【摘要】 目的:以Mass ARRAY质谱i PLEX分析技术为平台建立适合中国肺癌人群多基因同步检测的方法。方法:通过文献检索并结合国内外研究进展,确定与中国肺癌人群发病、耐药以及转移等密切相关的13个靶基因或相关传导通路基因(EGFR、KRAS、ALK、FGFR1、FGFR2、FGFR3、PIK3CA、BRAF、PTEN、MET、ERBB2、AKT1、STK11)。对目的基因突变进行筛选并确定99个热点突变。按Mass ARRAY突变位点标示方法和引物设计固定格式,引物设计软件在线设计297条引物(正、反向扩增引物和延伸引物各99条)。以8个肺癌细胞系以及6例肿瘤组织样本建立检测方法,与Lung Carta试剂盒对比验证。扩大检测100例肺癌组织样本,与EGFR和KRAS直接测序法比较敏感度与特异度。结果:使用本方法检测肺癌细胞系的基因突变,其中1例肺癌组织新检测到FGFR2基因突变,其他结果与Lung Carta试剂盒一致。与直接测序法相比较灵敏度为100%,特异度为96.3%。结论:成功建立Mass ARRAY质谱分析肺癌多基因突变检测方法,适合中国肺癌人群且具有临床应用前景。更多还原

【Abstract】 Objective: To establish a method based on the i PLEX analysis of Mass ARRAY mass spectrometry platform to detect multiplex genetic mutations among Chinese lung cancer patients. Methods: We reviewed the related literature and data of lung cancer treatments. We also determined 99 mutation hot spots in 13 target genes, namely, EGFR, KRAS, ALK, FGFR1, FGFR2, FGFR3, PIK3 CA,BRAF, PTEN, MET, ERBB2, AKT1, and STK11, which are closely related to the pathogenesis, drug resistance, and metastasis of lung cancer and are associated with relevant transduction pathways. A total of 297 primers comprising 99 paired forward and reverse amplification primers and 99 matched extension primers were designed by using Assay Design in accordance with the mutation label and format requirements of the MassA RRAY platform. The detection method was established by analyzing eight cell lines and six lung cancer specimens; the proposed method was then validated through comparisons with a LungC arta kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results: The proposed method could detect multiplex genetic mutations in the lung cancer cell lines, and this finding is consistent with that observed using previously reported methods. The proposed method could also detect such mutations in clinical lung cancer specimens; this result is also consistent with that observed by using the LungC arta kit. However, an FGFR2 mutation was detected only by using the proposed method. The measured sensitivity and specificity were100% and 96.3%, respectively. Conclusion: The proposed MassA RRAY technology-based method could detect multiplex genetic mutations among Chinese lung cancer patients. Indeed, the proposed method can be potentially applied to detect mutations in cancer cells.更多还原