Study Of FLT3 Gene Mutations In Patients With Acute Promyelocytic Leukemia

【Objective】To investigate the clinical features and the efficient therapy of acute promyelocytic leukemia (APL) with high initial white blood cell(WBC) count. To evaluate the prevalence of Fms-Like tyrosine kinase 3 (FLT3) gene mutations including internal tandem duplication(ITD) of juxtamembrane region and point mutation in the second tyrosine kinase domain(TKD) in APL and its clinical significance. To develop an assay to identify both FLT3 and (Nucleophosmin) NPM1 gene mutations in a single multiplex polymerase chain reaction (PCR).【Methods】The clinical features of 66 newly diagnosed APL patients with high initial WBC and 152 patients without high initial WBC between October 1993 and August 2006 were retrospectively analyzed. The therapeutic effects were compared according to the different therapy in 66 patients with high initial WBC.Bone marrow mononuclear cells of 160 newly diagnosed APL patients between January 2000 and May 2007 were analyzed. PCR was used to detect FLT3-ITD mutations, FLT3-ITD-positive samples were further analyzed for the ITD allelic ratio (ITD-AR, mutant-wild type ratio). The FLT3-TKD mutation was analyzed by PCR amplification of exon 20 followed by EcoRV digestion and sequence analysis.Bone marrow mononuclear cells of 25 newly diagnosed acute leukemia (AL) patients between July 2004 and December 2007 were analyze for both FLT3 and NPM1 mutations by a PCR and capillary electrophoresis (CE) assay All samples were from the First Affiliated Hospital of Soochow University.【Results】 The early death rate and the incidence of disseminated intravascular coagulation(DIC) and retinoic acid syndrome(RAS) were respectively 30.3%,57.6% and 31.8% in the group with high initial WBC, which were higher than the indexes of 7.2%,38.1% and 21.05%, respectively, in the group without high initial WBC (P<0.05), whereas complete remission (CR) rate (63.6%) for the group with high initial WBC was lower than that (88.2%) in the group without high initial WBC (P<0.05). 61 of 66 patients with high WBC accepted induction therapy: 31 patients treated with all-trans retinoic acid(ATRA) alone, 21 patients treated with the combination of ATRA and arsenic trioxide (ATO), 9 treated with ATO alone, the early death rates were respectively 27.3%,14.3%,55.6%, CR rates were respectively 67.7%,81.0%,44.4%, the result showed that the group with combination therapy had the lowest rate of early death and the highest rate of CR. Of 61 patients, 41 cases were given low-dose chemotherapy, the CR rate was 80.5%, total death rate was 19.5%, that of 20 patients without chemotherapy were 45.0%,55.0%, respectively. The differences between two groups had statistical significance (P<0.05).Out of 160 patients, FLT3 aberrations were detected in 40 cases (ITD: 24, TKD: 14, ITD+TKD: 2 ). Both mutations were associated with higher presenting WBC counts and short type (S-type) PML-RARαtranscript. The FLT3-ITD was also associated with the M3b/M3m subtype. For FLT3-ITD-positive patients, the RAS and DIC were 41.7% and 65.4%, respectively, higher than that of wild-type FLT3 (FLT3-wt) patients, while the CR rate was lower (69.2% vs 90.3%,P < 0.05); whereas, for FLT3-TKD-positive patients, the incidence of RAS,DIC and CR rate were not significantly different from FLT3-wt patients (P > 0.05). FLT3-ITD-positive patients had a shorter overall survival (OS) compared with FLT3-wt patients (P < 0.05), but not disease-free survival (DFS) (P > 0.05); There were no significant difference on either OS or DFS between FLT3-TKD-positive and FLT3-wt patients (P > 0.05). The ITD-AR of 26 FLT3-ITD positive patients varied from 0.11 to 6.55, with a median of 1.0. Neither diagnostic characteristics nor clinical outcome were significantly different between the patients with ITD-AR above 1.0 and those under 1.0 (P > 0.05).Of 25 AL samples analyzed, 9 were FLT3-ITD positive, 5 were FLT3-TKD positive, 6 were NPM1 positive. Among these patients with FLT3 or NPM1 berrations, one case was both FLT3-ITD and FLT3-TKD mutations, three were both FLT3-ITD and NPM1 mutations. There were no patients with both FLT3-TKD and NPM1 mutations or with all of three mutations.【Conclusions】Compared with APL without high initial WBC, APL with high initial WBC has lower CR rate, higher early death rate, higher incidence of DIC and RAS. The combination of ATRA,ATO and chemotherapy is the most efficient therapeutic approach to APL patients with high initial WBC, which can significantly reduce the early death rate and improve the CR rate.FLT3 mutations (FLT3-ITD or FLT3-TKD) are frequently identified in patients with newly diagnosed APL, both mutations are associated with high presenting WBC and S-type PML-RARαtranscript. FLT3-ITD mutation is more frequent than FLT3-TKD mutation, and predicts a poorer prognosis, whereas FLT3-TKD mutation does not show the same unfavorable prognostic effect on APL patients.Multiplex PCR followed by capillary electrophoresis can be used for a fast and reliable diagnosis of both FLT3 and NPM1 mutations.