A Novel Mass Spectrometry-Based Assay for Diagnosis of EML4-ALK-Positive Non-Small Cell Lung Cancer
A Novel Mass Spectrometry-Based Assay for Diagnosis of EML4-ALK-Positive Non-Small Cell Lung Cancer
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DOI：
10.1097/jto.0b013e31824c7f7a
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发表时间：
2012-05-01
影响因子：
20.4
通讯作者：
Nishio, Kazuto
中科院分区：
文献类型：
--
作者：
Sakai, Kazuko;Okamoto, Isamu;Nishio, Kazuto
简介：非小细胞肺癌（NSCLC）中存在转化融合基因棘皮动物微管相关蛋白样4（EML 4）-间变性淋巴瘤激酶（ALK）是间变性淋巴瘤激酶抑制剂疗效的预测标志物。然而，目前可用于检测EML 4-ALK的不同变体的检测方法具有limitations.Methods：我们开发了一种检测系统，用于检测从福尔马林固定的石蜡包埋（FFPE）NSCLC组织标本中获得的总RNA中EML 4-ALK变体1，2，3a，3b，4，5a，5 b，6或7的转录物。该试验基于EML 4-ALK互补DNA的区域特异性聚合酶链反应扩增，随后进行特异性单碱基引物延伸，并通过基质辅助激光解吸/电离飞行时间质谱法分析延伸产物。荧光原位杂交和亚克隆和测序的聚合酶链反应products.Results的结果证实了该测定法进行了验证：评价的分析灵敏度与含有EML 4-ALK互补DNA序列的质粒的系列稀释液，揭示它是能够可靠地检测每个反应的每个质粒的一个拷贝。该检测试剂盒还在3份手术切除的NSCLC FFPE样本中检测到EML 4-ALK变体1或3，通过荧光原位杂交显示间变性淋巴瘤激酶重排阳性。此外，该测定在来自晚期NSCLC患者的20份FFPE活检样本中的3份中鉴定出EML 4-ALK的变体1。所有阳性标本均经亚克隆和测序证实。结论：该方法灵敏度高，可用于FFPE标本中EML 4-ALK的检测。
Introduction: The presence of the transforming fusion gene echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations.Methods: We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by subcloning and sequencing of polymerase chain reaction products.Results: Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing.Conclusions: Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.