Size of FLT3 internal tandem duplication has prognostic significance in patients with acute myeloid leukemia

FLT3 internal tandem duplications (FLT3/ITDs) in the juxtamembrane domain are found in approximately 25% of acute myeloid leukemia (AML) patients, ranging in size from 3 to hundreds of nucleotides. We examined whether the sizes of FLT3/ITDs were associated with clinical outcomes in 151 AML patients enrolled in Southwest Oncology Group studies: S9333 and S9500. FLT3/ITDs were identified in 32% of patients (median ITD size = 39 nucleotides; range, 15-153 nucleotides). The CR rates were 35%, 67%, and 52% for patients with large (>or= 40), small (< 40), and no ITDs, respectively (P = .19). Increasing ITD size was associated with decreasing OS (estimated 5-year OS: large = 13%, small = 26%, and no ITD = 21%, P = .072) and RFS (estimated 5-year RFS: large = 13%, small = 27%, and no ITD = 34%, P = .017). These studies suggest that ITD size may have prognostic significance.     

A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 have been found in 20% to 30% of patients with acute myeloid leukemia (AML). These mutations constitutively activate the receptor and appear to be associated with a poor prognosis. Recent evidence that this constitutive activation is leukemogenic renders this receptor a potential target for specific therapy. In this study, dose-response cytotoxic assays were performed with AG1295, a tyrosine kinase inhibitor active against FLT3, on primary blasts from patients with AML. For each patient sample, the degree of cytotoxicity induced by AG1295 was compared to the response to cytosine arabinoside (Ara C) and correlated with the presence or absence of a FLT3/ITD mutation. AG1295 was specifically cytotoxic to AML blasts harboring FLT3/ITD mutations. The results suggest that these mutations contribute to the leukemic process and that the FLT3 receptor represents a therapeutic target in AML. (Blood. 2001;98:885-887)     

Incidence and prognostic value of FLT3 internal tandem duplication and D835 mutations in acute myeloid leukemia

BACKGROUND AND OBJECTIVES: Cytogenetics is the most important prognostic factor in acute myeloid leukemia (AML). However, a high proportion of patients show normal or intermediate-risk karyotypes. In these patients, other determinants could help to identify those with a higher risk of relapse. Recently, internal tandem duplications (ITD) and D835 mutations in FLT3 tyrosine kinase receptor have been shown to confer a bad prognosis in AML. DESIGN AND METHODS: We analyzed the incidence of these mutations in a total of 208 patients of different AML subsets and their prognostic relevance in non-promyelocytic de novo AML. RESULTS: FLT3 mutations were detected in 24% of de novo AML, 42% of acute promyelocytic leukemia (APL) and 17% of secondary AML. Four patients showed both ITD and D835 mutations. Ninety-four per cent of the patients with FLT3 alterations were classified into the intermediate-risk group. There was no association between the presence of FLT3 alterations and response to induction while the alterations were associated with a worse disease-free survival and event-free survival in both the overall and intermediate-risk patients. INTERPRETATION AND CONCLUSIONS: Our data confirm that any of the mutations in FLT3 confer a bad prognosis in AML. Because of the high prevalence of these mutations within the intermediate-risk group, their detection could be useful to identify patients with a poor prognosis.     

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.     

FLT3 internal tandem duplication in childhood acute myeloid leukaemia: association with hyperleucocytosis in acute promyelocytic leukaemia

We evaluated the incidence of FLT3/internal tandem duplication (ITD) mutation in childhood acute myeloid leukaemia (AML) diagnosed over 15 years. FLT3/ITD was found in 10 of 45 (22.2%) non-acute promyelocytic leukaemia (non-APL) patients. The 5-year event-free survival of non-APL patients was higher in FLT3/ITD-negative versus -positive patients (48.9%, SE 8.9, vs 20.0%, SE 16.1, P = 0.03). In childhood APL, FLT3/ITD incidence was higher than in non-APL, although not statistically significant (10 out of 29 patients, 34.5%, P = 0.29). In APL patients, FLT3/ITD was strongly correlated to a higher white blood cell count at diagnosis and the M3 French-American-British subtype.     

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.     

Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia

The Flt3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the Flt3 gene (Flt3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with poor prognosis. We analyzed diagnostic bone marrow specimens from 91 pediatric patients with AML treated on Children's Cancer Group (CCG)-2891 for the presence of the Flt3/ITD and correlated its presence with clinical outcome. Fifteen of 91 samples (16.5%) were positive for the Flt3/ITD. Flt3/ITD-positive patients had a median diagnostic white count of 73 800 compared with 28 400 for the Flt3/ITD-negative patients (P =.05). The size of the duplication ranged from 21 to 174 base pairs (bp). Nucleotide sequencing of the abnormal polymerase chain reaction products demonstrated that all duplications involved exon 11 of the Flt3 gene and also preserved the reading frame. Lineage restriction analysis revealed that Flt3/ITD was not present in the lymphocytes, suggesting a lack of stem cell involvement for this mutation. None of the Flt3/ITD-positive patients had unfavorable cytogenetic markers, and there was no predominance of a particular FAB class. The remission induction rate was 40% in Flt3/ITD-positive patients compared with 74% in Flt3/ITD-negative ones (P =.005). The Kaplan-Meier estimates of event-free survival at 8 years for patients with and without Flt3/ITD were 7% and 44%, respectively (P =.002). Multivariate analysis demonstrated that presence of the Flt3/ITD was the single most significant, independent prognostic factor for poor outcome (P =.009) in pediatric AML.     

FLT3 inhibitors in acute myeloid leukemia

The fms-like tyrosine kinase 3 (FLT3) plays an important role in both normal and malignant hematopoiesis. Activating mutations in the FLT3 receptor can be detected in approximately 30% of acute myeloid leukemias (AMLs) and are associated with a distinctly poor clinical outcome for patients. There are now several classes of FLT3 inhibitors in development with varying degrees of potency and selectivity for the target, including several in late-phase clinical trials in combination with chemotherapy. Major clinical responses in AML patients receiving single-agent FLT3 inhibitors have been rare, although transient peripheral blood blast reduction is common. Given such biological suggestion and preclinical activity, FLT3 inhibitors hold promise in improving the outcome of patients with mutant FLT3 AML. This review summarizes the current attempts to target this molecule, with emphasis on the validity of the target, the results of the clinical trials evaluating the FLT3 inhibitors in AML, the optimal use of these compounds and the mechanisms of resistance.     

Myeloid precursors and acute myeloid leukemia cells express multiple CD33-related Siglecs

OBJECTIVES: CD33 is a cell surface marker of committed myelomonocytic precursors and circulating monocytes, and is also found on acute myeloid leukemia (AML) cells. CD33 belongs to a family of sialic acid-binding cell surface proteins named Siglecs, among which there are 7 other functional CD33-related Siglecs (CD33rSiglecs). We sought to characterize the spectrum of expression of the other CD33rSiglecs on bone marrow precursors and AML cells and asked if they can potentially serve as targets for therapy. METHODS: Cell surface CD33rSiglecs were analyzed by flow cytometry. The ability of certain anti-Siglec antibodies to target toxin-mediated cell killing of Siglec-expressing cell lines was characterized and compared. RESULTS: We demonstrate that Siglecs-3, -5, -6, -7, and -9 are expressed on subsets of normal bone marrow precursors, including promonocytes and myelocytes. Furthermore, most AML (but not ALL) cells express these Siglecs. There is substantial variability in Siglec type and expression level between cases, with each having a unique "CD33rSiglec fingerprint." Individual anti-Siglec antibodies along with a saporin toxin-conjugated secondary antibody can target myelomonocytic leukemia cells for death, and targeting of multiple Siglecs improves cell killing. Cytotoxicity was further enhanced by sialidase treatment of target cells, which improves antibody binding. We also confirmed that antibody binding induced rapid internalization of Siglecs from the cell surface, which is a requirement for cell killing via saporin. CONCLUSIONS: Multiple CD33rSiglecs are expressed on normal and malignant myelomonoyctic cells. Targeting these Siglecs, possibly in combinations, could improve anti-CD33 antibody therapy or be used as an alternative to anti-CD33.     

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.     

Clinical impact of internal tandem duplications and activating point mutations in FLT3 in acute myeloid leukemia in elderly patients

BACKGROUND: The FLT3 gene is frequently mutated in acute myeloid leukemia (AML), either by an internal tandem duplication (ITD) of the juxtamembrane domain or by activating point mutations in the second tyrosine kinase domain (ATKD). Only a few investigations have focused on the prognostic significance of FLT3 alterations in AML among the elderly, yielding conflicting results. In the present study, the frequency and clinical relevance of FLT3 abnormalities were ascertained in a cohort of elderly AML patients. PATIENTS AND METHODS: A total of 109 AMLs, occurring in patients above the age of 60 yr (median 71.5), were investigated. DNA was extracted from fresh bone marrow cells or from cells in fixative and investigated for the presence of ITD of exons 14 and 15 and the ATKD D835 in exon 20. RESULTS: ITDs and ATKDs were identified in 20 (18%) and 11 (10%) of the cases, respectively. Three cases displayed both an ITD and an ATKD. FLT3 abnormalities were associated with leukocytosis (ITD P < 0.01; ATKD P = 0.069), and the monocytic FAB subtypes M4 and M5 [ITD (P < 0.05), ATKD (P = 0.05)], and ITD and ATKD were significantly (P < 0.05) more common in cases with a normal karyotype. There was no correlation between the presence of FLT3 abnormalities and complete remission rates or overall survival. CONCLUSION: A correlation was observed between FLT3 abnormalities and leukocytosis, a normal karyotype, and the M4/M5 subtypes of leukemia. However, no clear-cut prognostic impact of FLT3 abnormalities was identified in elderly AML patients.