Structural and numerical variation of FLT3/ITD in pediatric AML

FLT3 internal tandem duplication (FLT3/ITD) is a common somatic mutation in acute myeloid leukemia (AML) with significant variation in the position, length, and number of duplications of the FLT3 gene. We evaluated these physical characteristics in FLT3/ITD-positive patients who were treated on CCG-2941/2961 and correlated them with clinical outcome. Fiftynine of 77 FLT3/ITD-positive patients (77%) had a single ITD, 16 (21%) had 2 ITDs, and 2 (3%) had 3 ITDs. The length of the duplicated region varied from 6 to 51 amino acids, and in all cases amino acid residues Y591–Y597 were duplicated. Structural analysis demonstrated that Y591–Y597 encodes the switch and zipper regions of the juxtamembrane domain of FLT3. In addition, 24 of 77 patients (31%) had duplication of the critical STAT5 docking sites Y589/591. Patients with longer ITDs had a worse relapse-free survival (19% vs 51%, P = .035), while the presence of more than 1 ITD was not clinically significant. Physical characteristics including the length of FLT3/ITD may influence FLT3 activation state by altering its structure and may impact response to therapy.      

KIT mutations, and not FLT3 internal tandem duplication, are strongly associated with a poor prognosis in pediatric acute myeloid leukemia with t(8;21): a study of the Japanese Childhood AML Cooperative Study Group

Patients with t(8;21) acute myeloid leukemia (AML) are considered to have a good prognosis; however, approximately 50% of them relapse. The genetic alterations associated with a poor outcome in t(8;21) AML remain unknown. Recently, aberrations of receptor tyrosine kinases (RTKs) were frequently found in patients with AML. However, the prevalence and prognostic impact of RTK aberrations in pediatric t(8;21) AML remains undetermined. Here, we found the kinase domain mutations of the KIT gene in 8 (17.4%) of 46 patients with t(8;21) AML among newly diagnosed pediatric patients with AML treated on the AML99 protocol in Japan. Significant differences between patients with or without KIT mutations were observed in the 4-year overall survival (50.0% versus 97.4%, P = .001), disease-free survival (37.5% versus 94.7%, P < .001) and relapse rate (47.0% versus 2.7%, P < .001). Furthermore, FLT3 internal tandem duplication was found in only 2 (4.3%) patients. These results suggested that KIT mutations are strongly associated with a poor prognosis in pediatric t(8;21) AML.     

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is associated with poor outcome in childhood AML regardless of FLT3‐ITD status: a report from the Children's Oncology Group

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is a rare subtype associated with FLT3‐internal tandem duplication (ITD) and poor outcomes. The clinical outcomes of paediatric patients with t(6;9) with and without FLT3‐ITD treated on six consecutive cooperative trails were evaluated. In contrast to patients without t(6;9), those with t(6;9) had a significantly lower complete remission rate, higher relapse rate (RR), and poor overall survival (OS). Within t(6;9) patients, those with and without FLT3‐ITD had an OS of 40% and 27% respectively (P > 0·9), demonstrating that t(6;9) is a high‐risk cytogenetic feature in paediatric AML and its clinical impact is independent of the presence of FLT3‐ITD.     

Internal tandem duplication of the FLT3 gene confers poor overall survival in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based chemotherapy: an International Consortium on Acute Promyelocytic Leukemia study

Activating internal tandem duplication (ITD) mutations in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor outcome in acute myeloid leukemia, but their prognostic impact in acute promyelocytic leukemia (APL) remains controversial. Here, we screened for FLT3-ITD mutations in 171 APL patients, treated with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy. We identified FLT3-ITD mutations in 35 patients (20 %). FLT3-ITD mutations were associated with higher white blood cell counts (P?P?=?0.0007), higher hemoglobin levels (P?=?0.0004), higher frequency of the microgranular morphology (M3v) subtype (P?=?0.03), and the short PML/RARA (BCR3) isoform (P?FLT3-ITD^positive patients had a lower 3-year overall survival rate (62 %) compared with FLT3-ITD^negative patients (82 %) (P?=?0.006). The prognostic impact of FLT3-ITD on survival was retained in multivariable analysis (hazard ratio: 2.39, 95 % confidence interval [CI] 1.17–4.89; P?=?0.017). Nevertheless, complete remission (P?=?0.07), disease-free survival (P?=?0.24), and the cumulative incidence of relapse (P?=?0.94) rates were not significantly different between groups. We can conclude that FLT3-ITD mutations are associated with several hematologic features in APL, in particular with high white blood cell counts. In addition, FLT3-ITD may independently predict a shorter survival in patients with APL treated with ATRA and anthracycline-based chemotherapy.     

Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse

Analysis of internal tandem duplications of FLT3 (FLT3/ITD) was performed on bone marrow samples obtained at diagnosis and relapse from 108 adult patients with de novo acute myeloid leukemia (AML) to determine the role of this mutation in leukemic relapse. Eighty-three patients had wild-type FLT3 at both diagnosis and relapse, 16 had FLT3/ITD at both stages, whereas 8 had acquired the mutation and 1 had lost it at relapse. Using Genescan analysis, we found that FLT3/ITD levels at first relapse were significantly higher than those at diagnosis (mean +/- SE, 40.5% +/- 4.8% versus 17.9% +/- 3.6%, P <.001). The increase in mutation levels at relapse as compared with diagnosis did not correlate with the difference in blast cell percentages at both stages (P =.777). A hemizygous deletion of wild-type FLT3 was found in 4 patients at relapse compared to none at diagnosis. Nine of the 11 patients carrying a single mutation at diagnosis relapsed with an identical mutation. All 6 patients with more than one FLT3/ITD mutation at diagnosis showed changes in mutation patterns and levels at first relapse; however, each patient retained at least one mutation in the relapse sample. The changes of mutation patterns had implications for the monitoring of minimal residual disease. Our results suggest that FLT3/ITD may contribute as the initial transforming event in AML, and relapse can reflect the selection and outgrowth of a mutant clone or evolution of a new clone harboring this mutation.     

Activating internal tandem duplication mutations of the fms-like tyrosine kinase-3 (FLT3-ITD) at complete response and relapse in patients with acute myeloid leukemia

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are among the most frequent molecular aberrations in patients with acute myeloid leukemia. We retrospectively analyzed 324 patients with acute myeloid leukemia treated with front-line induction chemotherapy between October 2004 and March 2010. Fifty-six patients had FLT3-ITD mutation at diagnosis. Fifty-one (91%) patients with FLT3-ITD achieved complete remission. Thirteen patients had FLT3 analysis at complete remission. None had FLT3-ITD. Twenty-five (49%) patients with FLT3-ITD relapsed. Of these, 13 (52%) had FLT3-ITD at relapse (3 negative and 9 not done). Among the 201 patients without FLT3-ITD at diagnosis who achieved complete remission, 77 (38%) relapsed among whom 8 (10%) patients acquired FLT3-ITD clone. We conclude that FLT3-ITD mutations are unstable at follow up and may occur for the first time at relapse. Therefore, FLT3-ITD is not a reliable marker for minimal residual disease in acute myeloid leukemia.     

Role of autologous hematopoietic stem cell transplantation according to the NPM1/FLT3‐ITD molecular status for cytogenetically normal AML patients: A GOELAMS study

The choice of postremission therapy for acute myeloid leukemia (AML) patients is now based on the blasts' cytogenetic and molecular profile. However, the potential benefit of autologous hematopoietic stem cell transplantation (auto‐HSCT) according to the NPM1/FLT3‐ITD status has been poorly studied in AML patients with a normal karyotype (NK). Therefore, we evaluated the NPM1/FLT3‐ITD molecular status in 135 NK‐AML patients treated by allogeneic HSCT (allo‐HSCT), auto‐HSCT, or chemotherapy as consolidation therapy within the GOELAMS LAM‐2001 trial. In univariate analyzes, 4‐year leukemia‐free survival (LFS) and overall survival (OS) were significantly higher for NPM1+/FLT3‐ITD? patients compared with patients presenting another molecular profile (61 vs. 43% and 72 vs. 48%, P = 0.02 and P = 0.01, respectively). In the NPM1+/FLT3‐ITD? subgroup, there was no benefit for allo‐HSCT or auto‐HSCT vs. chemotherapy (4‐year LFS: 71, 56, and 60%; 4‐year OS: 73, 71, and 60%, respectively; P = NS). For patients with other NPM1/FLT3‐ITD molecular profiles, allo‐HSCT was found to be the best consolidation therapy, whereas auto‐HSCT was associated with a better outcome when compared with chemotherapy (allo‐HSCT‐, auto‐HSCT‐, and chemotherapy‐related 4‐year LFS: 68, 44, and 36%, P = 0.004; 4‐year OS: 68, 52, and 29%, respectively, P = 0.02). Our study indicates that allo‐HSCT and auto‐HSCT provide similar outcomes compared with chemotherapy as consolidation for NPM1+/FLT3‐ITD? NK‐AML patients. For NK‐AML patients with an adverse molecular profile, auto‐HSCT could represent an alternative therapeutic approach when no human leukocyte antigen–matched allogeneic donor is available. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.     

FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB M5. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P =.01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P =.0046) and overall survival (32% vs 58%; P =.037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%; P =.037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P =.26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P =.01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the poor outcomes in FLT3/ITD-positive patients. We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITD-positive patients could not be attributed to increased in vitro cellular drug resistance.     

The evolution of targeted therapy in pediatric AML: gemtuzumab ozogamicin,FLT3/IDH/BCL2 inhibitors,and other therapies

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Mutation topography and risk stratification for de novo acute myeloid leukaemia with normal cytogenetics and no nucleophosmin 1 (NPM1) mutation or Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)

About 25% of patients with newly diagnosed acute myeloid leukaemia (AML) have normal cytogenetics and no nucleophosmin 1 (NPM1) mutation or Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD). The prognosis and best therapy for these patients is controversial. We evaluated 158 newly diagnosed adults with this genotype who achieved histological complete remission within two cycles of induction therapy and were assigned to two post-remission strategies with and without an allotransplant. Targeted regional sequencing at diagnosis was performed and data were used to estimate their prognosis, including relapse and survival. In multivariable analyses, having wild-type or mono-allelic mutated CCAAT/enhancer-binding protein alpha (CEBPA) [hazard ratio (HR) 2·39, 95% confidence interval (CI) 1·08–5·30; P = 0·032), mutated NRAS (HR 2·67, 95% CI 1·36–5·25; P = 0·004), mutated colony-stimulating factor 3 receptor (CSF3R) (HR 2·85, 95% CI 1·12–7·27; P = 0·028) and a positive measurable residual disease (MRD)-test after the second consolidation cycle (HR 2·88, 95% CI 1·32–6·30; P = 0·008) were independently correlated with higher cumulative incidence of relapse (CIR). These variables were also significantly associated with worse survival (HR 3·02, 95% CI 1·17–7·78, P = 0·022; HR 3·62, 95% CI 1·51–8·68, P = 0·004; HR 3·14, 95% CI 1·06–9·31, P = 0·039; HR 4·03, 95% CI 1·64–9·89, P = 0·002; respectively). Patients with ≥1 of these adverse-risk variables benefitted from a transplant, whereas the others did not. In conclusion, we identified variables associated with CIR and survival in patients with AML and normal cytogenetics without a NPM1 mutation or FLT3-ITD.     

Age-dependent frequencies of NPM1 mutations and FLT3-ITD in patients with normal karyotype AML (NK-AML)

Prognosis of AML in elderly patients is poor due to adverse patient characteristics and comorbidities. In addition, disease-associated parameters reveal differences between older and younger patients with AML. Survival in normal karyotype AML (NK-AML) is influenced by different clinical and molecular markers. The aim of this work was to investigate the frequencies of molecular markers in patients with NK-AML with a focus on NPM1 mutations and FLT3-ITD in different age groups. In the present study, we analyzed the frequencies of mutations of NPM1 and FLT3-ITD in a cohort of 1,321 adult patients and 148 children with AML treated within the AMLCG99, the AML98, and AML04 trials and their distribution in different age groups. Additionally, the frequencies of mutations in CEBPA genes, FLT3-TKD, and MLL-PTD were analyzed in the cohort with NK-AML (n?=?729). Our data show that the presence of mutations of NPM1 (from 60% to 40%) and FLT3-ITD (from 50% to 20%) significantly decreased with age in adult AML. Consequently, the proportion of NPM1?/FLT3-ITD? patients increased with age. The decreasing frequency of NPM1 mutations in elderly patients was paralleled by a reduced complete remission (CR) rate in the elderly of 55% compared to 80% in the younger patients. By contrast, the frequencies of other gene mutations, like FLT3-TKD and MLL-PTD, and mutations in CEBPA were not age-dependent. The decreasing frequency of the favorable NPM1 mutations with increasing age may partially explain the worse outcome in the elderly patients. Furthermore, the increasing amount of elderly patients without NPM1 mutations or FLT3-ITD suggests that other molecular and clinical risk factors may influence prognosis in this age group.     

Mild chronic graft‐versus‐host disease may alleviate poor prognosis associated with FLT3 internal tandem duplication for adult acute myeloid leukemia following allogeneic stem cell transplantation with myeloablative conditioning in first complete remission: a retrospective study

Internal tandem duplication (ITD) of the FLT3 gene (Fms‐like tyrosine kinase 3) is the most commonly found mutation in acute myeloid leukemia (AML). The significance of FLT3‐ITD at diagnosis was retrospectively estimated for allo‐HSCT (allogeneic hematopoietic stem cell transplantation) outcomes in 140 patients, median age of 38, undergoing allo‐HSCT after myeloablative conditioning in first complete remission of AML. FLT3‐ITD was detected at AML diagnosis in 42/140 (30%) of included into this study patients. At 3 years, relapse incidence (RI) following allo‐HSCT in AML patients with intermediate or normal karyotype was significantly higher in those FLT3‐ITD positive than FLT3‐ITD negative [52.9 vs. 20.4%, P = 0.002]. Additionally, patients with mild chronic graft‐versus‐host disease (cGvHD) had significantly lower RI compared to patients with moderate or severe grade cGvHD or those not experiencing cGvHD, respectively, 4.8 vs. 36.0 vs. 27.8%, P = 0.032. FLT3‐ITD was harboring a poor prognosis in AML with intermediate or normal karyotype and significantly increased risk of relapse following allo‐HSCT. It appears that allo‐HSCT does not cure patients with FLT3‐ITD, unless they develop symptoms of mild cGvHD and graft versus leukemia, which may decrease RI.