re associated with unfavorable OS. Whereas the effect of a FLT3-TKD mutation was not significant with respect to RFS, the presence of FLT3-ITD in NPM1-mutated patients, significantly worsened RFS. Older age and a high initial WBC had an independent negative impact on PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632594 RFS. Independent prognostic Sunset Yellow FCF factors leading to a significantly prolonged RFS included mutations of NPM1 and biCEBPA. Performance status, de novo AML, platelet counts, hemoglobin level, LDH level, initial BM blasts and MLL-PTD did not significantly influence RFS. Analysis of FLT3-ITD mRNA level in a multivariate Cox regression model for OS and RFS revealed the same independent prognostic factors as analysis of FLT3-ITD in multivariate Cox regression models. The effect of a high FLT3-ITD mRNA level was restricted to NPM1-mutated patients Chemiluminescent Sandwich ELISA, Cell Signaling Technology was used according to the manufacturer’s instruction. Statistical Analysis of In Vitro Data Statistics were implemented with SigmaPlot 11.0 and two sided t-test as well as Mann-Whitney Rank Sum test were used. Heatmap was created using R Development Core Team. R: A language and environment for statistical computing. Three patients were positive for both types of mutation and one patient had an ITD and a point mutation in the JM region of FLT3. 10 of 113 patients with FLT3-WT at diagnosis acquired a FLT3 mutation during relapse. 7 of 113 acquired a FLT3ITD, 3 of 113 acquired a FLT3-TKD. 27 of 35 patients with a FLT3-ITD at initial diagnosis displayed a FLT3ITD at relapse. None of those eight patients that had lost the FLT3-ITD expressed a FLT3-TKD mutation at relapse. In contrast, FLT3-TKD mutations present at diagnosis were lost in the majority of patients at the time of relapse. 4 of 11 patients displayed the same FLT3-TKD mutation at diagnosis and relapse. One patient with a stable FLT3-TKD had acquired an additional FLT3-ITD at the time of relapse. In all three patients with simultaneous FLT3-ITD and FLT3-TKD mutation as well as FLT3-ITD and FLT3-V592 mutation at first diagnosis only the FLT3-ITD was present at relapse. Patients with FLT3-ITD at diagnosis and relapse showed significantly higher FLT3-ITD mRNA levels at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632393 the time of relapse. significantly with respect to their IL-3 independence. Defining the average growth of FLT3-ITD expressing cells as 100%, FLT3WT, -V592A and -I867S transfected cells displayed growth rates of 2.8%, 7.8% and 8.2%, respectively. FLT3-TKD mutants grew on a range between 14.5% and 42.5%. All FLT3 mutants conveyed protection from apoptosis to different degrees. FLT3V592A and -I867S achieved 59.7% and 58.9%, respectively. FLT3-TKD apoptosis rates ranged between 26.935.6% and FLT3-ITD cell lines showed apoptosis rates similar to IL-3 controls . We evaluated the effects of FLT3 ligand stimulation using Tyr591 phosphorylation as readout. In FLT3-WT, -I867S and V592A expressing cells a distinctive effect of FL on receptor phosphorylation was seen. FLT3-TKD mutants showed intermediate response, whereas no effect was seen in FLT3-ITD compared to FLT3-WT expressing cells . Taken together, all FLT3 mutations analyzed in this cell line model expressed a distinct gain-of-function phenotype. FLT3 Receptor Mutants Show Distinct Glycosylation and Cell Surface Expression Patterns To further analyze the differences in cell growth we performed a Western blot assay for the FLT3 protein. As reported before, FLT3 is detectable as two bands, representing the strong glycosy