Multiplex Reverse Transcription-PCR Screening for EML4-ALK Fusion Transcripts
Multiplex Reverse Transcription-PCR Screening for EML4-ALK Fusion Transcripts
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DOI：
10.1158/1078-0432.ccr-08-1018
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发表时间：
2008-10-15
影响因子：
11.5
通讯作者：
Mano, Hiroyuki
中科院分区：
文献类型：
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作者：
Takeuchi, Kengo;Choi, Young Lim;Mano, Hiroyuki
目的：EML 4-ALK是一种融合型蛋白酪氨酸激酶，由inv（2）（p21 p23）在非小细胞肺癌（NSCLC）基因组中产生。为了能够灵敏地检测EML 4-ALK融合转录物，我们现在开发了一种多重逆转录-PCR（RT-PCR）系统，该系统能够捕获两种基因之间的所有框内融合。设计引物以检测EML 4与ALK外显子20的所有可能的框内融合，采用单管多重RT-PCR方法检测了656例肺实体瘤（n = 364）和10例其他器官实体瘤的总RNA。从连续的肺腺癌病例（n = 253）中，我们确定了11个标本（4.35%）融合转录阳性，其中9个是阳性的先前确定的变体1，2和3。其余2份标本含有新型转录物亚型，其中EML 4的外显子14（变体4）或外显子2（变体5）与ALK的外显子20连接。对于其他类型的肺癌（n = 111）或来自10个其他器官的肿瘤（n = 292），未检测到融合转录物。通过基因组PCR分析和荧光原位杂交证实了NSCLC细胞中导致融合事件的基因组重排。EML 4-ALK的新亚型表现出显着的致癌活性，他们产生了一个模式的细胞质染色与细颗粒病灶的NSCLC标本的免疫组化analysis.Conclusions：这些数据加强了EML 4-ALK阳性肿瘤的准确诊断的重要性，为优化治疗策略。
Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2) (p21p23) in the genome of non - small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes.Experimental Design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs.Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens.Conclusions: These data reinforce the importance of accurate diagnosis of EML4-ALK - positive tumors for the optimization of treatment strategies.