急性髓系白血病FMS样酪氨酸激酶3-激酶结构域突变的高灵敏度检测方法的建立及应用

Development and application of a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain of acute myeloid leukemia

目的 建立一种急性髓系白血病(acute myeloid leukemia,AML)FMS样酪氨酸激酶3(FMS-like tyrosine kinase-3,FLT3)-激酶结构域突变(tyrosine kinase domain,TKD)的高灵敏度检测方法,并将该方法应用于AML微小残留病灶(minimal residual disease,MRD)的监测。方法 构建FLT3野生型质粒和FLT3-D835Y突变型质粒,按一定比例混合后制备FLT3-D835Y突变率为50%、1%、0.1%和0%的质粒标准品,以其为检测对象,联合限制性片段长度多态性(restriction fragment length polymorphism,RFLP)识别法和一代测序法建立新的FLT3-TKD检测方法。用建立的方法检测含不同FLT3-D835Y突变率的质粒DNA标准品和血液样本DNA标准品,以验证方法的灵敏度;用建立的方法检测AML患者治疗前后的基因组DNA样本,以监测MRD。结果 构建的FLT3野生型质粒和FLT3-D835Y突变型质粒经测序证明构建正确。在纯化的质粒DNA和收集的血液样本DNA中,建立的方法通过一代测序结合EcoRⅤ/XhoⅠ双酶切和回收突变片段的步骤,将灵敏度提高至0.1%;用该方法在1名AML患者完全缓解期外周血样本中检测到MRD,而直接一代测序法未检测到MRD。结论 本研究开发了一种高灵敏度检测FLT3-TKD突变的方法,该方法对指导AML治疗以及监测其完全缓解期内MRD具有重要临床意义。

ObjectiveTo develop a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain(FLT3-TKD)of acute myeloid leukemia(AML)and apply to the monitor of minimal residual disease(MRD).MethodsRecombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed respectively and mixed at certain ratios.The obtained standard plasmids with mutation rates of 50%,1%,0.1% and 0% respectively were determined by restriction fragment length polymorphism(RFLP)in combination with Sanger method.The plasmid DNA standards and blood DNA standards,at various FLT3-D835Y mutation rates,were determined by the developed method to verify the sensitivity.The genomic DNA samples of patients with AML before and after treatment were determined by the developed method to monitor the MRD.ResultsSequencing proved that both the recombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed correctly.The sensitivity of developed method increased to 0.1% through Sanger method combined with digestion with EcoR Ⅴ/Xho Ⅰ and recovery of mutant fragments in determination of purified plasmid DNA and collected blood DNA samples.MRD was detected in the peripheral blood sample of a patients with AML in complete remission period by the developed method but not by Sanger method.ConclusionA highly sensitive method for detection of FLT3-TKD mutation was developed,which was of an important clinical significance in guiding the treatment of AML and monitoring the MRD in complete remission period.