FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina.

Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting.

Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis.

Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1^mut: 4.6% and CEBPA^mut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1^mut: 25.0% and CEBPA^mut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1^mut: 12 years and CEBPA^mut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPA^mut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1^mut and 4/4 patients with CEBPA^mut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITD^mut: M3 (n:10/20; p<0.00001), FLT3-TKD^mut: M5 (n:8/16; p=n.s.), NPM1^mut: M2 (n:4/9; p=0.062) and CEBPA^mut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITD^mut, NPM1^mut and CEBPA^mut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITD^mut:68 (12)%, FLT3-TKD^mut:46 (17)%, NPM1^mut: 75 (15)%, CEBPA^mut: 100 (0)% and NPM1^mut/CEBPA^mut/FLT3-ITD^neg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITD^neg and NPM1^mut or CEBPA^mut was 88 (12)% (p=0.066).

Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1^mut and CEBPA^mut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1^mut/FLT3-ITD^neg and CEBPA^mut/FLT3-ITD^neg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity.