for solid tumors (11). extracted from a paraffin-embedded tissue block and peripheral The response could be evaluated in 80 patients who blood buffy coat. After deparaffinization in xylene, DNA was maintained treatment for more than 4 weeks. By the time the extracted using a Gene All Tissue DNA Purification kit (General patients began to take gefitinib, 16 patients were in poor Biosystem, Seoul, South Korea). condition and could not maintain treatment for more than For the PCR, three sets of previously reported primers (10) 4 weeks. Adverse events were graded using the Common were used. Using a Perkin-Elmer 2400 thermal cycler (Perkin- Terminology Criteria for Adverse Events, version 3.0 (December Elmer Corp., Norwalk,