Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

中文翻译：

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开发用于检测 FLT3D835Y 的高灵敏度方法。

急性髓细胞性白血病（AML）是髓系祖细胞的恶性血液肿瘤。在约 8% 的 AML 患者中发现 FLT3 的酪氨酸激酶结构域 (FLT3-TKD) 突变，其中 D835Y 是最常见的替代物。这种突变激活了驱动疾病的生存信号，并且对第一代 FLT3 抑制剂具有抗性。开发一种检测 FLT3D835Y 的高灵敏度方法对于指导治疗选择、预测预后和监测 AML 患者的微小残留疾病非常重要。在本研究中，我们利用限制性片段嵌套等位基因特异性 PCR 技术开发了一种高灵敏度的 FLT3D835Y 检测方法。该方法包括三个步骤：1) 使用 FLT3D835Y 突变位点周围的 PCR 引物对 DNA 样本进行初始扩增，2) 用仅切割野生型等位基因的限制酶 EcoRV 消化 PCR 产物，和 3) 用嵌套引物通过等位基因特异性 PCR 检测 FLT3D835Y。通过使用含有已知比例的 FLT3D835Y 的纯化质粒 DNA 和血细胞 DNA，我们能够以 0.001% 的灵敏度检测 FLT3D835Y。我们分析了 64 名 AML 患者的血细胞 DNA 样本，发现了 6 例 FLT3D835Y 阳性病例，其中 2 例无法通过常规 DNA 测序方法检测到。重要的是，该方法能够在从复发患者收集的样本中检测 FLT3D835Y，而该患者处于完全缓解状态，通过流式细胞术确定 MRD 为阴性。因此，我们的 RFN-AS-PCR 检测到了常规方法在流式细胞术和 Sanger DNA 测序中遗漏的治疗后的 MRD。我们开发了一种简单且高度灵敏的方法，可以在非常低的水平上检测 FLT3D835Y。这种方法可能对 AML 的治疗具有重要的临床意义。