The Significance of FLT3-ITD Mutation and Nucleophosmin Mutation in Acute Leukemia

Over the past decade, rapid advances have been made in elucidating some of the key molecular lesions that lead to AML, and understanding these alterations is guiding the design of new therapeutic strategies. Mutations within the FLT3 and NPM1 genes, ranked within the most frequent recurrent known genetic markers in AML and show apparently opposite prognostic significance. Objectives: This prospective study was designed to detect the frequency of FLT3-ITD mutation and NPM1mutations including the detection of the most common type (type A NPM1 mutation) in adult and pediatric AML patients using molecular techniques (conventional polymerase chain reaction) , and to correlate the prevalence of these mutations with the clinical presentation of the patients and their response to induction therapy . Also to correlate immunohistochemical staining of mutant NPM1 protein with the molecular findings of the mutation. Patients , Materials and Methods: In the period from March 2010 to July 2011, 140 individuals were recruited from 4 different hospitals in Baghdad , 88 of them had acute leukemia [75 acute myeloid leukemia patients(55 adult ;20 child) , 13 acute lymphoblastic leukemia(5 adult; 8 children)] and 2 patients had lymphoprolifrative disorders .The remaining 50 individuals (34 healthy adult persons ; 16 children with reactive bone marrow) representing age and sex matched control group. Hematological parameters including WBC count, platelet count and hematocrit percent were measured by automated hematology analyzer for each individual enrolled in the study. The blood film and bone marrow aspirate were evaluated by 2 hematology specialists. The AML and ALL cases were classified according to the FAB classification. Molecular analysis was done on bone marrow or peripheral blood samples from patients and control group according to the availability. The frequency of FLT3-ITD mutation in DNA samples from AML, ALL ,LPD patients and control individuals was studied by applying conventional polymerase chain reaction using specific primers. Nucleophosmin mutations were screened in all patients and controls using SSCP-RT-PCR with specific primers, whereas type A NPM1 mutation was studied in all patients and control group by ASO-RT-PCR using specific primers. Immunohistochemical staining for mutant NPM protein was used to detect the mutant protein in bone marrow biopsies from adult AML using anti NPM polyclonal antibody. Results: FLT3-ITD mutations was found in 14.54% of adult AML patients and in 10% of pediatric AML. The FLT3-ITD mutation showed no significant correlation with age and gender in adult and pediatric AML (P>0.05) . The mean peripheral and bone marrow blast cell percent in adult and pediatric AML mutated patients was higher than in non mutated patients,(P >0.05). Moreover, the FLT3-ITD mutation in adult and pediatric AML was mostly detected in M3, (P=0.153; 0.580), respectively. Furthermore, higher failure rate of induction therapy 62.5% in adult AML with FLT3-ITD mutation compared to non mutated patients 59% , (P= 0. 619) and 50% of pediatric AML patients with FLT3-ITD mutation had failed to achieve complete hematological remission compared to 17% pediatric patients without the mutation, (P= 0.619). Also in this study the FLT3-ITD mutation was detected in 7.69% of ALL patients whereas, the two lymphoproliferative cases and control group showed absence of this mutation. Regarding NPM1 gene analysis, the NPM1 mutations were found in 46.88% of adult AML patients and 25% of pediatric AML patients, whereas the ALL cases and control group did not had the mutations. The NPM1mutations showed no significant correlation to age and gender in adult and pediatric AML patients, (P>0.05). The relation of the NPM1 mutations to the hematological parameters in adult and pediatric AML patients showed no significant difference between mutated and non mutated cases, (P> 0.05) and the NPM1mutations were detected mainly in FAB M1and M3 (33.33% for each) subtypes in adult AML , (P= 0.191) whereas in pediatric patients ,the NPM1 mutations were equally detected in M2, M5 and M3v(33.33% for each) , (P= 0.384).NPM1 mutations were detected more in denovo than in secondary adult and pediatric AML patients ; and more in newly diagnosed cases than in relapsed cases , (P >0.05). Furthermore, higher failure rate of induction therapy was found in adult AML patients without NPM1 mutations 55.55% as compared to NPM1 mutated patients 44.44% , (P= 0.453) and 66.66% of mutated pediatric cases had achieved complete hematological remission ,(P= 0.626).The combining status of FLT3-ITD and NPM1 mutations in adult AML patients showed that FLT3-ITD mutation was detected in 20% of all NPM1 mutated cases and the highest remission rate was found in the group that had NPM1 mutations but lacking FLT3-ITD whereas, the lowest remission rate was found in the group had FLT3-ITD with and without NPM1 mutations, (P= 0.180) showing the poor prognostic effect of FLT3-ITD mutation that overcome the good prognostic effect of NPM1 mutations. In pediatric AML patients, 33.33% of NPM1mutated pediatric patients had in addition FLT3-ITD mutation .That child with both mutations had highest peripheral blood and BMA blast cell percent compared to the rest of children. NPM1 type A mutation was found in 34.37% adult AML patients representing 73.33% of all adult patients with NPM1 mutations; whereas in pediatric patients, NPM1 type A was found in 8.33% cases representing 33.33% of all pediatric patients with NPM1 mutations.
By immunohistochemical staining of NPM mutant protein, the percentage of positive blast cells ranged from 30%-100%. Seventeen out of 30 (56.66%) of adult patients who had bone marrow biopsy were positive for the mutant protein and a significant relation was found between RT-PCR analysis and mmunohistochemical staining, (P=0.009).The sensitivity of Immunohistochemical staining in detection of mutated NPM1 cases was 90% whereas, the specificity of this technique was 87.5%. Conclusions: It is a noval study for molecular detection of FLT3-ITD mutation and NPM1 mutations in Iraqi acute leukemia patients where the frequency FLT3-ITD mutation in adult AML patients was comparable to the incidence in nearby countries and lower than the incidence reported worldwide, whereas , the frequency of FLT3-ITD mutation in pediatric AML patients was comparable to the incidence worldwide. On the other hand, the prevalence of NPM1 mutations in pediatric and adult AML patients was higher than the incidence reported in different studies worldwide.
Regarding ALL patients ,the frequency of FLT3-ITD mutation was higher than the incidence that found in many studies worldwide and no NPM1 mutations was detected in those patients. FLT3-ITD mutation and NPM1 mutations in adult and pediatric AML patients were detected in all FAB subtypes but mostly presented in M3 with no relation to the clinical presentation. FLT3-ITD mutation was associated with poor prognostic parameters, whereas NPM1 mutations were associated with good prognostic factors but FLT3-ITD mutation had a poor prognosis independent on the presence of NPM1 mutations, indicating the overwhelming poor effect of FLT3-ITD over the good effect of NPM1 mutation. FLT3-ITD was detected in 20% of adult AML patients with NPM1 mutations and in 33.33% of pediatric AML with NPM1 mutated. Most frequent type of NPM1 mutations in adult AML patients was type A whereas in pediatric AML patients third of the cases was type A . Immunohistochemical staining for the mutant NPM1 protein can be used as a screening test for the detection of NPM1 mutations.