Detection of EML4-ALK fusion genes in a few cancer cells from transbronchial cytological specimens utilizing immediate cytology during bronchoscopy

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 x 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 x 106 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. (C) 2012 Elsevier Ireland Ltd. All rights reserved.