组织样本保存状态及时间对EGFR基因突变检测结果的影响

Influences of storage status and time on the mutations of EGFR gene

目的探讨不同组织保存状态(原始样本或基因组DNA)及保存时间对非小细胞肺癌(NSCLC)中表皮生长因子受体(EGFR)基因突变检测结果的影响。方法收集EGFR基因突变阳性NSCLC患者的新鲜冰冻组织和石蜡包埋(FFPE)组织各10例,每保存1个月提取1次DNA(共12次),设为冰冻新提取组和FFPE新提取组;同时分别选取初检后剩余基因组DNA作对照,设为冰冻预留组和FFPE预留组。分别采用检测试剂盒定性和定量检测上述4组EGFR基因的突变情况,同时采用荧光定量PCR技术检测内参基因的Ct值。比较4组不同保存时间的内参基因Ct值和EGFR基因突变频度情况。结果 20例原始组织样本均可以检出EGFR基因突变,与临床信息相符。新鲜冰冻组织、FFPE组织的DNA浓度均>50ng/μl,且A260/A280为1.8±0.2。新鲜冰冻组织保存10个月后,冰冻预留组的内参基因Ct值高于冰冻新提取组,差异有统计学意义(P<0.05);FFPE保存12个月内,FFPE新提取组和FFPE预留组内参基因Ct值的差异均无统计学意义(P>0.05)。冰冻新提取组保存6个月后和FFPE新提取组保存4个月后的EGFR基因突变频度高于对应预留组,差异均有统计学意义(P<0.05)。结论原始样本相对于预留基因组DNA更适合用于回顾性研究的材料。

Objective To explore the influences of storage status （original sample or genomic DNA） and time on the muta- tions of EGFR gene in non-small cell lung cancer （NSCLC）. Methods Ten fresh frozen tissues and 10 formalin-fixed paraffin-embed- ded （FFPE） tissues of NSCLC harboring EGFR mutations were collected. During the twelve months after the first detection, DNA was drawn from fresh frozen tissues （ new frozen extraction group） and FFPE tissues （ new FFPE extraction group ） at an interval of one month. The remaining genomic DNA from fresh frozen tissues （reserved frozen group） and FFPE tissues （reserved FFPE group） were chosen as control after the first detection. The detection kits were used to qualitatively and quantitatively measure the mutations of EG- FR gene in four groups, and real time PCR was performed to quantify the Ct value of reference gene to reflect the contents of genomie DNA. The mutation frequencies of EGFR gene and Ct values of reference gene were compared among 4 groups. Results The mutation of EGFR gene could be detected in 20 tissues of original samples, which were consisting with the clinical data. The concentrations of DNA drawn from fresh frozen tissues and FFPE tissues were above 50ng/μl with A260/A2s0 around 1.8±0. 2. The Ct values were high in the reserved frozen group versus the new frozen extraction group after 10 months （P〈0. 05）. No significant difference of Ct value was observed between the new FFPE extraction group and the reserved FFPE group （P〉0. 05）. The mutation frequencies of EGFR gene in the new frozen extraction group after 6 months and the new FFPE extraction group after 4 months were significant higher than those in the corresponding reserved group （P〈0. 05）. Conclusion Original samples are better resources than reserved genomic DNA in retro- spective studies.