非小细胞肺癌EML4-ALK融合方式的多样性

Diversity of EMIA-ALK fusion variants in non-small cell lung cancer

目的探讨在非小细胞肺癌（NSCLC）标本中棘皮动物微管相关蛋白样4与间变淋巴瘤激酶融合基因（EMIA-ALK）变异的复杂性，分析其酪氨酸激酶（TK）结构域是否存在突变。方法在用eDNA末端快速扩增联合测序法检出1例NSCLC患者标本存在EML4-ALK的新变体V3e基础上，进一步采用逆转录（RT）-PCR分析39例NSCLC患者标本，针对阳性产物采用T载体克隆测序技术分析潜在的EML4-ALK序列多样性。同时用RT—PCR和测序分析EML4．ALK酪氨酸激酶结构域序列信息。结果从40例NSCLC患者肿瘤标本中检出3例NSCLC患者存在EML4-ALK变异。1例患者标本中EML4-ALK存在V3e（64．6％）、V3d（25．0％）、V3e（2．1％）、V3f（4．2％）、V3g（2．1％）和V3h（2．1％）6种融合变体，但不存在V3a、V3b2种常见融合方式；1例为V1变体；1例为V2和V3a、V3b变体共存。3例阳性标本中未发现ALK基因的TK结构域有耐药基因突变。结论NSCLC患者中可同时共存多种EML4-ALK变体，即EML4-ALK融合方式存在多样性与序列复杂性，临床分子检测中应全面关注EMIA-ALK的多种融合变体，以提高检出率。

Objective To investigate the fusion sequence complexity of EML4-ALK in non-small cell lung cancer （NSCLC） patients, and the potential mutation in tyrosine kinase （TK） domain of ALK gene. Methods In routine practice, a novel echinoderm microtubule-associated protein-fike4 and anaplastic lymphoma kinase （EMIA-ALK） V3c variant was detected by rapid amplification of cDNA ends-polymerase chain reaction （RACE-PCR）-sequencing technology in a patient with NSCLC. The further consecutive 39 cases（ total of 40 cases） were screened by use of reverse transcription （ RT）-PCR for EML4-ALK fusion. Positive PCR products were purified and cloned into T vectors, transformed into DHSa germ cells and colony picked up and sequenced for sequence complexity analysis. Tyrosine kinase domain of ALK was amplified by RT-PCR and sequenced. Results Three out of 40 cases had EML4-ALK fusion. One case had six novel variants of EML4-ALK co-existing, termed as V3c （64. 6% ） , V3d （ 25.0% ） , V3e （ 2. 1% ）, V3f （4.2%） ,V3g（2.1% ）and V3h（2. 1% ） variants, whereas without common V3a and V3b variants. In other two positive cases, one was V1 variant, another was concurrent V2, V3a and V3b variants. No mutations were detected in the TK domain of EML4-ALK in any case. Conclusions Several EML-ALK variants could co-exist in a given lung cancer tissue, which suggest that the diversity and sequence complexity of EML4- ALK fusion are exist. Attentions should be paid to screen all the variants in clinic to improve the pick-up rate. （ Chin J Lab Med, 2012,35：593-597）