Activating FLT3 Mutations Research Articles

FLT3 Mutations in Normal Karyotype Acute Myeloid Leukemia in First Complete Remission Treated with Autologous Peripheral Blood Stem Cell Transplantation.

Two types of activating FLT3 mutations have been described in AML. FLT3/ITDs can be detected in 20% to 30% of patients with AML. Point mutations of FLT3/D835 have been described in 7% of adult AML patients. Activating FLT3 mutations has been associated with the leukocytosis and poor prognosis. We retrospectively analyzed the significance of FLT3 mutations in AML patients of normal karyotype treated with autologous peripheral blood stem cell transplantation (auto-PBSCT).METHOD: We evaluated 34 consecutive patients with first CR AML of normal karyotype who received myeloablative therapy and auto-PBSCT to analyze clinical features and outcomes between patients with and without FLT3/ITDs and FLT3/D835. There were 16 males and 18 females with a median age of 41.5 years ranging 15–74 years. Cytogenetic G-banding analysis was performed with standard method. The distribution of morphologic types of AML according to the FAB classification was as follows; one patient was M0, twelve M1, eight M2, seven M4 and six M5. The pre-transplant conditioning regimen was G-CSF combined with the high-dose chemotherapy consisting of busulfan (16 mg/kg), etoposide (40 mg/kg) and Ara-C (3 g/m2x4) (BEA regimen). DNA PCR assay was used to detect FLT3/ITDs. All abnormal longer products by agarose gel electrophoresis were subsequently sequenced. DNA PCR assay followed by direct sequence were used to detect FLT3/D835.RESULT: FLT3/ITDs were detected in 8 of 34 patients (23.5 %). FLT3 D835 mutation was detected in 2 of 34 patients (5.9%). To define clinical differences between patients with and without FLT3/ITDs, clinical variables at the diagnosis were compared. WBC (p=0.013), LDH (p=0.0147), the percentage of PB and BM blasts (p=0.0422 and P=0.0021) were significantly higher in the FLT3/ITDs patients. Other clinical parameters such as age, sex, FAB classification, Hb, Plt, remission induction therapy, interval from diagnosis to auto-PBSCT, number of infused CD34+ cells and hematological recovery after auto-PBSCT are not associated with the presence or absence of FLT3/ITDs. We analyzed the clinical significance of FLT3/ITDs mutations. OS and DFS was similar in patients with or without FLT3/ITDs (5 years OS, 71.4% vs 78.9%, p=0.5746; 5 years DFS, 72.9% vs 68.6%, p=0.9273 by the log-rank test).DISCUSSION: As far as we know, this is the first report to describe the significance of FLT3 mutations in AML patients of normal karyotype treated with auto-PBSCT. We show that FLT3 mutations have no prognostic impact in autotransplanted AML 1CR patients of normal karyotype. Our data suggest dose escalation of chemotherapy may conquer the poor prognostic implications of FLT3 mutations. The prognostic significance of activating FLT3 mutations in AML patients with normal karyotype should be evaluated in relation with post-remission therapeutic modalities.

Activating FLT3 mutations are rare in children with juvenile myelomonocytic leukemia.

Activating mutations of FLT3 have been identified in multiple myeloid malignancies. Two types of activating mutations have been described: (1) the internal tandem duplication (FLT3-ITD) and (2) point mutations within the activating loop (FLT3-ALM). Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder of early childhood. Mutations and other genetic abnormalities of RAS, NF1, and PTPN11 have been implicated as causative events in JMML, but approximately 25% of JMML patients harbor none of these abnormalities. We investigated whether FLT3 mutations might also contribute to JMML pathogenesis, and if present, whether FLT3 status would correlate with disease natural history and prognosis. Genomic DNA was isolated from peripheral blood and bone marrow samples of 60 patients meeting international JMML diagnostic criteria. Samples were analyzed for FLT3-ITD and FLT3-ALM using polymerase chain reaction and restriction endonuclease digestion. FLT3-ALM was found in 1/60 (1.7%) patients analyzed. Direct sequencing confirmed a C836G mutation. Clinical and laboratory characteristics of the JMML patient with the FLT3-ALM did not differ from the remainder of the cohort. No FLT3-ITD mutations were detected. This first reported mutational analysis for both FLT3-ITD and FLT3-ALM performed in JMML documents the presence of FLT3 mutations within JMML, but at a sufficiently low prevalence as to be clinically insignificant for most patients. Despite the poor prognosis and limited therapeutic options for JMML patients with refractory disease, compassionate therapy with targeted FLT3 inhibitors should not be considered in this patient population until adequate safety and efficacy data become available.

Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia

AbstractActivating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with de novo acute myeloid leukemia (AML) and are associated with lower cure rates from standard chemotherapy-based treatment. Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is cytotoxic to cell lines and primary AML cells harboring FLT3 mutations. Successful FLT3 inhibition can also improve survival in mouse models of FLT3-activated leukemia. CEP-701 is an orally available, novel, receptor tyrosine kinase inhibitor that selectively inhibits FLT3 autophosphorylation. We undertook a phase 1/2 trial to determine the in vivo hematologic effects of single-agent CEP-701 as salvage treatment for patients with refractory, relapsed, or poor-risk AML expressing FLT3-activating mutations. Fourteen heavily pretreated AML patients were treated with CEP-701 at an initial dose of 60 mg orally twice daily. CEP-701–related toxicities were minimal. Five patients had clinical evidence of biologic activity and measurable clinical response, including significant reductions in bone marrow and peripheral blood blasts. Laboratory data confirmed that clinical responses correlated with sustained FLT3 inhibition to CEP-701. Our results show that FLT3 inhibition is associated with clinical activity in AML patients harboring FLT3-activating mutations and indicate that CEP-701 holds promise as a novel, molecularly targeted therapy for this disease.

CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations.

To assess the prognostic relevance of mutations in the CEBPA gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha) in a large prospective series of younger adults with acute myeloid leukemia (AML) and normal cytogenetics. The entire CEBPA coding region was sequenced in diagnostic samples from 236 AML patients 16 to 60 years of age with normal cytogenetics who were uniformly treated on two consecutive protocols of the AML Study Group Ulm, and CEBPA mutation status was correlated with clinical outcome. CEBPA mutations were detected in 36 (15%) of 236 patients. Twenty-one (9%) of 236 patients had mutations predicted to result in loss of C/EBP alpha function. Remission duration and overall survival (OS) were significantly longer for the 36 patients with CEBPA mutations (P =.01 and P =.05, respectively). On multivariate analysis, wild-type CEBPA was an independent prognostic marker affecting remission duration (hazard ratio, 2.85; P =.01) and OS (hazard ratio, 1.87; P =.04). Analysis of cooperating mutations (both types of activating FLT3 mutations and MLL partial tandem duplications) showed that FLT3 mutations had no significant prognostic influence in patients with CEBPA mutations. Furthermore, there was no significant overlap between the subgroup of patients with CEBPA mutation with predicted loss of C/EBP alpha function and patients with FLT3 or MLL mutations, suggesting that CEBPA loss-of-function mutations define a distinct biologic subclass of AML with normal cytogenetics. Mutant CEBPA predicts favorable prognosis and may improve risk stratification in AML patients with normal cytogenetics.

FLT3-activating mutations are associated with poor prognostic features in AML at diagnosis but they are not an independent prognostic factor.

FLT3: gene alterations (internal tandem duplications - ITDs - and D835 mutations) are thought to be associated with poor-risk acute myeloid leukemia (AML). However, not all studies confirm this association, so it is still a matter of debate. Moreover, their association with other molecular abnormalities is less studied. We have investigated the presence of FLT3-ITD and D835 mutations in AML patients and their correlation with clinical and biological disease characteristics. The presence of ITD was analyzed in diagnostic samples of 176 AML patients and the D835 mutation in 135 of these patients. In all these patients, the presence of four well-known molecular abnormalities were also simultaneously characterized: PML/RARalpha, AML1/ETO, CBFbeta/MYH11 and MLL rearrangements. In all, 41 (23%) patients harbored FLT3 mutations, with 34 (19.3%) of them positive for the ITD, and seven (5%) positive for the D835 mutation. Of the acute promyelocytic leukemia (APL) patients, 16 (27%) showed FLT3 mutations, more frequently in M3 hypogranular cases (62% versus 17%, P=0.001) and cases with the short (bcr3) PML-RARalpha isoform (69%, P=0.002). In contrast, FLT3 was never altered in patients with inv(16), t(8;21) or 11q23 abnormalities. FLT3 mutations were significantly associated with some negative prognostic features at diagnosis (leukocytosis, high blast-cell percentage, and elevated LDH values), but they were not associated with different disease-free or overall survival. Therefore, we confirm a high frequency of FLT3 mutations in APL and in adult AML without recurrent cytogenetic translocations. In addition, they were not found as independent prognostic factors although associated with several adverse features at diagnosis.

Rapamycin inhibits IL-4—induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo

AbstractRapamycin (RAPA) is a potent immunosuppressive macrolide hitherto believed to mediate its action primarily via suppression of lymphocyte responses to interleukin 2 (IL-2) and other growth factors. We show here that this view is incomplete and provide evidence that RAPA suppresses the functional activation of dendritic cells (DCs) both in vitro and in vivo. In vitro, RAPA inhibits IL-4—dependent maturation and T-cell stimulatory activity of murine bone marrow—derived DCs. These effects are associated with posttranscriptional down-regulation of both subunits of the IL-4 receptor complex (CD124, CD132) and are mediated via binding of RAPA to its intracellular receptor FK506-binding protein 12 (FKBP12). In vivo, RAPA impairs steady-state DC generation and fms-like tyrosine 3 kinase ligand (Flt3L)—induced DC mobilization. In addition, in vivo administration of RAPA impairs DC costimulatory molecule up-regulation, production of proinflammatory cytokines, and T-cell allostimulatory capacity. These novel findings have implications for RAPA-based therapy of chronic DC-triggered autoimmune diseases, transplant rejection, and hematologic malignancies with activating Flt3 mutations.

Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm

To assess the prognostic relevance of activating mutations of the FLT3 gene in homogeneously treated adults 16 to 60 years of age with acute myeloid leukemia (AML) and normal cytogenetics, pretreatment samples from 224 patients entered into 2 consecutive multicenter treatment trials were analyzed for FLT3 internal tandem duplications (ITDs) and Asp835 mutations. Treatment included intensive double-induction therapy and postremission therapy with high cumulative doses of high-dose cytarabine. ITDs were detected in 32% of the patients and were related to de novo AML and to high white blood cell (WBC) counts, percentages of peripheral blood (PB) and bone marrow (BM) blasts, and serum lactate dehydrogenase levels. Asp835 mutations were present in 14% of the patients and were associated with WBC counts and percentages of PB and BM blasts that were higher than those of patients without FLT3 mutations. With a median follow-up of 34 months, remission duration and overall survival (OS) were significantly shorter for patients with Asp835 mutations or an ITD than for those without FLT3 mutations (P =.03 and P =.0004, respectively). These results were attributable mainly to the negative prognostic effect of FLT3 ITDs. On multivariate analysis, mutant FLT3 was an independent marker affecting remission duration and OS (hazard ratio, 2.35 and 2.11, respectively). Fluorescence in situ hybridization did not detect monoallelic FLT3 deletions in ITD-positive patients. FLT3 mutations identify a subset of young AML patients with normal cytogenetics who do not benefit from intensive chemotherapy, including double-induction and postremission therapy with high-dose cytarabine.

Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis: Presented in part at the 42nd Annual Meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA (abstract 2334).

Constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (TKD), has been described in patients with acute myelogenous leukemia (AML). We analyzed the prevalence and the potential prognostic impact of FLT3 mutations in 979 AML patients. Results were correlated with cytogenetic data and the clinical response. FLT3-ITD mutations were found in 20.4% and FLT3-TKD mutations in 7.7% of the patients. Each mutation was associated with similar clinical characteristics and was more prevalent in patients with normal karyotype. Significantly more FLT3 aberrations were found in patients with FAB M5, and fewer were found in patients with FAB M2 and M6. Although less frequent in patients with cytogenetic aberrations, FLT3-ITDs were found in 13 of 42 patients with t(15;17) and in 9 of 10 patients with t(6;9). The prevalence of the ITD allele on the DNA level was heterogeneous, ranging from faint mutant bands in some patients to predominant mutant bands in others. Based on quantitative analysis, the mutant-wild-type (wt) ratio ranged from 0.03 to 32.56 (median, 0.78). Patients with a high mutant/wt ratio (ie, greater than 0.78) had significantly shorter overall and disease-free survival, whereas survival in patients with ratios below 0.78 did not differ from those without FLT3 aberrations. Multivariate analysis confirmed a high mutant/wt ratio to be a strong independent prognostic factor. Taken together, these data confirm that FLT mutations represent a common alteration in adult AML. Constitutive activation may be associated with monocytoid differentiation. A high mutant/wt ratio in ITD-positive patients appears to have a major impact on the prognostic relevance.