Imaging, Diagnosis, Prognosis A Prospective PCR-Based Screening for the EML4-ALK Oncogene in Non–Small Cell Lung Cancer Manabu Soda 1 , Kazutoshi Isobe 2 , Akira Inoue 6 , Makoto Maemondo 7 , Satoshi Oizumi 8 , Yuka Fujita 9 , Akihiko Gemma 3 , Yoshihiro Yamashita 1 , Toshihide Ueno 1 , Kengo Takeuchi 4 , Young Lim Choi 1,5 , Hitoshi Miyazawa 10 , Tomoaki Tanaka 10 , Koichi Hagiwara 10 , and Hiroyuki Mano 1,5,11 , for the North-East Japan Study Group and the ALK Lung Cancer Study Group Abstract Purpose: EML4-ALK is a lung cancer oncogene, and ALK inhibitors show marked therapeutic efficacy for tumors harboring this fusion gene. It remains unsettled, however, how the fusion gene should be detected in specimens other than formalin-fixed, paraffin-embedded tissue. We here tested whether reverse transcrip- tion PCR (RT-PCR)-based detection of EML4-ALK is a sensitive and reliable approach. Experimental Design: We developed a multiplex RT-PCR system to capture ALK fusion transcripts and applied this technique to our prospective, nationwide cohort of non–small cell lung cancer (NSCLC) in Japan. Results: During February to December 2009, we collected 916 specimens from 853 patients, quality filtering of which yielded 808 specimens of primary NSCLC from 754 individuals. Screening for EML4-ALK and KIF5B-ALK with our RT-PCR system identified EML4-ALK transcripts in 36 samples (4.46%) from 32 individuals (4.24%). The RT-PCR products were detected in specimens including bronchial washing fluid (n ¼ 11), tumor biopsy (n ¼ 8), resected tumor (n ¼ 7), pleural effusion (n ¼ 5), sputum (n ¼ 4), and metastatic lymph node (n ¼ 1). The results of RT-PCR were concordant with those of sensitive immuno- histochemistry with ALK antibodies. Conclusions: Multiplex RT-PCR was confirmed to be a reliable technique for detection of ALK fusion transcripts. We propose that diagnostic tools for EML4-ALK should be selected in a manner dependent on the available specimen types. FISH and sensitive immunohistochemistry should be applied to formalin- fixed, paraffin-embedded tissue, but multiplex RT-PCR is appropriate for other specimen types. Clin Cancer Res; 18(20); 5682–9. Ó2012 AACR. Introduction An oncogenic fusion between the echinoderm microtu- bule-associated protein–like 4 gene (EML4) and the ana- plastic lymphoma kinase gene (ALK) was discovered by functional screening with a non–small cell lung cancer (NSCLC) specimen (1). EML4 and ALK are located within a short distance (12 Mbp) of each other on the short arm of human chromosome 2, and a small inversion involving the 2 loci is responsible for generation of the EML4-ALK fusion in lung cancer. The EML4-ALK tyrosine kinase undergoes constitutive dimerization through a coiled-coil domain within EML4, resulting in kinase activation and conferring potent transforming ability (2, 3). Transgenic mice expres- sing EML4-ALK in lung alveolar cells develop multiple adenocarcinoma nodules soon after birth, but treatment with an ALK inhibitor results in the rapid clearance of such nodules, confirming the addiction of EML4-ALK–positive tumors to the kinase activity of the fusion protein (4). The therapeutic efficacy of ALK inhibitors has been confirmed in other transgenic mice expressing EML4-ALK (5). Several ALK inhibitors have already entered clinical trials or are under preclinical development (6–10). Marked ther- apeutic efficacy of one such compound, crizotinib, has been described in patients with NSCLCs positive for EML4-ALK, with an overall response rate of 57% (7), and crizotinib was recently approved as a therapeutic drug by the U.S. Food Authors' Affiliations: 1 Division of Functional Genomics, Jichi Medical University, Tochigi; 2 Department of Respiratory Medicine, Toho University Omori Medical Center; 3 Nippon Medical School Hospital; 4 Pathology Project for Molecular Targets, The Cancer Institute; 5 Department of Med- ical Genomics, Graduate School of Medicine, University of Tokyo, Tokyo; 6 Tohoku University Hospital; 7 Miyagi Cancer Center, Miyagi; 8 First Depart- ment of Medicine, Hokkaido University School of Medicine; 9 Asahikawa Medical Center, Hokkaido; 10 Saitama Medical University Hospital; and 11 CREST, Japan Science and Technology Agency, Saitama, Japan Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). The nucleotide sequence of the novel EML4-ALK variant cDNA from patient J-#189 has been deposited in the DDBJ/EMBL/GenBank databases under the accession number AB663645. Corresponding Author: Hiroyuki Mano, Division of Functional Genomics, Jichi Medical University, 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329- 0498, Japan. Phone: 81-285-58-7449; Fax: 81-285-44-7322; E-mail: hmano@jichi.ac.jp doi: 10.1158/1078-0432.CCR-11-2947 Ó2012 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 18(20) October 15, 2012 5682 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/18/20/5682/2006096/5682.pdf by guest on 13 March 2023