锁核酸扩增阻滞荧光聚合酶链反应技术检测结直肠癌患者血浆肿瘤K-ras基因突变

K -ras mutation detection using a novel amplification refractory mutation system -based quantitative polymerase chain reaction with locked nucleic acid blocker in plasma among colorectal cancer patients

目的建立锁核酸扩增阻滞突变体系荧光聚合酶链反应（LNA-ARMS-PCR）技术检测血浆K-ras突变方法，探讨该方法替代组织检测K-ras突变的可行性。方法应用锁核酸标记技术建立LNA-ARMS-PCR检测K-ras突变技术，收集155例结直肠癌患者肿瘤组织及其对应血浆标本，同时检测其中的K-ras突变，分析其符合率。结果建立的检测技术灵敏度高，质粒混合实验证实其灵敏度高达0.1%。肿瘤原发灶内的异质性分析结果显示本方法可以避免因肿瘤异质性导致的假阳性。血浆与肿瘤组织K-ras突变检测的阳性符合率为35.3%，阴性符合率均为100.0%；Ⅳ期患者血浆与组织突变阳性符合率高达83.3%（15/18）。血浆与组织突变检测符合率与TNM分期及循环游离DNA（cfDNA）水平密切相关。结论LNA-ARMS-PCR方法检测血浆K-ras突变，可以取代肿瘤组织用于检测K-ras突变，但仅限于晚期转移性患者临床价值高。

Objective To determine whether plasma circullating cell -free DNA （cfDNA） is a viable alternative approach for K - ras mutation testing in colorectal cancer （ CRC ） patients. Methods The study included 155 CRC patients. An a novel amplification refractory mutation system -based quanti- tative polymerase chain reaction with locked nucleic acid （ LNA - ARMS - PCR） was developed to assess the K - ras mutation in preoperative plasma and paired tumor tissues. Firstly intra - tumoral heterogeneity analysis was used to determine whether it was a robust mutation detection method for the detection of low - abundance K - ras mutations. Results The newly built LNA - ARMS - PCR was a robust mutation detection method to escape the most false negative caused by intra - tumoral heterogeneity. The positive consen- sus rate and negative consensus rate was 35.3% and 100. 0% , between plasma and tumor tissue K - ras mutation detection, respectively. The majority of K - ras mutations detected in tumors were also found in plasma（ 15/18 （83.3%）） in patients of stage Ⅳ. The accordance of plasma and tumor mutation are strongly correlated to TNM stage and efDNA level. ConcLusion K - ras analysis in plasma cfDNA is only a viable alternative to tissue analysis in mCRC （ stage Ⅳ）. Quantitative levels of cfDNA and plasma mutation positivity are strongly correlated.