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.
Lung Cancer. 2012 Aug;77(2):293-8. doi: 10.1016/j.lungcan.2012.03.018. Epub 2012 Apr 10.
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.
棘皮动物微管相关蛋白样 4 (EML4) 基因与间变性淋巴瘤激酶 (ALK) 之间融合基因的存在可用于确定非小细胞肺癌 (NSCLC) 患者的适当分子靶向治疗。NSCLC 的诊断通常根据经支气管细胞学标本进行判断。尚未研究 RT-PCR 检测经支气管细胞学标本中 EML4-ALK 融合基因的效果。在此，我们评估了在支气管镜检查期间即时细胞学检查中，经支气管细胞学标本呈阳性的 NSCLC 患者中 EML4-ALK 融合基因的检出率。将不同数量的携带 EML4-ALK 变体 3 的 H2228 细胞与 1×10(6) 个野生型 WBC 混合。提取 RNA，并用巢式 RT-PCR 确定检测 EML4-ALK 融合基因的灵敏度。共分析了 161 例细胞样本，这些细胞样本来自无组织样本的患者，通过支气管镜检查获得，用于即时细胞学检查。然后，使用巢式多重 RT-PCR 分析这些患者的 EML4-ALK 融合基因。即使存在 1×10(6) WBC，也可在少量 H2228 细胞 (10 个细胞) 中检测到 EML4-ALK 变体 3 (灵敏度：0.001%)。在患者细胞学样本中，在 161 例 NSCLC 中的 5 例 (3.1%) 和 88 例腺癌中的 4 例 (4.5%) 中检测到 EML4-ALK 融合基因。测序证实这些样本包括 3 个变体 1 基因、1 个变体 2 基因和 1 个变体 3 基因。在 161 例 NSCLC 中，使用相同的细胞学样本检测到 39 例 (24.2%) 和 88 例腺癌中的 36 例 (40.9%) EGFR 突变。没有同时检测到 EML4-ALK 融合和 EGFR 突变的病例。利用即时细胞学的支气管镜检查快速诊断有助于选择用于基因分析的最佳样本。通过巢式多重 RT-PCR，成功地从经支气管细胞学标本中的少量癌细胞中检测到 EML4-ALK 融合基因和 EGFR 突变。我们的策略可以整合到临床流程中，而无需对患者进行额外的侵入性检查。在 NSCLC 分子靶向治疗时代，支气管镜检查期间的快速诊断与储存样本作为 cDNA 的结合，可以进一步对应 NSCLC 中不断积累的驱动基因的遗传分析。
