FLT3 Gene Mutations Research Articles

Role of mutation independent constitutive activation of FLT3 in juvenile myelomonocytic leukemia

FLT3 gene mutations have been identified as prognostic factors in myeloid malignancies. Furthermore, FLT3 can be activated by wild type overexpression or ligand-dependent in leukemic cells co-expressing FLT3 ligand (FLT3L). So far no data are available on FLT3/FLT3L expression and activation in JMML. In 51 clinical JMML samples, activating mutations were screened, FLT3 and FLT3L mRNA levels were assessed and the sensitivity of JMML cells to the FLT3 inhibitor PKC412 was tested by MTT assays. No evidence for constitutively activation of FLT3/FLT3L was found in JMML, indicating that FLT3 inhibitors are unlikely to be effective in JMML.

Overexpression of CXCR4 predicts adverse overall and event‐free survival in patients with unmutated FLT3 acute myeloid leukemia with normal karyotype

CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. The prognostic significance of CXCR4 expression in patients with AML that have a normal karyotype and no evidence of FLT3 gene mutations was examined. The prognostic significance of CXCR4 expression in 122 AML patients with normal karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003 was analyzed. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy or clot specimens obtained before treatment were immunostained for CXCR4. There were 70 men and 52 women with a median age of 62 years (range, 22-82 years). Median follow-up was 18 months (range, <1-97 months). Seventy-six patients achieved complete remission (CR); 39 had recurrence. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (P = .09). Multivariate analysis demonstrated that CXCR4 expression, presence of multilineage dysplasia, and high creatinine level predicted poorer overall (OS) and event-free (EFS) survival. CONCLUSIONS.: The results suggest that CXCR4 expression is associated with poor prognosis in AML patients with an unmutated FLT3 gene.

Overexpression of CXCR4 in Acute Myeloid Leukemia Predicts Adverse Overall and Progression-Free Survival Independently of FLT3 Gene Mutation.

CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. We analyzed the prognostic significance of CXCR4 expression in 122 AML patients with diploid karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy specimens obtained at diagnosis from untreated patients were immunostained with an antibody specific for CXCR4 (Abcam, clone 2074; Cambridge, MA). Statistical analysis included the following variables: CXCR4 expression, age, sex, race, incorporation of cytarabine in the therapeutic regimen, presence of multilineage dysplasia, history of antecedent hematologic disorder, history of prior chemotherapy (for either hematological or non-hematological disorder), performance status, leukocytosis, hemoglobin, platelet count, bilirubin, and creatinine. There were 70 men and 52 women with a median age of 62 years (range, 22–82 years). Median follow-up was 18 months (range, <1–97 months). Seventy-six patients achieved complete remission (CR); 39 relapsed. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (p=0.09). In the univariate Cox model, CXCR4 expression was associated with shorter overall survival (OS), p=0.008, and disease-free survival (DFS), p=0.012, figure1. [Display omitted] Shorter OS was also associated with failure to achieve CR (p=<0.0001), age (p=0.01), presence of multilineage dysplasia (p=0.006), poorer performance status (p=0.049), higher creatinine level (p=0.00002), and lower hemoglobin level (p=0.042). Shorter DFS was associated with failure to achieve CR (p=0.0002), presence of multilineage dysplasia (p=0.02), history of prior chemotherapy (p=0.04), and higher creatinine level (p=0.0009).In multivariate analysis, shorter OS was associated with CXCR4 expression (p=0.047), age (p=0.029), presence of multilineage dysplasia (p=0.042), and high creatinine level (p=0.016). Shorter DFS was associated with CXCR4 expression (p=0.036), history of prior chemotherapy (p=0.044), presence of multilineage dysplasia (p=0.007), and high creatinine level (p=0.034). These results suggest that CXCR4 expression is associated with poor prognosis in AML patients and is independent of FLT3 gene mutation.

Gene Mutations as Predicitive Markers for Postremission Therapy in Younger Adults with Normal Karyotype AML.

Background: Mutations in the genes encoding NPM1, FLT3 (FLT3 ITD, FLT3 TKD), CEBPA, MLL (PTD) and NRAS have been identified as molecular markers in acute myeloid leukemia (AML) exhibiting a normal karyotype. Most recent studies focused on the prognostic value of single markers not taking into account their potential interactions. In addition, little is known about the predictive value of these markers on postremission therapy.Aims: To evaluate the prognostic impact of NPM1, FLT3, CEBPA, MLL and NRAS gene mutations on response to induction therapy, relapse-free (RFS) and overall survival (OS) as well as the predictive impact of these mutations on RFS and OS following different postremission therapies.Methods: Patients [16 to 60 years of age] were entered on four AMLSG treatment trials [AML-2/95, AML-1/99, AML HD93, AML HD98A]. As a consistent feature, in all four trials a genetic randomization was performed assigning all patients with an HLA-matched family donor (MRD) to allogeneic stem cell transplantation (SCT) in first complete remission (CR). Leukemia cells were analyzed for mutations in the above genes as previously described.Results: Between 1993 and 2004, 872 patients exhibiting a normal karyotype were registered. Median age was 48 years; median follow-up time was 49 months. Mutations were identified as follows (total number of samples analyzed; incidence of mutations): NPM1 (n=526; 53%), FLT3-ITD (n=531; 31%), FLT3-TKD (n=617; 11%), CEBPA (n=509; 14%), MLL-PTD (640; 8%), and NRAS (641; 13%). CR rate was 76%. In a logistic regression model, the NPM1+/FLT3-ITD- (p<.0001) and the CEBPA+ genotypes (p=0.05) were associated with induction success. Of 666 patients achieving a CR after induction therapy, 171 had a MRD and 143 (84%) of these received an allogeneic SCT in first CR. In survival analyses, the variable “availability of a MRD” was included on a strict intent-to-treat basis. Cox proportional hazard models for RFS and OS revealed age <48 years (hazard ratio (HR) 0.72 and 0.62, respectively), availability of a MRD (HR 0.62 and 0.75), the CEBPA+ (HR 0.42 and 0.36) and the NPM1+/FLT3-ITD- (HR 0.34 and 0.43) genotype as prognostic factors. To better define the role of allogeneic SCT in first CR, subgroup analyses were performed according to the combined FLT3-ITD and NPM1 mutation status (NPM1+/FLT3-ITD- versus all other combinations). These analyses revealed a marked difference in HR estimation for the variable “availability of a MRD”. In the NPM1+/FLT3-ITD- subgroup, HR for RFS and OS were 0.89 (95%-CI 0.49–1.64) and 0.93 (95%-CI 0.51–1.67) translating into a nearly equivalent RFS after 4 years of 61% (MRD) and 57% (no MRD). In contrast, in the subgroup defined by all other combinations of FLT3-ITD and NPM1 mutations, HR for RFS and OS were 0.56 (95%-CI 0.39–0.81) and 0.69 (95%-CI 0.48–0.98) translating into a marked difference in 4 years RFS of 47% (MRD) and 23% (no MRD).Conclusions: Specific genotypes emerge as highly significant prognostic factors for response to induction therapy and for survival in AML patients exhibiting a normal karyotype. The genotypic marker constellation NPM1+/FLT3-ITD- also fulfils the criteria of a predictive marker indicating a strong beneficial effect of allogeneic SCT in first CR only in the subgroup of patients without the NPM1+/FLT3-ITD- marker constellation.

Nucleophosmin gene mutations in acute myeloid leukemia.

Abstract Context.—Heterozygous mutation of the nucleophosmin gene (NPM1) has recently been described as one of the most frequent genetic lesions in acute myeloid leukemia (AML). Objective.—(1) To discuss the clinical, morphologic, immunophenotypic, and genetic features of AML with NPM1 gene mutations, along with various detection methods, (2) To explore the mechanisms by which NPM1 gene mutations contribute to leukemogenesis. Data sources/extraction.—Data were analyzed from 7 recently published papers. Results.—NPM1 gene mutations tend to occur more frequently in women, and also tend to be associated with a higher white blood cell count. There is no significant age difference. NPM1-mutated AML is preferentially associated with AML with monocytic differentiation (in particular FAB M5b), lack of CD34, normal cytogenetics, FLT3 gene mutations, and a trend toward favorable clinical outcome, especially in patients without FLT3 gene mutation. NPM1 gene mutations cause a frame shift in the C-terminus of exon 12,...

Nucleophosmin Gene Mutations in Acute Myeloid Leukemia

Heterozygous mutation of the nucleophosmin gene (NPM1) has recently been described as one of the most frequent genetic lesions in acute myeloid leukemia (AML). (1) To discuss the clinical, morphologic, immunophenotypic, and genetic features of AML with NPM1 gene mutations, along with various detection methods, (2) To explore the mechanisms by which NPM1 gene mutations contribute to leukemogenesis. DATA SOURCES/EXTRACTION: Data were analyzed from 7 recently published papers. NPM1 gene mutations tend to occur more frequently in women, and also tend to be associated with a higher white blood cell count. There is no significant age difference. NPM1-mutated AML is preferentially associated with AML with monocytic differentiation (in particular FAB M5b), lack of CD34, normal cytogenetics, FLT3 gene mutations, and a trend toward favorable clinical outcome, especially in patients without FLT3 gene mutation. NPM1 gene mutations cause a frame shift in the C-terminus of exon 12, disrupting the NPM nucleolar-localization signal or generating a leucine-rich nuclear export motif, resulting in abnormal cytoplasmic accumulation of NPM. Several methods are suitable for detecting NPM1 gene mutation, including molecular and immunohistochemical studies. These mutations may contribute to leukemogenesis, at least in part, through disruption of the p14(ARF) (alternative reading frame) MDM2-p53 pathway and centrosomal duplication. Detection of NPM1 gene mutations may allow dissection of the heterogeneous group of AML with normal karyotype into prognostically different subgroups. Exploring the mechanisms may lead to a better understanding of how mutant NPM protein becomes leukemogenic, thereby providing insights for the development of new chemotherapeutic agents.

RAS, KIT and FLT3 Gene Mutations in inv(16)/t(16;16)-Positive Acute Myeloid Leukemia (AML): Incidence and Relevance on Clinical Outcome.

Inversion or translocation of chromosome 16 inv(16)/t(16;16) [hereafter abbreviated inv(16)] represent common cytogenetic abnormalities in adult acute myeloid leukemia (AML). At the molecular level inv(16) result in the generation of the CBFb-MYH11 fusion protein that is known to interfere with the heterodimeric transcription factor RUNX1/CBFb and thereby contributes to impaired differentiation of hematopoietic cells. Patients (pts.) with inv(16) are considered to have a favorable outcome, in particular when treated with cytarabine-based consolidation regimens. However, a significant proportion of these pts. relapse and survival after 5 years is about 60%. These findings together with studies from murine models suggest that additional genetic lesions are underlying the clinical heterogeneity of inv(16)-positive AML. The recently described mutations in the signaling molecules FLT3, KIT and RAS represent potential secondary genetic lesions that might contribute to leukemic transformation through constitutive activation.In this study we determined the incidence of KIT (exons 8, 10, 11, and 17), FLT3 (ITD; TKD at D835/I836,) and RAS (NRAS/KRAS exon1, exon2) mutations in 94 adult AML pts. (16 to 60 years; median age 41 years) with inv(16) and evaluated their prognostic impact on clinical outcome.KIT and RAS mutation screening was performed using a sensitive DHPLC-based assay; samples with abnormal profile were confirmed by direct sequencing. FLT3 mutations were identified as previously described. Pts. were entered on 3 AMLSG treatment trials [AML HD93, AML HD98A, AMLSG 07–04]. Postremission therapy implied cytarabine-based (HiDAC n=57) regimens as well as autologous (n=23) or allogeneic (n=13) stem cell transplantation (SCT) in first CR.Mutations were identified in 84% of inv(16) AML with highest frequencies in NRAS (47%) followed by KIT (26%) and FLT3-TKD (15%); 10/24 KIT mutations affected exon17. KRAS and FLT3-ITD mutations were detected in 10% and 3%, respectively. Complete remission (CR) rate was 90% for the whole group. In univariable analyses, FLT3-TKD mutations were associated with a significant inferior relapse-free survival (RFS) (p=0.01). For the other mutations there was no significant difference in RFS when comparing mutated and unmutated pts. Multivariable analysis adjusted for postremission therapy revealed FLT3-TKD (HR 2.39, p=0.04) and in trend KIT exon17 mutations (HR 2.8, p=0.06) as adverse prognostic factors. Therefore, an explorative subgroup analysis was performed for KIT exon17 mutations for the different postremission strategies. In pts. treated with HiDAC, KIT exon17 mutations were associated with a significant inferior RFS (p<0.0001), in contrast to pts. receiving SCT (p=0.70). For overall survival (OS) none of the tested variables were significantly associated with prognosis.KIT, FLT3, or RAS gene mutations can be detected in 84% of inv(16)-positive AML further sustaining the model of cooperating gene mutations. Although the numbers are still quite small, FLT3-TKD and KIT exon17 mutations are of prognostic relevance; the prognostic impact of KIT exon17 mutations seems to be abrogated by SCT strategies. Thus, KIT and FLT3 mutation status might reach clinical importance with regard to the availability of specific inhibitors and the type of postremission therapy.

Detection of RAS, KIT and FLT3 Gene Mutations in t(8;21)-Positive Acute Myeloid Leukemia (AML): Evaluation of the Clinical Relevance.

Acute myeloid leukemia (AML) with translocation t(8;21)(q22;q22) creating the AML1-ETO fusion gene is a distinct type of AML generally associated with a favorable prognosis. However, a significant proportion of these patients (pts.) relapse and survival after 5 years is approximately 50%. These findings together with studies from murine models suggest that additional genetic lesions are underlying the clinical heterogeneity of t(8;21)-positive AML. The recently described mutations in the signaling molecules FLT3, KIT and RAS represent potential secondary genetic lesions that might contribute to leukemic transformation through their constitutive activation.In this study we determined the incidence of KIT (exons 8, 10, 11, and 17), FLT3 (internal tandem duplications, ITD; tyrosine kinase mutations at D835/I836, TKD) and RAS (NRAS and KRAS exon1 and exon2) mutations in 65 adult pts. (median age 49 years) with t(8;21)-positive AML and evaluated their prognostic impact on clinical outcome.KIT and RAS mutation screening was performed using a sensitive DHPLC-based assay; samples with abnormal profile were confirmed by direct sequencing. FLT3 mutations were identified as previously described (Fröhling et al., Blood 2002). Pts. were entered on 3 AMLSG treatment trials [AML HD93, AML HD98A, AMLSG 07–04]. Postremission therapy was high-dose cytarabine-based (Ara-C) in all trials.Mutations were identified in 51% of the t(8;21)-positive AML, with highest frequencies in NRAS (18%) followed by KIT (17%) and FLT3 (ITD; 9%;); 10/11 KIT mutations affected exon17. FLT3-TKD and KRAS mutations were detected in 3% and 1.5%, respectively. Complete remission (CR) rate was 88% for the entire group. In univariable analyses, KIT exon 17 mutated pts. had a significantly inferior event-free (EFS) (p=0.04), overall (OS) (p=0.05), but not relapse-free survival (RFS) (p=0.43). In addition, the presence of FLT3-ITD mutations was in trend associated with a shorter EFS (p=0.09) and OS (p=0.07), and significantly with a shorter RFS (p=0.04). For the other mutations there was no significant or in trend difference in EFS, OS and RFS when comparing mutated and unmutated pts. Multivariable analysis for OS and RFS revealed FLT3-ITD (HR 3.16, p=0.04) and KIT exon17 mutations (HR 2.89, p=0.03) as adverse prognostic factors for OS, whereas only FLT3-ITD showed a significant prognostic impact on RFS (HR 3.32, p=0.04). Since FLT3-ITD and KIT exon 17 mutations both represent potential targets for tyrosine kinase inhibitors, an explorative analysis combining both gene mutations versus unmutated patients was performed revealing a significant inferior RFS (p=0.04) and OS (p=0.006).Mutations in the genes KIT, FLT3 or RAS were detected in 51% of t(8;21)-positive AML. Univariable and multivariable analyses showed a significant or in trend negative prognostic impact for FLT3 and KIT mutations on clinical outcome. Thus, information on the mutation status might reach clinical importance for identifying pts. who are eligible for molecular targeted therapies.

The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling

We studied antitumor effects of receptor tyrosine kinase inhibitor sunitinib (formerly SU11248) against a variety of hematologic malignancies including the following leukemias: eosinophilic (EOL-1), acute myeloid (THP-1, U937, Kasumi-1), biphenotypic (MV4-11), acute lymphoblastic (NALL-1, Jurkat, BALL-2, PALL-1, PALL-2), blast crisis of chronic myeloid (KU812, Kcl-22, K562), and adult T-cell (MT-1, MT-2, MT-4), as well as non-Hodgkin's lymphoma (KS-1, Dauji, Akata) and multiple myeloma (U266). Thymidine uptake studies showed that sunitinib was active against EOL-1, MV4-11, and Kasumi-1 cells, which possessed activating mutations of the PDGFRalpha, FLT-3, and c-KIT genes, respectively, with IC(50)s of <30 nmol/L. In addition, sunitinib inhibited the proliferation of freshly isolated leukemia cells from patients possessing mutations in FLT3 gene. Annexin V staining showed that sunitinib induced apoptosis of these cells. Sunitinib inhibited phosphorylation of FLT3 and PDGFRalpha in conjunction with blockade of mammalian target of rapamycin signaling in MV4-11 and EOL-1 cells, respectively. Interestingly, rapamycin analogue RAD001 enhanced the ability of sunitinib to inhibit the proliferation of leukemia cells and down-regulate levels of mammalian target of rapamycin effectors p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in these cells. Taken together, sunitinib may be useful for treatment of individuals with leukemias possessing activation mutation of tyrosine kinase, and the combination of sunitinib and RAD001 represents a promising novel treatment strategy.

ActivatingFLT3Mutations Are Detectable in Chronic and Blast Phases of Chronic Myeloproliferative Disorders Other Than Chronic Myeloid Leukemia

FLT3 gene mutations, either internal tandem duplication (ITD) or D835 point mutations, have been studied extensively in acute myeloid leukemia and myelodysplastic syndrome (MDS). Little is known about FLT3 mutations in chronic myeloproliferative diseases (CMPDs) or their relationship with V617F JAK2 mutations. We analyzed bone marrow samples from 142 patients with Philadelphia (Ph) chromosome- CMPD or CMPD/MDS and from 119 patients with Ph+ chronic myeloid leukemia (CML) using a multiplex polymerase chain reaction assay. FLT3 mutations, 11 ITD and 2 D835, were detected in 13 (9.2%) patients with CMPD or CMPD/MDS, 7 in blast phase and 6 in chronic phase. Analyses for JAK2 mutations in 11 of 13 cases were all negative. By contrast, no FLT3 mutations were detected in CML, including 108 chronic and 11 blast phase cases. FLT3 mutations occur in approximately 10% of CMPD and CMPD/MDS but are not observed in JAK2+ CMPD or in CML.

Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML)

In core binding factors (CBF) acute myeloid leukemia (AML), the disruption of CBFalpha/beta genes impairs normal hematopoietic differentiation and is supposed to cooperate with additional mutations promoting proliferation. The incidence and the prognosis of receptor tyrosine kinase (RTK) c-Kit and FLT3 mutations and Ras mutations were evaluated in 103 pediatric and adult patients with CBF-AML. c-Kit mutations were present in 17% patients. c-Kit exon 8 mutations were more frequent in inv(16) than in t(8;21) subset (20 versus 6%). Only one patient had FLT3-ITD but FLT3-D835 was as frequent as reported in AML population (7%). Ras mutations were significantly more frequent in inv(16) than in t(8;21) subset (36 versus 8%, P=0.001). RTK mutations were associated with a higher white blood cell count (WBC) (36 versus 21 G/L, P=0.05). FLT3 mutations were significantly associated with a shorter EFS and survival (P<0.0001 and P=0.0002) owing to an excess of early events. c-Kit mutations were associated with a shorter EFS and RFS (P=0.002 and P=0.003) in t(8;21) but not inv(16) patients. As previously observed, Ras mutations did not affect prognosis. Screening for RTK mutations may help to identify patients with a more adverse outcome and thus susceptible to benefit from intensified protocols or RTK inhibitors.

Distinct Profile of FLT3 Mutations in Brazil.

Mutations in the tyrosine kinase receptor FLT3 are the most common molecular abnormality in acute myeloid leukemia (AML) being detected in about 30% of AML cases. According to the protein domain altered FLT3 mutations may be classified as juxtamembrane or activation loop. The former are caused by internal tandem duplications (ITD) in exons 14 and 15 and is detected in 20–27% of AML patients. Mutations in the activation loop are mainly due to point mutations in exon 20 and is present in 5–7% of AML patients. Most of these mutations lead to changes in the aspartate in position 835 (D835), which have been detected in about 7% of AML cases. Both types of mutations cause the constitutive activation of FLT3 and are associated with bad prognosis. AML characteristics in Latin America are different from those in Europe and US. Namely, there is a higher frequency of acute promyelocytic leukemia (APL) and the clinical outcome of adult patients with other subtypes of AML treated with standard protocols is poorer. The worse prognosis seems to be related to the biology of the disease, rather than socio-economic features, based on studies of other hematological malignancies. In order to test if a higher frequency or different FLT3 mutations might explain these observations, we performed a screening for mutations in FLT3 using PCR and single strand conformation polymorphism (SSCP) techniques to evaluate exons 12 to 20, which encode for the intracytoplasmatic domains of the protein. Ninety-nine consecutive patients with AML (90 adults and 9 children) and 55 blood donors (controls) were analyzed. Two synonyms mutations that have not been previously described were detected: one in exon 12 (T526T) and the other in exon 17 (G697G). ITD mutations were detected in 23 (23.2%) patients with AML, therefore within the expected frequency based on the studies in developed countries. On the other hand, D835 mutations were absent, and except for a mutation detected in one patient causing the deletion of the aminoacid in the position 836 no other abnormality was detected in exon 20. No mutations were detected in exons 13, 16, 18 and 19. In adults, mutations were more frequent in acute promyelocitic leukemia and in women. There were no associations between FLT3 mutations and CBC values. Interestingly, FLT3 mutations were less frequently detected in patients whose leukemic cells expressed the CD56 marker. As described for other countries, overall survival was worse in patients with FLT3 mutations. In conclusion, this study demonstrates that the frequency of ITD mutations in FLT3 gene in Brazilian patients with AML was similar to the frequency described in the European and North American populations, whereas activation loop mutations were rarely detected, especially those in D835. Thus, our results suggest that Brazilian patients with AML have a distinct profile of genetic abnormalities. In addition, this is the first study to demonstrate a negative association between FLT3 mutations and the expression of CD56 by leukemic blasts. This is a relevant observation, since CD56 expression per se has been identified as prognostic factor by other investigators. Finally, our study demonstrates that the SSCP method may be useful for screening mutations of FLT3 gene.

Acute myeloid leukaemia with FLT3 gene mutations of both internal tandem duplication and point mutation type

FLT3 gene mutations, either internal tandem duplication or point mutation type, are common in acute myeloid leukaemia (AML). We describe 21 AML cases with both types of gene mutations, so-called dual mutations, representing approximately 1% of all cases. Most newly diagnosed AML with FLT3 dual mutations had monocytic differentiation and a normal karyotype. Over the disease course, changes in FLT3 mutation status were seen in 89% of cases, and were associated with cytogenetic changes. We conclude that FLT3 dual mutations occur rarely in AML, and appear to be related to clonal evolution.

Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group

Internal tandem duplications (ITDs) of the FLT3 gene have been observed in about 35% of APL cases. If FLT3-ITD is associated with a worse outcome in patients with acute myeloid leukemia (AML) in general, its prognostic value in acute promyelocytic leukemia (APL) is still a matter of debate. We investigated incidence, associated clinical features, and prognostic implication of FLT3-ITD, but also FLT3-D835 point mutation and N-Ras or K-Ras mutations in 119 APL patients, all prospectively enrolled in the two consecutive APL-93 and APL-2000 trials. Mutation incidences were 38, 20, and 4%, for FLT3-ITD, FLT3-D835, and Ras, respectively. The presence of FLT3-ITD was associated with high white blood cell count, high Sanz index, M3-variant subtype, and V/S PML-RAR alpha isoforms. Complete remission (CR), induction death, and death in CR rates were not affected by FLT3 or Ras mutations, as well as cumulative incidence of relapse. However, a trend for a shorter overall survival (P=0.09) was observed in FLT3-ITD patients, because of a very poor postrelapse survival (P=0.02). This feature, which has been also reported in patients with AML in general, is suggestive of an underlying genetic instability in FLT3-ITD patients, leading to the acquisition of additional unknown bad-prognosis gene mutations at relapse.

Blockade of p110delta Isoform Activity of Phosphoinositide 3-Kinase Inhibits Blast Cell Proliferation in Acute Myeloblastic Leukemia.

AMLs constitute an heterogeneous group of hematopoietic stem cell disorders of rather poor prognosis. Abnormal activation of signaling pathways including STAT, MAPK and NF-KB have been described in AMLs and could constitute new targets for therapy. In this study, we focused on the PI3K/AKT and mTOR/P70S6K activity in primary blast cells purified from the bone marrow of patients with primary (n = 54) or secondary (n =10) AMLs. Patients with de novo AMLs were issued from the AML2001 French Multicenter Trial directed by the GOELAMS. The FAB classification was the following: M0 (n = 8), M1 (n = 10) M2 (n = 21), M4 (n = 10), M4eo (n = 2), M5 (n = 11), unknown AML (n = 2). In 58% of these patients, a constitutive activation of PI3K/AKT was detected in blast cells by western blot analysis showing AKT and FOXO3a phosphorylation, and confirmed by immunofluorescence confocal microscopy and flow cytometry analysis in CD34+ blast cells. Constitutive GAB1/2 phosphorylation was detected in all PI3K+ patients. However, we did not find any correlation between PI3K activation and FLT3 gene mutations. PTEN and SHIP-1 expression was normal in all tested PI3K+ patients. Proliferation (3H thymidine incorporation) was significantly increased in PI3K+ samples compared to PI3K- samples (p=0.001). The mTOR/P70S6K pathway was activated and the mTOR inhibitor rapamycin selectively reduced proliferation of PI3K+ samples. We determined the expression of the alpha, beta and delta isoforms of the catalytic sub-unit of PI3K (p110) in leukemic cells and found that p110 delta was the only consistently expressed isoform. In 8 PI3K+ samples tested, IC87114 (ICOS Corporation), a specific p110delta inhibitor compound used at 10μM totally suppressed AKT and FOXO3a phosphorylation. IC87114 inhibited proliferation of PI3K+ leukemic cells whereas it did not induce apoptosis. IC87114 did not significantly impaired the proliferation of PI3K- blast cells. Interestingly, IC87114 did not inhibit the proliferation or clonogenicity of CD34+ cord blood cells cultured in SCF, FLT3L and TPO used as controls.Overall, our results report, a/ a constitutive activation of PI3K in 58% of AML, b/ no correlation between FLT3 gene mutations and PI3K positivity, c/ no implication of PTEN or SHIP-1 phosphatases in PI3K activation, d/ a major role of the p110 delta isoform of PI3K in leukemic cell proliferation, e/ a potential therapeutic value of inhibiting mTOR and P110 delta activity.

Acute Myeloid Leukemia With t(6;9)(p23;q34) Is Associated With Dysplasia and a High Frequency offlt3Gene Mutations

We report 12 cases of t(6;9)(p23;q34)-positive acute myeloid leukemia (AML), all classified using the criteria of the World Health Organization classification. There were 10 women and 2 men with a median age of 51 years (range, 20-76 years). Dysplasia was present in all cases (9 previously untreated), and basophilia was present in 6 (50%). Immunophenotypic studies showed that the blasts were positive for CD9, CD13, CD33, CD38, CD117, and HLA-DR in all cases assessed. CD34 was positive in 11 (92%) of 12, and terminal deoxynucleotidyl transferase was positive in 7 (64%) of 11 cases. The t(6;9) was the only cytogenetic abnormality detected in 7 cases (58%), and 5 cases had additional chromosomal abnormalities. Of 8 cases assessed, 7 (88%) had flt3 gene mutations. We conclude that t(6;9)-positive AML cases have distinctive morphologic features, an immunophenotype suggesting origin from an early hematopoietic progenitor cell, and a high frequency of flt3 gene mutations.

Evolution of FLT3-ITD and D835 activating point mutations in relapsing acute myeloid leukemia and response to salvage therapy

Internal tandem duplications (ITDs) of the juxtamembrane region of the FLT3 tyrosine kinase receptor are the most frequent genetic alterations in acute myeloid leukemia (AML). The presence of this mutation has been recognized as an independent poor prognostic factor. In this study, we compared the FLT3 mutational status between diagnosis and subsequent relapses in 31 patients with AML. At diagnosis, seven patients were identified to contain FLT3-ITD mutations and one patient to harbor the D835 mutation. Five patients remained FLT3-ITD positive throughout the disease course (+/+). Three patients lost the FLT3 gene mutation at first (one FLT3-ITD, one D835 mutation), or second relapse (one FLT3-ITD) (+/−). One additional patient lost a small FLT3-ITD positive clone at relapse and at the same time gained an apparently unrelated FLT3-ITD positive clone. One patient without FLT3 mutation at diagnosis relapsed with an FLT3-ITD mutation (−/+). A shorter median duration of first remission (6 months versus 11.5 months) and a higher relapse rate after salvage therapy (e.g. allogeneic peripheral blood stem cell transplantation) resulted in a lower leukemia-free survival in the FLT3 mutated group (11% versus 31%). The loss of a clone with a mutation in the FLT3 gene at relapse did not improve the prognosis.

Acute myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high frequency of flt3 gene mutations.

We report 12 cases of t(6;9)(p23;q34)-positive acute myeloid leukemia (AML), all classified using the criteria of the World Health Organization classification. There were 10 women and 2 men with a median age of 51 years (range, 20-76 years). Dysplasia was present in all cases (9 previously untreated), and basophilia was present in 6 (50%). Immunophenotypic studies showed that the blasts were positive for CD9, CD13, CD33, CD38, CD117, and HLA-DR in all cases assessed. CD34 was positive in 11 (92%) of 12, and terminal deoxynucleotidyl transferase was positive in 7 (64%) of 11 cases. The t(6;9) was the only cytogenetic abnormality detected in 7 cases (58%), and 5 cases had additional chromosomal abnormalities. Of 8 cases assessed, 7 (88%) had flt3 gene mutations. We conclude that t(6;9)-positive AML cases have distinctive morphologic features, an immunophenotype suggesting origin from an early hematopoietic progenitor cell, and a high frequency of flt3 gene mutations.

Abnormalities of p51, p53, FLT3 and N-ras genes and their prognostic value in relapsed acute myeloid leukemia.

To clarify the role of the genetic mutations in the clinical course of acute myeloid leukemias (AML), we analyzed for p51, p53, FLT3 and N-ras gene mutations and the expression of the p51 gene in relapsed AML. Paired samples obtained from patients with AML at both stages of diagnosis and first relapse were analyzed. Twenty-four patients with relapsed AML survived for 6 to 81 months (median 24 months) from diagnosis. In one patient, no point mutation of the p51 gene was detected, but loss of the p51 gene expression was observed at both stages. Point mutations of the p53 gene were positive at both stages (+/+) in two patients and negative at diagnosis and positive at relapse (-/+) in two patients. Tandem duplication of the FLT3 gene was detected in five patients at both stages (+/+). N-ras gene mutations at both stages (+/+) were detected in three patients. Mutant p53 at relapse was associated with short survival in patients with relapsed AML (P<0.014). Our findings show that p53 mutations were, at least in part, associated with the mechanism of relapse in AML, while new p51, FLT3 and N-ras gene alterations did not occur at relapse. Loss of the p51 gene expression in de novo AML has not been reported yet.

Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.

Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A-->G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7.2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A-->G change was shown to be a polymorphism, with an allelic frequency of 0.24 for the G and 0.76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.