Molecular Epidemiological Study in Pediatric Acute Myeloid Leukemia.

It is hypothesized that AML arises from two cooperative types of mutations: type I mutations mainly induce proliferation and type II mutations involved in the maturation arrest. AML is a rare disease in children and few molecular data are available on pediatric AML. We therefore studied N-RAS, K-RAS, FLT3-ITD, FLT3 , C-KIT mutations (type I), and CEBPA mutations (type II) as well as FLT3, EVI-1 and WT1 gene expression in 77 de novo AML.

Patients and methods: All the patients (aged 1 month-17 years, median age: 6.9 years, male/female ratio 1.26) were treated for de novo AML between 1995 and 2003 in two French institutions and prospectively enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: M0:6.5%, M1: 5.2%, M2: 22%, M3: 13%, M4 :14.3%, M5 :30%, M7: 6.5% and unclassified :2.5%. Cytogenetics features according to the MRC classification were favorable, intermediate or poor in 25% (t(8;21) n=5; t(15,17) n=8, inv(16) n=5), 65% (normal n=20 and 11q23 abnormalities n=15) and 10% (−7, n=4) respectively. With a median follow-up of 26 months (range 2–98 months), Complete Remission was obtained in 92% (71/77) of patients, OS was 71% and EFS 61%. CEBPA, N-RAS, K-RAS, C-KIT and FLT3 mutations detection was performed by direct sequencing. FLT3, EVI-1 and WT1 transcripts were quantified by RQ-PCR.

Results: (1) Frequency of N-RAS and K-RAS mutations were 11% (8/75) and 16% (12/75) respectively. RAS-mutated patients belonged to favorable (30%), intermediate (60%) and poor (10%) cytogenetic subgroups. In univariate analysis only N-RAS mutations is associated with adverse outcome (OS 37% vs 79%, p<0.05). (2) CEBPA mutations were found in 8% (6/75), mostly belonged to the intermediate cytogenetic risk subgroup (66%). (3) C-KIT mutations were observed in 4% (4/75) always associated with fusion CBFb/MYH11 transcript and excellent outcome. (4) FLT3-ITD and FLT3 Asp835 mutations were obtained in 12% (9/74) and 4% (3/74) of patients respectively. Cytogenetic subgroups were favorable (33%) and intermediate (67%). (5) At diagnosis FLT3 overexpression was detected in 34% (24/70) and 11q23 abnormalities were associated in 7/24 patients. (6) EVI-1 overexpression was found in 21% (16/76), belonged to intermediate (85%) and poor (15%) cytogenetic subgroups with a significant frequency in monosomy 7 (p<0.025). The EVI-1 expression was specifically expressed (p<0.001) in M5 and M7-FAB subtypes. (7) WT1 overexpression was detected in 81% (62/76).

Conclusion: In total, 48% of de novo AML in children had a mutation in N-RAS, K-RAS, FLT3-ITD, FLT3 Asp835, C-KIT or CEBPA with a high frequency of RAS mutations (27%) compared to adult AML and a significantly bad survival. Additional gene expression quantification of EVI-1, FLT3 and WT1 allows MDR detection in 95% of patients.