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

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• 作者：Nobuhiro Kanaji ; Shuji B ; oh ; Tomoya Ishii ; Akira Tadokoro ; Naoki Watanabe ; Takayuki Takahama ; Reiji Haba ; Osamu Imataki ; Hiroaki Dobashi ; Takuya Matsunaga
• 关键词：EML4-ALK ; EGFR ; Bronchoscopy ; RT-PCR ; Cytological sample ; Immediate cytology ; Rapid diagnosis ; Ultrafast Papanicolaou
• 刊名：Lung Cancer
• 出版年：2012
• 出版时间：August, 2012
• 年：2012
• 卷：77
• 期：2
• 页码：293-298
• 全文大小：496 K

文摘

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 ¡Á 10⁶ 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 ¡Á 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.