The FLT3-D324N Variant Is a Functionally Silent Polymorphism in the FLT3 Gene and May Be Associated with a Higher Risk for AML.

Abstract

Mutations within the FLT3-gene are of growing importance for classification, risk assessment and therapeutic targeting in acute myeloid leukemia (AML). An increasing number of activating mutations have been reported during the last few years. A D324N variant located in the extracellular region of the FLT3 protein has been described recently in 4/94 (4.3%) of AML patients (Ley TJ et al., PNAS 2003). We have analyzed 705 de novo AML for D324N using a LightCycler based screening assay and found a gac to aac change in codon 324 in 43 cases (6.1%). This is approximately the same frequency that has been described for tyrosine kinase domain mutations in FLT3. However, in contrast to other FLT3 mutations the D324N was associated with a low leucocyte count (6.700/μl) and had no association to any AML subtype nor a prognostic impact regarding overall survival and event free survival (235 D324N- cases vs. 13 D324+ cases with normal karyotype analyzed). To analyze whether this FLT3 variant might be a polymorphism we analyzed peripheral blood of 329 healthy donors with a similar ethnic background. In this population we could also detect the D324N variant, but only in 4 cases (1.2%). This difference between AML and healthy donors was statistically significant (p=.0001). Three of the cases were heterozygous and one was homozygous for the D324N variant. Of one of the heterozygous cases a buccal smear was evaluated and the same heterozygous pattern could be detected in this material. In addition, of three D324N positive AML at diagnosis a sample from any time point in CCR was available that was negative for the leukemia clone with a sensitivity of 10−4 to 10−6 as assessed by quantitative PML-RARA- (1 case) or CBFB-MYH11- (1 case) specific PCR or by immunophenotyping (1 case). In these remission samples again a 50% ratio of the normal and the D324N variant was detectable. To functionally characterize the FLT3-D324N in vitro, FLT3-WT, FLT3-D324N, and FLT3-ITD cDNA were retrovirally transduced into IL-3 dependent Ba/F3 cells. Stably expressing cell lines were grown for 72h in the absence of IL-3 with varying doses of human FLT3 ligand (FL) and the number of viable cells was assessed by trypan blue exclusion. In contrast to FLT3-ITD expressing cells, FLT3-D324N transduced cells were not able to grow in the absence of IL-3. The growth of FLT3-WT and FLT3-D324N, but not vector expressing cell lines could be stimulated by exogenous FL in a dose-dependent manner. No significant difference could be demonstrated between FLT3-WT and FLT3-D324N cells. In apoptosis assays using annexin-V-PE and 7-AAD staining FL stimulation protected both D324N mutant and FLT3-WT expressing Ba/F3 cells from apoptotic cell death to a similar degree. These results strongly support the hypothesis that the D324N variant in the FLT3 gene represents a functionally silent polymorphism. The fivefold higher frequency in patients with AML compared to healthy donors raises the question whether this FLT3 variant is associated with a higher risk for AML.