FLT3-TKD mutation in childhood acute myeloid leukemia.

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. An internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence of the FLT3 gene (FLT3-ITD) is found in 20-25% of adult acute myeloid leukemia (AML) and at a lower frequency in childhood AML. FLT3-ITD is associated with leukocytosis and a poor prognosis, especially in patients with normal karyotype. Recently, there have been three reports on point mutations at codon 835 of the FLT3 gene (D835 mutations) in adult AML. These mutations are located in the activation loop of the second tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD). The clinical and prognostic relevance of the TKD mutations is less clear. To the best of our knowledge, there has been no report to describe FLT3-TKD mutations in childhood AML. In this pediatric series, FLT3-TKD mutations occurred in three of 91 patients (3.3%), an incidence significantly lower than that of FLT3-ITD (14 of 91 patients, 15.4%) in the same cohort of patients. None of them had both FLT3-TKD and FLT3-ITD mutations. Sequence analysis showed one each of D835 Y, D835 V, and D835 H. Of the three patients carrying FLT3-TKD, two had AML-M3 with one each of L- and V-type PML-RARalpha, and another one had AML-M2 with AML1-ETO. None of our patients with FLT3-TKD had leukocytosis at diagnosis. At bone marrow relapse, one of the four patients examined acquired FLT3-ITD mutation and none gained FLT3-TKD mutation.     

FLT3 and NPM1 gene mutations in childhood acute myeloblastic leukemia

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. Mutations in fms-like tyrosine kinase 3 (FLT3) gene including internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain (TKD) as well as in nucleoplasmin (NPM1) gene are associated with pathogenesis of acute myeloblastic leukemia (AML). Several reports have demonstrated high incidences of the FLT3 and NPM1 mutations in adult AML patients. Since the pathogenesis of pediatric AML is different from that of adult and the FLT3 and NPM1 mutations have not been well characterized in childhood AML. Therefore, the objective of this study was to determine the frequencies of FLT3 and NPM1 mutations in 64 newly diagnosed childhood AML patients. All blood and bone marrow samples were previously diagnosed with AML by using flow cytometry and/or cytochemistry. FLT3-ITD and FLT3-TKD were detected by PCR and PCR-RFLP methods, respectively. The NPM1 mutation was analyzed by PCR and direct DNA sequencing. The FLT3 mutations were detected in 7 of 64 (11.1%), including FLT3-ITD in 4 of 64 (6.3%) and FLT-TKD in 3 of 62 (4.8%). The NPM1 mutation was not detected in this cohort. By multivariate analysis, white blood cell counts, peripheral blood and bone marrow blast cell counts at diagnosis were significantly higher in children with FLT3-ITD (P<0.05). In addition, the median percentage of CD117 was significantly higher in leukemic blast cells with FLT3-ITD than those with wild type (P=0.01). We did not find any FLT3 mutations in children aged less than 5 years. The AML M3 cell type was most frequently associated with FLT3 gene mutations (50%). In conclusion, the FLT3 mutations was found in 11.1% but none of NPM1 mutation was detected in Thai children with AML. These data support the hypothesis of different biology and pathogenesis between adult and childhood AML.     

FLT3突变急性淋巴细胞白血病的临床特征

目的：探讨急性淋巴细胞白血病（acute lymphoblastic leukemia,ALL）患者FLT3（Fms-like tyrosine kinase）基因内部串联重复（internal tandem duplication,ITD）与其ASP835（D835）突变（mutation in the tyrosine kinase domain,FLT3-TKD）的发生情况及临床意义。方法采用聚合酶链反应法（polymerase chain reaction, PCR）扩增产物,采用限制性酶切产物分析法分析初诊患者基因易突变区的突变情况。结果147例ALL患者中,11例（7.5%）存在FLT3突变。FLT3突变患者与无突变患者在年龄、性别、骨髓原始细胞计数和外周血原始细胞计数的比较均无统计学意义（均P＞0.05）。T细胞型ALL（T-ALL）患者中FLT3突变的发生率为17.2%,其他类型ALL患者中FLT3突变的发生率为5.1%,差异具有非常显著的统计学意义（P＜0.001）。结论 ALL患者存在FLT3突变,且T-ALL患者的FLT3突变率高于其他类型患者。     

A novel prognostic scoring model for newly diagnosed FLT3-ITD-positive acute myeloid leukemia

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML). However, the molecular structure characteristics and widely accepted prognostic factors for FLT3-ITD are still not well described. This study aimed to retrospectively examine 81 patients with FLT3-ITD-positive AML diagnosed and treated at the First Affiliated Hospital of Zhejiang University from December 2013 to March 2018 using the next-generation sequencing 185-gene platform. High variant allele frequency (VAF) [> 0.48, P = 0.0089 for overall survival (OS), P = 0.13 for relapse-free survival (RFS)], multiple ITDs (> 1 ITDs, P = 0.011 for OS, P = 0.033 for RFS) and longer insertion length (> 69 bp, P = 0.14 for OS, P = 0.0078 for RFS) predicted poor survival. The study further proposed an easily applicable scoring model for OS using the Least Absolute Shrinkage and Selector Operation (LASSO) Cox regression model. Also, an independent cohort of 30 patients was used for external model validation. The mode was expressed as follows: 0.659 × FLT3-ITD VAF + 0.375 × FLT3-ITD number + 0.807 × Age + 0.688 × DNMT3A + 1.939 × U2AF1 (FLT3-ITD VAF > 0.48 scored 1; FLT3-ITD number scored 1 if carried 1 ITD, 2 if carried ≥ 2 ITDs; age > 44 years scored 1, the presence of DNMT3A or U2AF1 scored 1; 0 for other conditions). It categorized patients into low-risk (L-R, score < 1, n = 20) and high-risk (H-R, score ≥ 1, n = 61) groups based on the risk score with a significant difference in survival (3-year OS, P < 0.0001; 3-year RFS, P = 0.0005). A prognostic nomogram that integrated these five factors was developed with a concordance index calculation [OS: 0.68, 95% CI (0.64-0.72)].     

Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells.

PURPOSE: Activating length mutations in the juxtamembrane domain (FLT3-LM) and mutations in the tyrosine kinase domain (FLT3-TKD) of FLT3 represent the most frequent genetic alterations in acute myeloid leukemia (AML). However, the functional role of active FLT3 mutants in primary AML blast cells is not well characterized. EXPERIMENTAL DESIGN: We analyzed the transforming potential and the signaling of FLT3-ITD mutants in Ba/F3 cells and in primary AML blasts. RESULTS: FLT3-ITD mutants induce an autophosphorylation of the receptor, interleukin 3-independent growth in Ba/F3 cells, and a strong STAT5 and mitogen-activated protein kinase (MAPK) activation. In contrast to the FLT3-ITD mutants, the ligand-stimulated FLT3-WT receptor was unable to transduce a fully proliferative response in Ba/F3 and monocytic OCI-AML5 cells. The ligand-stimulated FLT3-WT receptor activated AKT and MAPK, but not STAT5. In primary blast cells from 60 patients with AML, FLT3 was expressed in 91.9% of patients carrying a FLT3-LM/TKD mutation compared with 77.8% in FLT3-LM/TKD-negative patients. STAT3 and STAT5 were constitutively activated in 76 and 63% of patients, respectively. In accordance with the results in Ba/F3 cells, a high FLT3 expression and the presence of a FLT3-LM was strongly associated with the STAT5 but not with the STAT3 activation in primary AML blast cells. Moreover, the constitutive tyrosine phosphorylation of STAT5 was efficiently down-regulated by a FLT3 protein tyrosine kinase inhibitor in AML cells expressing an active FLT3 mutant. CONCLUSIONS: Active FLT3 receptor mutants have transforming potential in hematopoietic cells and induce a strong activation of STAT5 in primary AML cells. The FLT3-STAT5 pathway contributes to the malignant phenotype and represents a promising molecular therapeutic target structure in AML.     

FLT3 mutations in canine acute lymphocytic leukemia

Background  

FMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit FLT3 ITD mutations.     

Chemoimmunotherapy for maintenance in acute myeloblastic leukemia.

Of 30 adult patients with acute myeloblastic leukemia, 14 achieved complete remission. Eight of these were given chemoimmunotherapy for maintenance. The immunotherapy consisted of intradermal pooled allogeneic leukemic cells (snap-frozen irradiated) and BCG vaccine given by Heaf gun, given twice in 4 weeks. The chemotherapy was given for 1 week in 4 weeks. The median duration of remission in these eight patients was 115+ weeks and the median duration of survival was 147+ weeks. The other six patients who were given chemotherapy only for maintenance had a median duration of remission of 15 weeks and a median survival of 52 weeks. The two groups cannot be compared properly, however, as allocation of patients was not random, and the chemotherapy differed significantly.     

急性髓系白血病患者FLT3表达及TKD点突变的研究

  目的 研究急性髓系白血病（AML）患者与正常人FMS样酷氨酸激酶3（FLT3）基因在骨髓中表达的差异以及FLT3基因酪氨酸激酶结构域（TKD）点突变与AML的关系。方法 将AML初治患者和正常人的骨髓分别用CD135抗体标记后，应用三色流式细胞术检测并分析；采用RT-PCR结合限制性内切酶酶切，检测30例AML初治患者FLT3基因的TKD点突变。结果 AML患者骨髓中CD135阳性率均＞20 ％，而正常人＜2 ％，二者差异有统计学意义（P＜0.05）；另外，30例AML患者中3例存在FLT3-TKD点突变，阳性率为10 ％，其中2例化疗后完全缓解（CR），缓解率66.7 ％，其他27例非点突变患者化疗后完全缓解率为84 ％，二者比较差异无统计学意义（P＞0.05）。结论 AML患者骨髓中FLT3基因表达显著高于正常人。AML患者FLT3-TKD点突变的阳性率为10 ％左右，FLT3-TKD点突变对患者化疗后CR率的影响有待进一步研究。     

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.Subject terms: Acute myeloid leukaemia, Cell signalling     

Clonal expansion and progression in acute myeloblastic leukemia.

Diseaes originating in pluripotent stem cells, then developing through clonal expansion and clonal progression may properly be grouped together because of their common features. Among these, AML appears to be unique by reason of the presence within the clone of a blast cell population. Studies of cellular composition and regulation in AML clones require assays that measure not only myelopoiesis but also blast cell proliferation. The relation of the blast population to other components of AML clones remains uncertain. Resolution of the uncertainty is important in considering therapeutic strategies.     

FLT3 inhibitors in the treatment of acute myeloid leukemia

Despite recent modest improvements in the chemotherapy regimens used to treat acute myeloid leukemia (AML), many patients diagnosed with AML ultimately die of the disease. Commonly occurring genetic alterations have been identified that strongly affect the prognosis for patients with AML. These alterations represent possible targets for investigational therapies that could act to specifically halt the aberrant growth of AML cells while limiting damage to normal cells. One such gene is the Fms-like tyrosine kinase 3 (FLT3) gene, which is mutated in approximately 30% of adult patients with AML and has a significant impact on prognosis. In particular, internal tandem duplications in FLT3 confer a poor prognosis to this large subgroup of patients with AML. Agents that target FLT3 are in development for the treatment of patients who have AML and offer a potential paradigm change in the current standard treatment of AML. For this report, the authors reviewed the prognostic significance of genetic alterations observed in AML with a focus on the therapeutic implications of targeting FLT3. The introduction of such agents may be the next major step toward the era of personalized therapy in AML.     

FLT3抑制剂在急性髓细胞白血病中的研究进展

 FLT3是一种受体酪氨酸激酶（RTK）,是急性髓细胞白血病（AML）中最常见的突变基因之一,有内部重复串联序列和激活环点突变两种形式,FLT3基因突变与AML的发病密切相关,作为一项独立指标,在疾病的发生中具有重要的意义,并且是AML的独立预后因素。以FLT3为靶点的研究已成为当今AML治疗的热点。文章阐述了FLT3的临床意义、FLT3抑制剂的作用机制以及数种新型药物的最新研究进展,并针对目前FLT3抑制剂存在的困难及前景进行了讨论。     

急性髓系白血病患者外周血细胞中FLT3基因表达与FLT3／ITD突变的关系及其意义

背景与目的：研究表明FLT3／ITD突变的急性髓性白血病（acutemyeloid leukemia,AML）患者预后差,但关于AML患者FLT3基因的表达水平在预后中的作用及其与FLT3／ITD突变关系的研究尚不充分。本研究探讨初治AML患者FLT3基因的表达水平与FLT3／ITD突变的关系及其临床意义。方法：建立实时荧光定量PCR检测FLT3基因表达水平及PCR检测FLT3／ITD突变的方法。分析79例初治AML患者FLT3基因水平、FLT3／ITD突变及与预后的关系。结果：22．7％（18／79）的AML患者存在FLT3／ITD突变。92．4％（73／79）的患者标本中可检测到FLT3基因表达,FLT3基因表达水平为0-7320,中位数为312,正常对照组未检测到FLT3基因的表达。FLT3基因高表达及FLT3／ITD突变AML组的白细胞计数及骨髓白血病细胞均显著高于低表达和无突变AML组（P〈0．05）。FLT3基因高表达的AML组FLT3／ITD突变率（25．6％）同低表达的AML组（20．0％）相比,差异无统计学意义（P〉0．05）,FLT3／ITD突变AML患者FLT3基因表达中位数与无突变组比较差异也无统计学意义。FLT3／ITD突变组的完全缓解率（58．8％）显著低于无FLT3／ITD突变组（82．1％）（P〈0．05）;FLT3基因高表达AML组FLT3／ITD的完全缓解率（68．6％）同低表达AML组（84．2％）相比差异无统计学意义（P〉0．05）,但无FLT3／ITD突变组中,FLT3基因高表达组完全缓解率（69．2％）显著低于低表达组（93-3％）（P〈0．05）。结论：FLT3高表达与FLT3／ITD突变之间无明显相关性,FLT3高表达对于无FLT3／ITD突变AML患者可能是一个预后不良的指标。     

慢性粒细胞性白血病FLT3基因及FLT3/ITD基因突变的检测及意义

背景与目的:大部分急性髓细胞性白血病(acutemyeloidleukemia,AML)患者出现FLT3基因异常表达,20%～30%AML患者会出现FLT3/ITD基因突变并与临床预后相关。本研究旨在了解慢性粒细胞白血病(chronicmyeloidleukemia,CML)患者FLT3基因及FLT3/ITD基因突变情况。方法:采用聚合酶链反应(polymerasechainreaction,PCR)检测53例CML慢性期和34例CML加速期或急变期患者DNA水平FLT3基因及FLT3/ITD基因突变。结果:53例CML慢性期患者3例(5.7%)出现FLT3基因阳性,34例加速期或急变期患者有19例(55.9%)出现FLT3基因阳性,CML加速期和急变期患者FLT3基因阳性率显著高于慢性期患者(P<0.001),87例CML患者,仅2例(2.3%)出现FLT3/ITD基因突变。结论:在CML患者中,FLT3基因表达主要见于加速期或急变期患者;CML很少发生FLT3/ITD基因突变;FLT3基因及FLT3/ITD基因突变阳性CML患者可能提示预后不佳,此方面研究尚需深入。     

Novel AXL-targeted agents overcome FLT3 inhibitor resistance in FLT3-ITD+ acute myeloid leukemia cells

AXL receptor tyrosine kinase (AXL) upregulation mediates drug resistance in several types of human cancer and has become a therapeutic target worthy of exploration. The present study investigated AXL antigen expression and the effects of novel AXL-targeted agents in acute myeloid leukemia (AML) cells. AXL antigen expression in drug-sensitive and drug-resistant human AML cell lines, and AML blast cells from 57 patients with different clinical characteristics, was analyzed by flow cytometry and compared. Furthermore, the effects of the novel AXL antibody DAXL-88, antibody-drug conjugate DAXL-88-monomethyl auristatin E (MMAE), AXL small molecule inhibitor R428 and their combination with FMS-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) in AML cells were analyzed by Cell Counting Kit-8 assay, flow cytometry and western blotting. The present study revealed that AXL antigen expression was upregulated in FLT3-internal tandem duplication (ITD)/tyrosine kinase domain mutation-positive (TKD)⁺ AML blast cells compared with FLT3-ITD/TKD⁻ AML cells. Additionally, AXL antigen expression was markedly upregulated in the AC220-resistant FLT3-ITD⁺ MV4-11 cell line (MV4-11/AC220) and in FLT3 inhibitor-resistant blast cells from a patient with FLT3-ITD⁺ AML compared with parental sensitive cells. The AXL-targeted agents DAXL-88, DAXL-88-MMAE and R428 exhibited dose-dependent cytotoxic effects on FLT3-mutant AML cell lines (THP-1, MV4-11 and MV4-11/AC220) and blast cells from patients with FLT3-ITD⁺ AML. Combinations of AXL-targeted agents with AC220 exerted synergistic cytotoxic effects and induced apoptosis in MV4-11/AC220 cells and FLT3 inhibitor-resistant blast cells. The antileukemic effect of DAXL-88 and DAXL-88-MMAE may rely on their ability to block AXL, FLT3 and their downstream signaling pathways. The present study demonstrated the association between AXL antigen expression upregulation and drug resistance in FLT3-ITD⁺ AML, and proposed a method for overcoming FLT3 inhibitor resistance of FLT3-ITD⁺ AML using novel AXL-targeted agents.     

Double minute chromosomes in acute myeloblastic leukemia

Two patients with acute myeloblastic leukemia (AML) with double minute chromosomes (dmins) are described. One patient had dmins in approximately one-third of bone marrow cells examined at diagnosis; no other karyotypic changes were observed. The dmins disappeared when the patient achieved a complete remission. The second patient developed acute leukemia as a second cancer, having previously received radiotherapy and chemotherapy for a breast carcinoma. At the time of diagnosis of AML, the patient exhibited dmins in 12% of bone marrow cells; other complex karyotypic changes were observed. Data on the clinical and cytogenetic features of these cases are compared with those of other reported cases of acute leukemia with dmins. The possible biologic and clinical significance of dmins in acute leukemia is discussed.