FLT3 mutations. There are two types of FLT3 mutations, FLT3-ITD and FLT3-KDM. FLT3-ITD occurs between exons 14 and 15. When leukemia cells have FLT3-ITD, PCR products give a wild-type band and a larger ITD band (A). D835 and I836 codons are encoded by the nucleotide GATATC which forms the Eco RV restriction site. The amplified products of wild type FLT3 are digested to two bands by the Eco RV. When amplified products contain D835-mutations (FLT3-KDM), undigested bands are observed (B). 

Context in source publication

Context 1

... (FMS-like tyrosine kinase 3) belongs to a type III receptor tyrosine kinase together with KIT, FMS and PDGF-receptor, and consists of five immunoglobulin-like domains in the extracellular region, a juxtamembrane (JM) domain, a tyrosine kinase (TK) domain separated by a kinase insert (KI) domain and a C-terminal domain in the intracellular region. 5-7) FLT3 expresses on the surface of normal hematopoietic stem/progenitor cells. FLT3 ligand (FL) is expressed by bone marrow stroma cells, and FL-FLT3 interaction plays an important role in the survival, proliferation and differentiation of normal hematopoietic stem/progenitor cells. [8][9][10][11][12][13] In addition, FLT3 expresses in most AML and ALL cells, and FL-stimulation enhances proliferation and reduces apoptosis of leukemia cells. 14) In 1996, an internal tandem duplication mutation in the JM domain-coding sequence of the FLT3 gene (FLT3-ITD) was first identified in AML cells. 15) Subsequently, a missense point mutation at the D835 residue and point mutations, deletions and insertions in the codons surrounding D835 within a TK domain of FLT3 (FLT3-KDM) were identified (Figure 1). [16][17][18][19][20][21][22] FLT3 mutations are identified in about 30% of the adult patients with AML, and are highly associated with leukocytosis and poor prognosis. [23][24][25][26][27][28] The WHO and European LeukemiaNet, therefore, recommend that FLT3 mutations should be analyzed at the diagnosis of AML. 2,29) BIOLOGICAL EFFECTS OF FLT3 MUTATIONS The binding of FL to the extracellular domain of FLT3 leads to the dimerization and transphosphorylation of the A-loop, resulting in the activation of FLT3, followed by induction of multiple intracellular signaling pathways, such as MAPK-and AKT-signals, leading to cell proliferation and activation. In contrast, mutant FLT3 is ligand-independently dimeralized and activated. Of note is that mutant FLT3 activates STAT5 in addition to MAPK-and AKT-signals, indicating that the phosphorylation level of STAT5 is a surrogate marker of the mutant FLT3 activation. [30][31][32][33][34] The constitutively active mutant FLT3 kinase induces autonomous proliferation to cytokine-dependent cell lines, such as Ba/F3 and 32D cells. Furthermore, when mutant FLT3-transfected hematopoietic stem cells are transplanted, the mice develop an oligoclonal myeloproliferative disorder (MPD), but not AML, suggesting that mutant FLT3 is sufficient to induce a MPD, and thus additional mutations that impair hematopoietic differentiation and/or proliferation might be necessary for the development of monoclonal AML. 35) FIRST-GENERATION FLT3 INHIBITORS Since FLT3 mutation is the most frequent genetic alteration and an independent poor prog- nostic factor in AML, mutant FLT3 serves as an important molecular target in the treatment of AML. At first, tyrosine kinase inhibitors (TKIs), which have a potency to inhibit the FLT3 kinase, were subjected to clinical trials ( Figure 2). Tandutinib (Millenium) is a derivative of quinazoline and inhibits the kinase activity of FLT3- ITD, but not FLT3-KDM. 36) Although this compound also has potency against PDGFRB and KIT, the inhibitory spectrum is relatively selective to FLT3. 37) In a Phase 1 study of 40 patients with relapsed or refractory AML, three patients had 40 to 50% reduction in the number of bone marrow (BM) blasts. In this study, no adverse effects were observed, while the peak plasma concentration of this compound did not reach to the biologically effective level. In a Phase 2 study of 25 patients with relapsed or refractory AML harboring FLT3-ITD, decrease of peripheral and BM leukemia cells was observed in seven of the 15 patients, while clinical response could not be evaluated in eight patients because of rapid disease progression or the toxicity, such as ptosis and QTc prolongation. 38) Therefore, further clinical development was discontinued. Although this compound has a high selectivity against FLT3, a lower inhibitory effect (IC50 is 220 nM) might cause clinically unimpressive ...