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Abstract：
The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.
摘要：
FMS样酪氨酸激酶3(FLT3-TKD)的酪氨酸激酶结构域在急性髓性白血病(AML)中反复突变。其检测中常用的分子技术包括PCR和毛细管电泳,Sanger测序和下一代测序具有公认的灵敏度限制。本研究旨在验证液滴数字PCR（ddPCR）在检测涉及常见FLT3-TKD突变（D835Y，D835H,D835V,D835E)。22个诊断样本,六个捐赠者控制，和商业D835Y阳性对照使用商业Bio-rad®ddPCR测定法进行测试。确定了所有已知的变异体,并且在野生型对照中未检测到假阳性(100%特异性和敏感性)。该测定在0.01%变体等位基因分数下达到适合于MRD测试的检测限。测试了来自7名在诊断时具有FLT3-TKD变体的强化治疗患者的系列样品。五名患者表现出FLT3-TKD克隆的清除，但2例患者在原发难治性疾病的情况下存在FLT3-TKD持续性.总之,ddPCR适用于MRD环境中FLT3-TKD突变的检测和定量；然而，MRD阳性的临床意义和最佳管理需要进一步探索.
