title: Detection of EML4-ALK fusion genes in a few cancer cells from transbronchial cytological specimens utilizing immediate cytology during bronchoscopy.
abstract: The presence of fusion genes between the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) genes is useful for determining appropriate molecular-targeted therapies in patients with non-small cell lung cancer (NSCLC). The diagnosis of NSCLC is often judged from transbronchial cytological specimens. The efficacy of RT-PCR for detection of EML4-ALK fusion genes in transbronchial cytological specimens has not been studied. Here, we evaluated the detection rate of EML4-ALK fusion genes in transbronchial cytological specimens positive for NSCLC by immediate cytology during bronchoscopic examination. Various numbers of H2228 cells carrying EML4-ALK variant 3 were combined with 1×10(6) wild-type WBCs. The RNA was extracted and the sensitivity of detection of the EML4-ALK fusion gene was determined using a nested RT-PCR. A total of 161 cell samples, from cases without available tissue samples, obtained by bronchoscopic examinations utilized for immediate cytology in patients with NSCLC were subsequently analyzed for EML4-ALK fusion genes using a nested multiplex RT-PCR. EML4-ALK variant 3 was detected in a small number of H2228 cells (10 cells), even in the presence of 1×10(6) WBCs (sensitivity: 0.001%). In the patient cytological samples, EML4-ALK fusion genes were detected in five of 161 NSCLCs (3.1%) and four of 88 adenocarcinomas (4.5%). Sequencing confirmed that these samples included three variant 1 genes, one variant 2 gene and one variant 3 gene. Using the same cytological samples, EGFR mutations were detected in 39 of 161 NSCLCs (24.2%) and 36 of 88 adenocarcinomas (40.9%). There was no case in which both EML4-ALK fusion and EGFR mutation were simultaneously detected. Rapid diagnosis during bronchoscopy utilizing immediate cytology contributed to the selection of the best samples for genetic analysis. EML4-ALK fusion genes as well as EGFR mutations were successfully detected in a small number of cancer cells from transbronchial cytological specimens using a nested multiplex RT-PCR. Our present strategy can be integrated into the clinical process without additional invasive examination of patients. In the era of molecular-targeted treatments for NSCLC, the combination of rapid diagnosis during bronchoscopic examination and stocking samples as cDNA could further correspond to genetic analyses of accumulating driver genes in NSCLC.
authors.affiliation: Department of Internal Medicine, Division of Endocrinology and Metabolism, Hematology, Rheumatology and Respiratory Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. kanaji@med.kagawa-u.ac.jp
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Kanaji
authors.forename: Shuji
authors.initials: S
authors.lastname: Bandoh
authors.forename: Tomoya
authors.initials: T
authors.lastname: Ishii
authors.forename: Akira
authors.initials: A
authors.lastname: Tadokoro
authors.forename: Naoki
authors.initials: N
authors.lastname: Watanabe
authors.forename: Takayuki
authors.initials: T
authors.lastname: Takahama
authors.forename: Reiji
authors.initials: R
authors.lastname: Haba
authors.forename: Osamu
authors.initials: O
authors.lastname: Imataki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Dobashi
authors.forename: Takuya
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D001999:Bronchoscopy; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D003581:Cytodiagnosis; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D059368:RNA Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
chemical_list: C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; D059368:RNA Isoforms; D012333:RNA, Messenger; D066246:ErbB Receptors