FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina
Authors: Alonso Cristina N. , Rubio Patricia L. , Medina Adriana , Eandi Eberle Silvia , Bernasconi Andrea , Cocce Mariela , Gallego Marta , Zubizarreta Pedro , Rossi Jorge Gabriel , Felice Maria Sara 
Published: October 2019

Abstract
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%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. 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 NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut 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-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut 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 NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg 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.

Disclosures
No relevant conflicts of interest to declare.