Pathology Project for Molecular Targets, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan.
PLoS One. 2012;7(2):e31323. doi: 10.1371/journal.pone.0031323. Epub 2012 Feb 8.
The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5'-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.
间变性淋巴瘤激酶（ALK）抑制剂的可喜结果改变了几种癌症中 ALK 融合的意义。这些融合不再仅仅是研究靶点或诊断标志物，而是直接与患者的治疗获益相关。然而，临床中大多数可用的肿瘤组织都是经过福尔马林固定和石蜡包埋（FFPE）的，这在许多临床情况下极大地限制了对遗传的详细研究。尽管最近的技术改进允许对 FFPE 组织中的一些已知突变进行分析，但仅使用 FFPE 组织来鉴定未知的融合基因仍然很困难。我们开发了一种优化用于 FFPE 组织的 5' 快速扩增 cDNA 末端系统，并在具有 ALK 重排但没有 2 种已知的 ALK 融合 EML4-ALK 和 KIF5B-ALK 的肺癌组织上评估了该系统。使用该系统，我们成功鉴定了一种新型的 ALK 融合，即 KLC1-ALK。通过逆转录-聚合酶链反应和荧光原位杂交验证了该结果。然后，我们合成了假定的全长 KLC1-ALK cDNA，并使用 3T3 细胞进行了融合激酶的转化潜力测定。据我们所知，KLC1-ALK 是仅使用 FFPE 组织鉴定的第一个新型致癌融合。这一发现将拓宽存档 FFPE 组织的潜在价值，并为 ALK 阳性肺癌提供进一步的生物学和临床见解。
