Scholl Sebastian, Krause Claudia, Loncarevic Ivan F, Müller Rouven, Kunert Christa, Wedding Ulrich, Sayer Herbert G, Clement Joachim H, Höffken Klaus
Department of Internal Medicine II (Oncology and Hematology), Friedrich Schiller University, Jena, Germany.
J Lab Clin Med. 2005 Jun;145(6):295-304. doi: 10.1016/j.lab.2005.03.005.
Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.
在激活型III类受体酪氨酸激酶（Flt3）突变中，约25%的急性髓系白血病（AML）患者可检测到Flt3的内部串联重复（Flt3-ITD）。相比之下，Flt3酪氨酸激酶结构域内的突变（Flt3-TKD突变）频率较低（约7%），而关于最近发现的密码子592处激活型Flt3点突变（Flt3-V592A突变）的频率仅有有限的数据。我们在一组122例患者中评估了一种基于荧光共振能量转移（FRET）原理快速筛查Flt3-TKD和Flt3-V592A突变的新方法。基于个体Flt3-TKD突变，我们设计了患者特异性引物，以进行高灵敏度聚合酶链反应（PCR）检测，用于快速检测微小残留病（MRD）。我们还使用携带Flt3-V592A突变的MonoMac-6细胞模型系统，为这种分子畸变建立了一种突变特异性实时PCR方法。我们鉴定出9例（8%）Flt3-TKD突变（5例D835Y突变、3例D835H突变和1例Del836突变），未发现Flt3-V592A突变病例。用荧光探针筛查Flt3-TKD突变等同于使用标准PCR随后进行EcoRV酶切的传统筛查。我们提出了一种可用于基于个体Flt3-TKD突变进行MRD分析的实时PCR方案。本文给出了本研究中鉴定出的所有3种Flt3-TKD突变亚型的MRD分析示例。总之，我们展示了用于快速筛查Flt3点突变以及基于患者特异性Flt3-TKD突变检测MRD的新方法。
