Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer Junichi Soh 1 , Shinichi Toyooka 1 * , Keisuke Aoe 2 , Hiroaki Asano 1 , Syuji Ichihara 1 , Hideki Katayama 2 , Akio Hiraki 2,3 , Katsuyuki Kiura 4 , Motoi Aoe 1 , Yoshifumi Sano 1 , Kazuro Sugi 2 , Nobuyoshi Shimizu 1 and Hiroshi Date 1 1 Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan 2 NHO Sanyo National Hospital Respiratory Disease Center, Ube, Yamaguchi, Japan 3 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan 4 Department of Hematology, Oncology, and Respiratory Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan The importance of epidermal growth factor receptor (EGFR) gene mutation has been recognized in nonsmall cell lung cancer (NSCLC), requiring the standardization of mutation screening system including the kind of samples. Here, we examined the EGFR mutation status in 61 pleural fluid samples from NSCLC cases using direct sequencing, nonenriched PCR, mutant-enriched PCR and peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay. The mutant-enriched PCR assay detected 16 mutant cases. Among them, the nonenriched PCR assay failed to detect 3 mutant cases. Regarding the discrepancy between mutant-enriched PCR and PNA-LNA PCR clamp assays, 3 cases of exon19-deletions were detected only by mutant-enriched PCR assay and no difference at the L858R mutation. There was no dif- ference in results between direct sequencing and nonenriched PCR assay. We also correlated the EGFR mutation with clinical outcome of gefitinib-treated 29 cases. EGFR mutations were pres- ent in 10 cases, revealing 7 partial response and 3 no change (NC). In EGFR wild-type cases, 10 revealed NC and 9 progressive dis- ease. The responders were significantly more frequent among the EGFR mutant cases than among the wild-type (p < 0.0001). Over- all survival (p 5 0.0092) and progression-free survival (p 5 0.018) were significantly longer among the EGFR mutant cases than among the wild-type. In summary, we evaluated the utility of EGFR mutation screening in pleural fluid using 4 assays that showed some discrepancies arising from the designs of the assays. As clinical importance, the EGFR mutation status in pleural fluid can be a biomarker for the favorable outcome of gefitinib-treated NSCLC cases. ' 2006 Wiley-Liss, Inc. Key words: EGFR; lung cancer; pleural fluid; mutant-enriched PCR; gefitinib Mutations in the epidermal growth factor receptor (EGFR) gene have been reported in lung cancer, especially in adenocarcinoma of the lung, a subtype of nonsmall cell lung cancer (NSCLC). 1–4 Of particular interest, EGFR mutations or gene amplification are considered to be the indicators of a better clinical outcome in patients treated with EGFR tyrosine kinase inhibitors, like gefiti- nib and erlotinib. 1–8 These findings have led to the next stage for NSCLC treatment, where the EGFR status plays a crucial role in determining the therapeutic strategy. Thus, reliable assays to detect EGFR mutations in various kinds of clinical samples are now needed. Recent advances in biotechnology have enabled us to develop convenient assays to detect mutations in DNA, and we previously established a sensitive PCR-based assay to detect EGFR exon19- deletions and an exon21 point-mutation (L858R). 9 Several studies have also reported unique assays to detect EGFR mutations with high sensitivity, including a peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay developed by Nagai and col- leagues. 10,11 Although the individual assays seem to be well designed and useful, the cross-validation of each assay is an impor- tant process in standardizing mutation screening using these assays. Malignant pleural effusions can be observed in patients with various types of neoplasms, but lung cancer is the leading cause of this complication, accounting for approximately one-third. 12 Ma- lignant effusions are observed in 7–15% of all bronchogenic carci- nomas, most frequently in lung adenocarcinomas, at some time during the course of illness. 12 Pleural effusion is often present in patients with advanced disease, and thoracentesis may be neces- sary for diagnosis and treatment, suggesting its usefulness for mo- lecular analysis. Indeed, mutations in TP53, KRAS and EGFR have been detected in pleural effusion samples. 13,14 In addition, although the cytological examination of pleural effusion is useful for diagnosing cancer, it is not routinely warranted for light mi- croscopy studies alone (sensitivity, 40–87%). 15 Thus, genetic analyses of pleural effusion samples might also be helpful for di- agnosis, especially since EGFR mutations exhibit a high specific- ity for lung adenocarcinomas. We previously demonstrated that EGFR mutations were detectable in soluble DNA from the pleural fluid that is usually discarded after the removal of cell component for cytological examination, indicating the usefulness of such clin- ical samples. 9 In this study, we examined the presence of EGFR exon19-dele- tions and the L858R mutation in pleural fluid samples using direct sequencing, nonenriched PCR, mutant-enriched PCR and PNA- LNA PCR clamp assays performed at independent laboratories. In addition, we examined the correlation between the EGFR muta- tion status and the clinical outcome, including the responsiveness and prognosis of NSCLC patients treated with gefitinib. Material and methods Clinical samples, DNA extraction and EGFR direct sequencing Sixty-one samples of pleural fluid from lung cancer patients were analyzed by direct sequencing, nonenriched PCR, mutant- enriched PCR and PNA-LNA PCR clamp assays. The patient characteristics are shown in Table I. The pleural fluid was col- lected from pleural effusion after the removal of cells. The permis- sion of the institutional review board and informed consent from each patient were obtained. Soluble DNAs in 1 ml from each of the 61 pleural fluid samples were extracted using the QIAmp DNA blood kit (Qiagen), according to the manufacturer’s protocol for blood and body fluid. The concentration of DNA was quanti- Grant sponsor: Ministry of Education, Science, Sports, Culture and Technology of Japan; Grant number: 18790993. *Correspondence to: Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Fax: 181-86-235-7269. E-mail: s_toyooka@nigeka2.hospital.okayama-u.ac.jp Received 22 May 2006; Accepted 14 June 2006 DOI 10.1002/ijc.22190 Published online 18 August 2006 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 119, 2353–2358 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer