Peng Rongxue, Zhang Rui, Lin Guigao, Yang Xin, Li Ziyang, Zhang Kuo, Zhang Jiawei, Li Jinming
National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China.
National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China; Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People's Republic of China.
J Mol Diagn. 2017 Sep;19(5):766-775. doi: 10.1016/j.jmoldx.2017.06.003. Epub 2017 Jul 18.
The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non-small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity. Herein, we developed novel reference materials for various kinds of EML4-ALK testing. CRISPR/Cas9 was used to edit various NSCLC cell lines containing EML4-ALK rearrangement variants 1, 2, and 3a/b. After s.c. inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts were prepared and tested for suitability as candidate reference materials by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing. Sample validation and commutability assessments showed that all types of FFPE samples derived from xenograft tumors have typical histological structures, and EML4-ALK testing results were similar to the clinical ALK-positive NSCLC specimens. Among the four methods for EML4-ALK detection, the validation test showed 100% concordance. Furthermore, these novel FFPE reference materials showed good stability and homogeneity. Without limitations on variant types and production, our novel FFPE samples based on CRISPR/Cas9 editing and xenografts are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of EML4-ALK detection.
棘皮动物微管相关蛋白样4与间变性淋巴瘤激酶（ALK）受体酪氨酸激酶（EML4-ALK）重排是一种重要的生物标志物，在非小细胞肺癌（NSCLC）患者的治疗决策中起着关键作用。通过荧光原位杂交、免疫组织化学、逆转录聚合酶链反应（RT-PCR）和下一代测序确保EML4-ALK检测的准确性和可重复性，需要可靠的参考材料来监测检测的敏感性和特异性。在此，我们开发了用于各种EML4-ALK检测的新型参考材料。利用CRISPR/Cas9编辑含有EML4-ALK重排变体1、2和3a/b的各种NSCLC细胞系。皮下接种后，制备来自异种移植瘤的福尔马林固定、石蜡包埋（FFPE）样本，并通过荧光原位杂交、免疫组织化学、RT-PCR和下一代测序测试其作为候选参考材料的适用性。样本验证和互换性评估表明，来自异种移植瘤的所有类型的FFPE样本都具有典型的组织结构，并且EML4-ALK检测结果与临床ALK阳性NSCLC标本相似。在四种EML4-ALK检测方法中，验证测试显示一致性为100%。此外，这些新型FFPE参考材料表现出良好的稳定性和均一性。基于CRISPR/Cas9编辑和异种移植瘤的新型FFPE样本不受变体类型和生产的限制，适合作为EML4-ALK检测的验证、核查、内部质量控制和能力验证的候选参考材料。
