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Mutation of FLT3 gene in acute myeloid leukemia with normal cytogenetics and its association with clinical and immunophenotypic features
Abstract and Figures
Acute myeloid leukemia (AML) with normal karyotype represents a clinically and molecularly heterogeneous disease. Molecular markers with prognostic significance have been examined to improve risk profile characterization of this group. Activating mutations on FLT3 receptor are one of the most common genetic alterations reported. However, the prevalence and prognostic significance of FLT3 genetic alterations in AML patients with cytogenetically normal karyotype is still controversial. In this study, FLT3/ITD and FLT3/D835 mutations were analyzed in 133 patients with de novo AML with normal cytogenetics by genomic PCR assay. Of 133 patients with AML with normal cytogenetics, FLT3 internal tandem duplication (ITD) and FLT3/D835 mutations were detected in 27 (20%) and 4 (3%) samples, respectively. Although statistically insignificant, the frequency of FLT3/ITD was higher in >15 year age group when compared to <15 year group (23 vs. 13%, P = 0.2). The white blood count was found to be significantly higher in patients with FLT3/ITD mutation when compared to those without the mutation (40 × 10⁹/L vs. 20 × 10⁹/L, P = < 0.002) or those with FLT3/D835 mutations (30 × 10⁹/l). Aberrant expression of CD7 was observed more frequently in patients with FLT3/ITD mutation (P < 0.002). There was no significant difference in the response rate to chemotherapy in patients with or without FLT3/ITD mutation (67 and 64%, respectively). FLT3/ITD mutation was found to be associated with the age, leukocytosis and aberrant expression of CD7, although no influence of FLT3/ITD mutation was seen on the clinical outcome of AML patients with normal cytogenetics.
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... The myeloblastic populations included Table 1. Clinical and biological characteristics of patient populations according to FLT3-ITD and NPM1 mutational sta- The selection of specific antigens for quantitative analysis was based on an initial antigen expression screening (using qualitative flow cytometry data obtained from patient diagnostic reports, Supplementary data methods section, Supplementary Table 2), and on previously published reports regarding antigen expression in FLT3-ITD and/ or NPM1-MUT AML [7][8][9]11,15,[19][20][21][22][23]. The geometric mean fluorescence intensities (MFIs) of CD4, CD7, CD9, CD13, CD14, CD33, CD34, CD56, CD64, CD71, CD117, and CD123 were determined for the blasts populations and normalized to the MFIs of negative lymphocyte populations for the respective markers, as previously described (Supplementary Figure 1(D,E)) [9]. ...
... CD117 negtive populations were observed in the FLT3-WT/NPM1-MUT group (Fischer's exact test ¼ 7.747, p¼0.023). Based on these results and a review of the literature [7][8][9]11,15,[19][20][21][22][23], as described above, the following markers were selected for MFI analyses: CD4, CD7, CD9, CD13, CD14, CD33, CD34, CD56, CD64, CD71, CD117, and CD123. Antigen expression levels according to only the FLT3-ITD and Antigen expression patterns and levels according to FLT3-ITD and NPM1 mutation combinations ...
The most frequent mutations in acute myeloid leukemia (AML) – FLT3-ITD and NPM1 – are associated with a specific immunophenotype. We evaluated the levels of surface antigens in an uninvestigated AML patient population according to the combination of FLT3-ITD/NPM1 mutations. Antigen levels were calculated as the geometric mean fluorescence index (MFI) ratio between myeloblasts or monoblasts/monocytes and a negative population for the specific antigen. In myeloblastic populations, FLT3-ITD cases presented CD7high MFI values (p < .001), while NPM1-MUT cases presented CD33high (p < .001), and CD34low (p < .001) MFI values. Within the monoblastic/monocytic populations, CD56high expression was observed only in the FLT3-WT/NPM1-MUT population (p=.003). The single common antigen expression between myeloblasts and monoblasts/monocytes was CD123high expression only within the FLT3-ITD/NPM1-MUT subgroup. Our results present a subtle influence of FLT3-ITD/NPM1 mutations upon antigen expression profiles in myeloblasts vs monoblasts/monocytes, and we described a novel correlation between the presence of NPM1 and CD56high values within bulk leukemic monoblasts/monocytes.
... No remarkable differences between the control and the NaOCl could be found. The group treated with the bromelain enzyme displayed the maximum bond strength [8]. ...
... AML is more common in males than in women, with a male to female ratio of roughly 2:1. The current investigation's observation of a male majority (58.5% of patients) was consistent with other researches (16,17) . ...
... Activating mutations in FLT3, especially ITD, leads to proliferative signals and is commonly associated with high white blood cell counts, increased chances of central nervous system disease, short DOR, and poor survival outcomes in AML [16,[23][24][25][26]. The oncogenic impact of FLT3 ITD is also dependent on the mutated to wild-type allelic ratio, the mutation length and its interaction with cytogenetics, and co-occurring mutations [15,18,[27][28][29]. ...
Opinion statement
Treatment options in acute myeloid leukemia (AML) have improved significantly over the last decade with better understanding of disease biology and availability of a multitude of targeted therapies. The use of FLT3 inhibitors (FLT3i) in FLT3-mutated (FLT3mut) AML is one such development; however, the clinical decisions that govern their use and dictate the choice of the FLT3i are evolving. Midostaurin and gilteritinib are FDA-approved in specific situations; however, available data from clinical trials also shed light on the utility of sorafenib maintenance post-allogeneic stem cell transplantation (allo-SCT) and quizartinib as part of combination therapy in FLT3mut AML. The knowledge of the patient’s concurrent myeloid mutations, type of FLT3 mutation, prior FLT3i use, and eligibility for allo-SCT helps to refine the choice of FLT3i. Data from ongoing studies will further precisely define their use and help in making more informed choices. Despite improvements in FLT3i therapy, the definitive aim is to enable the eligible patient with FLT3mut AML (esp. ITD) to proceed to allo-SCT with regimens containing FLT3i incorporated prior to SCT and as maintenance after SCT.
... Therefore, it was concluded that rapid identification of specific translocations at diagnosis is important for prognostic purposes and their detection should be incorporated into routine assessment. In India, Chauhan et al 14 showed FLT3-ITD mutation to be present in 23% with association to young age less than 15 years and high WBC count. However, no significant difference was reported in the response rates to conventional chemotherapy in patients with or without FLT3/ITD mutation. ...
Background:
FLT-3 mutation is a valuable prognostic marker in patients of AML being related with bad prognosis and poor clinical response to conventional chemotherapeutic agents. Frequency of FLT-3 mutation in AML varies from 25% to 35%. The objective of this study was to determine prevalence of FLT-3 mutation in patients with Acute Myeloid Leukaemia.
Methods:
This observational cross-sectional Study was conducted in Department of Oncology, Jinnah Hospital Lahore from 1st October 2018 to 31st March 2019. Patients with acute myeloid leukaemia, aged 15-60 years, of both genders were included. After taking consent, demographic data was noted. Three ml of sample of blood was obtained from each patient and sent for detection of FLT-3 mutation. Data was analysed using SPSS version 20.0. Chi square test was applied, pvalue <0.05 significant.
Results:
A total of 180 patients were enrolled in this study. The mean±SD age of patients was 34.72±14.3 years, among which 38.3% were female and 61.7% male. The mean±SD duration of disease was 3.39±2.8 months. The mean±SD haemoglobin level, TLC and platelet counts were 7.2±2.3 g/dl, 30,913±63,573 per cm and 58.6±52.3×103 per cm. The blast cell (%) count was 69.6±19.8. FLT-3 mutation was present in 18.9%.
Conclusions:
We conclude that FLT-3 mutation to be present in only a minority of patients with Acute Myeloid Leukaemia having no significant association with age, sex, haemoglobin, WBCs and blast counts.
... Several individual markers have been studied for their association with prognosis, but reports on a possible association vary widely between studies. Expression of CD7, normally found on thymocytes and mature T-cells, on AML blasts has been associated with FLT3/ITD mutations (7,8) and double mutated CEBPa (9,10), while reports on the impact on prognosis vary (11)(12)(13)(14)(15)(16)(17). CD11b (MAC-1), a protein subunit that binds to ICAM1 and is involved in leukocyte adhesion and migration (18), has been related to presence of a monosomal karyotype (MK) (19) and poor prognosis (19)(20)(21), but this is disputed by others (22). ...
Background:
In acute myeloid leukemia controversy exists about the role of immunophenotyping of the blasts at diagnosis as a potential prognostic factor.
Methods:
We retrospectively analyzed immunophenotypic marker expression on blasts in relation to genetic aberrancies and survival data of 684 patients. All patients were included in different studies from the HOVON/SAKK Consortium.
Results:
Markers CD2, CD7, CD11b, CD19, CD22, and CD56 all appeared to be associated with one or more established prognostic genetic aberrancies. In the overall population, differences in univariate survival analyses were observed for CD2, CD11b, and CD22. After correcting these survival differences for currently used risk profile data, only CD11b expression remained of prognostic value for poor overall survival (OS) and event-free survival (EFS). CD11b expression turned out to be an independent factor for poor OS and EFS in the subgroup of patients who lacked cytogenetic and molecular aberrancies.
Conclusions:
In this study, we demonstrate that associations between immunophenotypic markers and genetic aberrancies interfere with the prognostic properties of immunophenotypic markers. Such may account for most of the previously reported prognostic impact of these markers. Only CD11b expression remained as an independent prognostic marker. © 2017 International Clinical Cytometry Society.
... (9) Surprisingly, the mean age of the patients in our study is 42 years, which is in concurrence to published reports from Pakistan (10,11) AML is slightly more common in men, with a male to female ratio of 2:1. (12) However, in our study female patients were slight dominant which may be coincidence. However, one of the study recently conducted in Iranian population showed same prevalence of male versus female which was seen in our patients. ...
Background:
Acute myeloid leukemia (AML) is a malignant disease of the bone marrow in which karyotypic analysis is the most important diagnostic and prognostic tool for predicting remission rate, relapse and overall survival. This study was carried out to determine the frequency and type of cytogenetic aberrations in de novo acute myeloid leukemia in adults at a tertiary care hospital.
Materials and methods:
This descriptive cross-sectional study was carried out in the Hematology Department, Liaquat National Hospital from November 2014 to April 2016.A total of 51cases were diagnosed with AML during the study period. Cytogenetic analysis was carried out by banding technique on bone marrow aspirate samples.
Results:
The mean age of the study subject was 42.03±17.70 years. Frequency of karyotyping abnormalities was observed in 47% of cases, in which most frequently occurring cytogenetic abnormalities were those of good cytogenetics including t(15;17) and t(8;21), seen in 23.5% and 9.8% of cases respectively. Intermediate risk cytogenetics including Del 9q was seen in 1.96% of cases. However, poor risk cytogenetics including complex cytogenetics, t(11;q23) and del (13) were seen in 7.8%, 1.96% and 1.96% of cases respectively. Normal cytogenetics was seen in 27 (52.9%) patients.
Conclusion:
Karyotyping is one of the most important diagnostic and prognostic tools and a maximum benefit could be attained through cytogenetic analysis. Cytogenetic aberrations in our series are more or less similar as reported at national level with preponderance of good risk cytogenetics in our setting.
Objectives
Various prognostically important genetic mutations are associated with acute myeloid leukemia (AML). Studies have found correlation between these mutations and expression of certain abnormal proteins in the tumor cells by immunohistochemistry (IHC). Common genetic mutations are nucleophosmin 1 (NPM1) and FMS like tyrosine kinase 3 (FLT3). This study aimed at studying the prognostic utility of surrogate IHC for these mutations-NPM1 IHC for NPM1 mutation, whereas C-X-C Chemokine Receptor type 4 (CXCR4) and Cluster of Differentiation 123 (CD123) IHC for FLT3 mutation in AML patients.
Material and Methods
This was a prognostic test accuracy study done in a tertiary care centre over a period of two years (2018–2020) under two subgroups: who attained remission (remission group) and who failed to achieve remission (not in remission group) after induction therapy. Prognostic IHC markers were performed on the diagnostic bone marrow biopsy.
Results
There were 70 cases in remission and 49 cases not in remission with median age of 32 and 31 years, respectively. Median total leucocyte count was significantly more in remission group ( P = 0.02). AML subtype and cytogenetics wise, remission group, had significantly more M3 and M4 subtypes and translocations, while not in remission group had more M2 and M1 and more of normal and complex cytogenetics ( P = 0.01 and 0.03, respectively). NPM1 and FLT3 mutation did not show significant association with remission status. IHC for NPM1, CXCR4, and CD123 was performed in the diagnostic bone marrow biopsy. Loss of nuclear localization of NPM1 and CXCR4 positivity by IHC was more in remission than not in remission (34.3% vs. 28.6% and 54.3% vs. 44.9%, respectively) which was not statistically significant. The expression of NPM1, CXCR4, and CD123 IHC had low sensitivity (34%, 54%, and 4.3%, respectively) to predict remission status. NPM1 IHC was highly significantly associated with NPM1 mutation and had high sensitivity (89%) and specificity (86%) to predict NPM1 mutation whereas CXCR4 and CD123 had low sensitivity, specificity to predict FLT3 mutation.
Conclusion
NPM1 IHC can be used as a surrogate to predict NPM1 mutation whereas CXCR4 and CD123 are not effective surrogates to predict FLT3 mutation.
Background:
T-cell lymphoblastic lymphoma (T-LBL) is an aggressive neoplasm closely related to T-cell acute lymphoblastic leukaemia (T-ALL). Despite their similarities, and contrary to T-ALL, studies on paediatric T-LBL are scarce and, therefore, its molecular landscape has not yet been fully elucidated. Thus, the aims of this study were to characterize the genetic and molecular heterogeneity of paediatric T-LBL and to evaluate novel molecular markers differentiating this entity from T-ALL.
Procedure:
Thirty-three paediatric T-LBL patients were analyzed using an integrated approach, including targeted next-generation sequencing, RNA-sequencing transcriptome analysis and copy-number arrays.
Results:
Copy number and mutational analyses allowed the detection of recurrent homozygous deletions of 9p/CDKN2A (78%), trisomy 20 (19%) and gains of 17q24-q25 (16%), as well as frequent mutations of NOTCH1 (62%), followed by the BCL11B (23%), WT1 (19%) and FBXW7, PHF6 and RPL10 genes (15%, respectively). This genetic profile did not differ from that described in T-ALL in terms of mutation incidence and global genomic complexity level, but unveiled virtually exclusive 17q25 gains and trisomy 20 in T-LBL. Additionally, we identified novel gene fusions in paediatric T-LBL, including NOTCH1-IKZF2, RNGTT-SNAP91 and DDX3X-MLLT10, the last being the only one previously described in T-ALL. Moreover, clinical correlations highlighted the presence of Notch pathway alterations as a factor related to favourable outcome.
Conclusions:
In summary, the genomic landscape of paediatric T-LBL is similar to that observed in T-ALL, and Notch signaling pathway deregulation remains the cornerstone in its pathogenesis, including not only mutations but fusion genes targeting NOTCH1.
Introduction:The FMS-like tyrosine kinase-3 (FLT3), a member of the Platelet-derived growth factor (PDGF-R) subfamily of receptor tyrosine kinases, expressed on early
hematopoietic progenitor cells play an essential role in survival and differentiation of stem cell. Majority of acute myeloid leukemia (AML) patients have mutation in this gene. Two types of frequent mutations are present in this gene. Both the types FLT3-ITD and D835 mutations play an important role in prognosis of AML patients.
Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, -5, del(5q), -7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.
We analyzed tandem duplication in the juxtamembrane (JM) domain of the FLT3 (FMS-like tyrosine kinase 3/FLK2, CD135) gene in 94 children with acute myeloid leukemia (AML) and evaluated its correlation with clinical features. Longer polymerase chain reaction (PCR) products were observed in five patients; 1/3 of M0, 119 of M1, 1/39 of M2, 1/9 of M3 and 1/12 of M5. The sequence analyses of abnormal PCR products showed that all the abnormal products were derived from tandem duplications involving the JM domain and that all the lengthened sequences were in-frame as we previously reported. Statistical analyses revealed a significantly lower incidence of the tandem duplication in childhood AML patients than in adult patients (P < 0.05), and significantly shorter disease-free survival in patients with mutant FLT3 than in patients with wild-type FLT3 (P < 0.05). Our results suggest that the tandem duplication in the JM domain of the FLT3 gene is not a frequent phenomenon but might be a factor of poor prognosis in childhood patients with AML.
The associations of cytogenetics with complete remission (CR) rates, overall survival (OS), and outcomes after CR were studied in 609 previously untreated AML patients younger than 56 years old in a clinical trial comparing 3 intensive postremission therapies: intensive chemotherapy, autologous transplantation (ABMT), or allogeneic bone marrow transplantation (alloBMT) from matched related donors. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes. CR rates varied significantly (P < .0001) among the 4 groups: favorable, 84% (95% confidence interval [CI], 77%-90%); intermediate, 76% (CI, 71%-81%); unfavorable, 55% (CI, 48%-63%); and unknown, 54% (CI, 33%-74%). There was similar significant heterogeneity of OS (P < .0001), with the estimated relative risk of death from any cause being 1.50 (CI, 1.10-2.05), 3.33 (CI, 2.43-4.55), and 2.66 (CI, 1.59-4.45) for the intermediate, unfavorable, and unknown risk groups, respectively, compared with the favorable group. In multivariate analyses, the effects of cytogenetic risk status on CR rate and OS could not be explained by other patient or disease characteristics. Among postremission patients, survival from CR varied significantly among favorable, intermediate, and unfavorable groups (P = .0003), with significant evidence of interaction (P = .017) between the effects of treatment and cytogenetic risk status on survival. Patients with favorable cytogenetics did significantly better following ABMT and alloBMT than with chemotherapy alone, whereas patients with unfavorable cytogenetics did better with alloBMT. Cytogenetic risk status is a significant factor in predicting response of AML patients to therapy; however, to tighten treatment correlates within genetically defined AML subsets, a significantly larger leukemia cytogenetic database is warranted.
Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.
Three receptor molecules, belonging to the class III of receptor tyrosine kinases, namely the receptors for colony-stimulating factor 1, CSF1R (product of the FMS proto-oncogene) and Steel factor, SLFR (product of the KIT proto-oncogene), as well as the recently identified FLT3/FLK2 gene product, appear to play distinct roles in normal hematopoietic differentiation. Their potential role in leukemic hematopoiesis has been approached by expression studies in hematopoietic malignancies, especially in acute leukemias of the myeloid and lymphoid lineages. We present here a review of available data, and discuss the possible significance and potential applications of these results.
Receptor-type tyrosine kinases (RTK) with five or seven immunoglobulin-like domains in their extracellular region are encoded by genes grouped in clusters. In human, two such clusters have been individualized, in chromosomal regions 4q11-q12 and 5q33-qter respectively. We define here a third cluster located on chromosome 13q and containing two contiguous RTK genes, FLT1 and FLT3. The former has recently been shown to encode a RTK of a new class while the latter codes for a hematopoietic receptor closely related to the products of the FMS and KIT genes. The physical linkage is also evidenced in mouse, where the two genes appear to lie within a 350 kb Mlu I fragment, on mouse chromosome 5.
The novel hematopoietic growth factor FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell factor), and FMS. the receptor for M-CSF (macrophage colony- stimulating factor). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced growth of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell growth underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean 20%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic growth factor- dependent cell lines increased their proliferation upon incubation with FL, whereas all growth factor-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic fibroblast growth factor), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming growth factor-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.
We analyzed mRNA expression of the flt3 gene in 30 patients with acute myeloid leukemia (AML) and 50 with acute lymphoblastic leukemia (ALL). Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of flt3 was observed in 61 patients; 22 (73%) with AML and 39 (78%) with ALL. Among these, five patients with AML (one M2, two M4, and two M5) showed unexpected longer transcripts with a primer combination which could amplify the transmembrane (TM) domain through the juxtamembrane (JM) domain. For those patients who expressed flt3 mRNA, the extracellular domain of the flt3 gene was also examined by RT-PCR, but no length abnormality was seen in this region. We further analyzed the TM domain through the second tyrosine kinase domain by genomic amplifications. The five patients who showed aberrant flt3 transcripts exhibited abnormal longer PCR products in addition to the germline products at a region corresponding to the JM through the first TK (TK1) domains. Sequence analyses of the abnormal RT-PCR products demonstrated that partial sequences were tandemly duplicated. Because all these altered transcripts were in-frame, deduced protein products could be expected. Sequence analyses of the genomic DNA revealed that three of the five patients showed a simple internal duplication within exon 11; one had an internal duplication (26 bp) with a 4-bp insertion; and in the fifth patient, a 136-bp sequence from the 3' part of exon 11 to intron 11 and the first 16-bp sequence of exon 12 were each duplicated with 1-bp insertion. In order to confirm the tumor specificity of these alterations, DNA samples obtained at complete remission were also analyzed in the three patients harboring an flt3 duplication, but no abnormal PCR product other than germline was detected in any of the samples. Our results suggest that an internal tandem duplication at the JM/TK1 domains of the flt3 gene is a somatic change detected preferentially in AML, possibly containing a monocytic component.
Internal tandem duplication of the FLT3 gene and point mutations of the N-RAS gene are the most frequent somatic mutations causing aberrant signal-transduction in acute myeloid leukemia (AML). However, their prognostic importance is unclear. In this study, their prognostic significance was analyzed in 201 newly diagnosed patients with de novo AML except acute promyelocytic leukemia. Three patients had mutations in both genes, 43 had only the FLT3 gene mutation, 25 had only the N-RAS gene mutation, and 130 had neither. These mutations seemed to occur independently. Both mutations were related to high peripheral white blood cell counts, and the FLT3 gene mutation was infrequently observed in the French-American-British (FAB)-M2 type. AML cases with wild FLT3/mutant N-RAS had a lower complete remission (CR) rate than those with wild FLT3/wild N-RAS, whereas the presence of mutant FLT3 did not affect the CR rate. Univariate analysis showed that unfavorable prognostic factors for overall survival were age 60 years or older (P =.0002), cytogenetic data (P =.002), FAB types other than M2 (P =.002), leukocytosis over 100 +/- 10(9)/L (P =.003), and the FLT3 gene mutation (P =.004). However, the N-RAS gene mutation was only a marginal prognostic factor (P =.06). For the subjects under 60 years old, multivariate analysis showed that the FLT3 gene mutation was the strongest prognostic factor (P =.008) for overall survival. The FLT3 gene mutation, whose presence is detectable only by genomic polymerase chain reaction amplification and gel electrophoresis, might serve as an important molecular marker to predict the prognosis of patients with AML.
We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.