Novel FLT3 point mutations within exon 14 found in patients with acute myeloid leukaemia

Internal tandem duplications in FLT3 are the most common mutation in acute myeloid leukaemia (AML), with agarose gel electrophoresis of polymerase chain reaction products (PCR/agarose) being the screening method of choice for these mutations. As PCR/agarose screening does not detect small mutations, single-stranded conformational polymorphism analyses (PCR/SSCP) were used in an attempt to identify previously unrecognized point mutations in FLT3 exons 14 and 15 of 140 AML patients, using newly designed primers that anneal within intron sequences. Novel missense point mutations were found in exon 14, suggesting additional investigations should be performed in AML and other haematopoietic malignancies, using this sensitive technique.     

Prognostic impact of FLT3-ITD mutation on NPM1+ acute myeloid leukaemia patients and related molecular mechanisms

The prognosis of acute myeloid leukaemia (AML) patients carrying NPM1 mutations is significantly worse when accompanied by FLT3-ITD mutations. However, accurate quantitative detection of FLT3-ITD mutations remains challenging. To identify a novel biomarker in NPM1⁺FLT3-ITD⁺ AML patients for more accurate stratification, we analysed the differential gene expression between the NPM1⁺FLT3-ITD⁺ and NPM1⁺FLT3-ITD⁻ groups in five public AML datasets and identified a biomarker by taking the intersection of differentially expressed genes. We validated this biomarker in bone marrow samples from NPM1⁺ AML patients at the Peking University Institute of Haematology and analysed its prognostic significance. BCAT1 expression was higher in the NPM1⁺FLT3-ITD⁺ group than in the NPM1⁺FLT3-ITD⁻ group in all seven cohorts. BCAT1 was able to predict the prognosis of NPM1⁺FLT3-ITD⁺ AML patients, and its predictive ability was superior to that of the FLT3-ITD allelic ratio (AR). FLT3-targeted inhibitor quizartinib reduced BCAT1 expression. BCAT1 knockdown using lentiviral vectors led to the downregulation of MYC expression. Thus, we identified BCAT1 as a novel biomarker for NPM1⁺FLT3-ITD⁺ AML patients. The FLT3-ITD/BCAT1/MYC signalling pathway may play a biological role in promoting the occurrence and development of AML in FLT3-ITD⁺ cell lines.     

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.     

FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform ‘UniCAR’ is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.     

A phase I/II study of sunitinib and intensive chemotherapy in patients over 60 years of age with acute myeloid leukaemia and activating FLT3 mutations

Acute myeloid leukaemia (AML) with FLT3 mutation has a dismal prognosis in elderly patients. Treatment with a combination of FLT3 inhibitors and standard chemotherapy has not been extensively studied. Therefore, we instigated a phase I/II clinical trial of chemotherapy with cytosine arabinoside (Ara‐C)/daunorubicin induction (7+3) followed by three cycles of intermediate‐dose Ara‐C consolidation in 22 AML patients with activating FLT3 mutations. Sunitinib was added at predefined dose levels and as maintenance therapy for 2 years. At dose level 1, sunitinib 25 mg daily continuously from day 1 onwards resulted in two cases with dose‐limiting toxicity (DLT), prolonged haemotoxicity and hand‐foot syndrome. At dose level ?1, sunitinib 25 mg was restricted to days 1–7 of each chemotherapy cycle. One DLT was observed in six evaluable patients. Six additional patients were treated in an extension phase. Thirteen of 22 patients (59%; 8/14 with FLT3–internal tandem duplication and 5/8 with FLT3‐tyrosine kinase domain) achieved a complete remission/complete remission with incomplete blood count recovery. For the 17 patients included at the lower dose level, median overall, relapse‐free and event‐free survival were 1·6, 1·0 and 0·4 years, respectively. Four out of five analysed patients with relapse during maintenance therapy lost their initial FLT3 mutation, suggesting outgrowth of FLT3 wild‐type subclones.     

Acute myeloid leukaemia blast cells with a tyrosine kinase domain mutation of FLT3 are less sensitive to lestaurtinib than those with a FLT3 internal tandem duplication

FLT3 tyrosine kinase domain mutations (FLT3/TKDs) are associated with a favourable prognosis in acute myeloid leukaemia (AML), unlike FLT3 internal tandem duplications (FLT3/ITDs) that have a poor prognosis. Whilst FLT3/ITD+ cells are more susceptible to the cytotoxic effects of FLT3 inhibitors than wild type (WT) cells, the sensitivity of FLT3/TKD+ cells to therapeutic agents is unclear, as is the importance of the mutant level. We therefore studied the effect of cytarabine and the FLT3 inhibitor lestaurtinib, either alone or in combination, on in vitro survival of blast cells from 36 cases of AML (14 FLT3/WT, 11 FLT3/ITD+ and 11 FLT3/TKD+). All three groups showed similar sensitivity to the cytotoxic effects of cytarabine but FLT3/ITD mutant level was inversely correlated with cytarabine cytotoxicity (P = 0.04) whereas FLT3/TKD mutant level had no impact. FLT3/TKD+ cells showed a similar response to lestaurtinib as FLT3/WT cells, whereas FLT3/ITD+ cells were more sensitive (P = 0.004). There was no correlation between mutant level and lestaurtinib sensitivity for either FLT3/ITD+ or FLT3/TKD+ cells. Synergistic cytotoxicity of lestaurtinib plus cytarabine was demonstrated in all three groups. These results suggest that FLT3/TKD+ and FLT3/WT cases should not be differentiated when considering patients for treatment with FLT3 inhibitors.     

Development of a human acute myeloid leukaemia screening panel and consequent identification of novel gene mutation in FLT3 and CCND3

A study was undertaken to develop an acute myeloid leukaemia (AML) screening panel to uncover novel recurring gene mutations. Analysis was performed on six genes known to be mutated in AML (RUNX1, FLT3, KIT, CEBPA, PTPN11 and NRAS) and an additional two candidate genes (CCND3 and FES) in a panel of 175 primary human AML samples that included all French-American-British types except M3, and all cytogenetic risk groups. One hundred and fifteen mutations were identified in 97 (55%) patients comprising 81 patients (46%) with one mutation, 14 patients (8%) with two mutations and two patients (1%) with three mutations. Fifty-five of 88 (63%) patients with normal karyotype AML had at least one mutation. Correlation was observed between KIT mutation and 'favourable risk' cytogenetics (P <0.001), CEBPA mutation and 'intermediate risk' cytogenetics (P=0.045), and PTPN11 mutation and 'poor risk' disease (P <0.001). The frequency of individual gene mutation was in accordance with previously published studies. Three novel mutations of FLT3 were detected (Y589D, D839G, Y842H) that would have been overlooked by conventional gel electrophoresis. A 51-bp deletion was detected in CCND3 in a patient with normal karyotype AML. This validated panel now provides an important tool to evaluate other candidate genes in the genesis of myeloid malignancy.     

Ponatinib may overcome resistance of FLT3-ITD harbouring additional point mutations, notably the previously refractory F691I mutation

Fms-like tyrosine kinase (FLT3) mutations are the most frequent mutations in patients with acute myeloid leukaemia (AML) that confer a poor prognosis. Constitutively active FLT3-ITD (internal tandem duplications) mutations define a promising target for therapeutic approaches using small molecule inhibitors. However, several point mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) have been identified to mediate resistance towards FLT3 tyrosine kinase inhibitors (FLT3-TKI), including secondary mutations of FLT3. We investigated the cellular effects of the recently characterised FLT3-TKI ponatinib (AP24534) on murine myeloid cells transfected with FLT3-ITD with or without additional point mutations of the FLT3-TKD including the (so far) multi-resistant F691I mutation. Ponatinib effectively induced apoptosis not only in the parental FLT3-ITD cell line but also in all stably transfected subclones harbouring additional FLT3-TKD point mutations (N676D, F691I or G697R). These observations correlated with a strong inhibition of FLT3-ITD and its downstream targets STAT5, AKT and ERK1/2 upon ponatinib incubation, as determined by Western blotting. We conclude that ponatinib represents a promising FLT3-TKI that should be further investigated in clinical trials. The targeted therapy of FLT3-ITD-positive AML with ponatinib might be associated with a lower frequency of secondary resistance caused by acquired FLT3-TKD mutations.     

Serial determination of FLT3 mutations in myelodysplastic syndrome patients at diagnosis, follow up or acute myeloid leukaemia transformation: incidence and their prognostic significance

The incidence of FLT3 mutations (internal tandem duplication and Asp835) was investigated in bone marrow samples from 97 patients with myelodysplastic syndrome [(MDS); excluding cases with refractory anaemia with excess blasts in transformation] at the time of diagnosis and several time points thereafter. Three patients had FLT3 mutations at presentation. Forty-two patients progressed to acute myeloid leukaemia (AML), including the three patients with FLT3 mutations at MDS diagnosis. Three additional patients acquired FLT3 mutations and progressed to AML in 1 month. FLT3 mutations seem to be a critical additional genetic event that transforms a minority of MDS patients to AML.     

Molecular evolution of acute myeloid leukaemia in relapse: unstable N-ras and FLT3 genes compared with p53 gene

Relapse is a major cause of treatment failure in acute myeloid leukaemia (AML), and is usually accompanied by resistance to chemotherapy. To study whether relapse is accompanied by genetic alterations, we compared N-ras, p53 and FLT3 gene mutations in paired samples obtained at initial diagnosis and first relapse. 28 patients with relapsed AML were studied, and their duration of complete remission ranged from 133 to 989 d (mean 318 d). Karyotype changes were observed at relapse in 11 patients. Point mutations of the N-ras gene were positive at both stages (+/+) in three patients, positive at initial diagnosis and negative at relapse (+/-) in three patients, and negative at initial diagnosis and positive at relapse (-/+) in two patients. Internal tandem duplications of the FLT3 gene (FLT3/ITD) were +/+ in five patients, +/- in one patient, and -/+ in six patients. The p53 gene mutations were +/+ in two patients, +/- in one patient, and -/- in 25 patients. FLT3/ITD and mutant p53 at relapse were associated with short survival after relapse. These results indicate that relapse is frequently accompanied by molecular alterations that include the loss and/or acquisition of mutations. Thus relapse can be understood as clonal shift or collateral succession rather than clonal progression.     

FLT3 inhibition in acute myeloid leukaemia

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that appears to play a significant role in leukaemogenesis. Activating mutations of FLT3 are present in approximately one-third of acute myeloid leukaemia patients and are associated with adverse clinical outcome, while many non-mutated cases also show evidence of FLT3 activation. FLT3 thus represents a potentially exciting molecular therapeutic target. A number of small-molecule tyrosine kinase inhibitors with anti-FLT3 activity have been developed and several of these compounds have entered early phase clinical trials where clinical anti-leukaemic activity has been demonstrated. The depth and duration of clinical responses to FLT3 inhibitor monotherapy have been modest, however, and a number of mechanisms by which blasts may acquire resistance have been proposed. Based on preclinical evidence of synergy with conventional chemotherapy, several combination trials are now underway. FLT3 inhibition may also be effective used in combination with other molecularly targeted agents, in postchemotherapy stem-cell-directed maintenance therapy and in MLL-rearranged infant acute lymphoblastic leukaemia.     

The role of FLT3 inhibitors in the treatment of FLT3‐mutated acute myeloid leukemia

FLT3 mutations are present in about one‐third of patients with acute myeloid leukemia (AML). Several FLT3 inhibitors have been used in clinical trials, and these include midostaurin, sorafenib, quizartinib, crenolanib, and gilteritinib. Monotherapy with early tyrosine kinase inhibitors (TKIs) did not have much success; however, later generation agents have shown more promising results. Combination with conventional chemotherapy may have benefit as evidenced by recently presented results, and data from ongoing trials are eagerly awaited. Several trials are also evaluating TKI given after HSCT, and a large international randomized trial is planned. We may be close to an era of targeted therapy where the standard of care for this biologically defined subset will involve incorporation of a FLT3 TKI during induction chemotherapy and after HSCT. It is important that our community continues to collaborate to conduct well‐designed clinical trials to properly define the role of FLT3 TKIs in therapy for FLT3‐mutant AML.     

Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.     

The expression of P-glycoprotein in AML cells with FLT3 internal tandem duplications is associated with reduced apoptosis in response to FLT3 inhibitors

P-glycoprotein (pgp), a membrane efflux pump, is recognized to have an anti-apoptotic function. Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) receptor are the most common mutations in acute myeloid leukaemia (AML). Both ITDs and pgp positivity confer an adverse clinical prognosis. FLT3 inhibitors induce variable apoptosis in cell lines transfected with FLT3 ITDs. We studied the effect of herbimycin A, AG1296 and PKC412 on primary AML blasts. All compounds showed significantly higher cell kill after 48-h incubation in samples with an ITD compared with wild type (Herbimicin P < 0.001; AG1296 P = 0.001, PKC412, P = 0.002). Pgp-positive samples were significantly less sensitive to herbimycin and AG1296 than pgp-negative samples, although neither molecule inhibited the efflux function of pgp. The concurrent incubation with the pgp inhibitor PSC833 resulted in an enhanced cell kill in 4/5 ITD pgp-positive samples versus two of nine ITD pgp-negative samples. PKC412 inhibited pgp function and induced cell death in FLT3 ITD/pgp-positive samples. We conclude that AML samples with a FLT3 ITD are more susceptible to these inhibitors than wild-type samples. However, the expression of pgp in cells with FLT3 ITDs can reduce their sensitivity to FLT3 inhibitors and therefore pgp expression should be assessed in clinical trials of FLT3 inhibitors.     

Mutation spectrum of FLT3 and significance of non-canonical FLT3 mutations in haematological malignancy

Fms-like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non-canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non-canonical FLT3 mutations depending on the affected protein structure: namely non-canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high-frequency (>1%) FLT3-NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3-deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3-ITD mutant of TKD2 had significantly higher kinase activity than wild-type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild-type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non-canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non-canonical mutations.     

Subclinical minute FLT3-ITD clone can be detected in clinically FLT3-ITD-negative acute myeloid leukaemia at diagnosis

Recent advances in next-generation sequencing (NGS) have enabled the detection of subclinical minute FLT3-ITD. We selected 74 newly diagnosed, cytogenetically normal acute myeloid leukaemia (AML) samples in which FLT3-ITD was not detected by gel electrophoresis. We sequenced them using NGS and found minute FLT3-ITDs in 19 cases. We compared cases with clinically relevant FLT3-ITD (n = 37), cases with minute FLT3-ITD (n = 19) and cases without detectable FLT3-ITD (n = 55). Molecular characteristics (location and length) of minute FLT3-ITD were similar to those of clinically relevant FLT3-ITD. Survival of cases with minute FLT3-ITD was similar to that of cases without detectable FLT3-ITD, whereas the relapse rate within 1 year after onset was significantly higher in cases with minute FLT3-ITD. We followed 18 relapsed samples of cases with clinically FLT3-ITD-negative at diagnosis. Two of 3 cases with minute FLT3-ITD relapsed with progression to clinically relevant FLT3-ITD. Two of 15 cases in which FLT3-ITD was not detected by NGS relapsed with the emergence of minute FLT3-ITD, and one of them showed progression to clinically relevant FLT3-ITD at the second relapse. We revealed the clonal dynamics of subclinical minute FLT3-ITD in clinically FLT3-ITD-negative AML. Minute FLT3-ITD at the initial AML can expand to become a dominant clone at relapse.     

Salvage therapy using FLT3 inhibitors may improve long‐term outcome of relapsed or refractory AML in patients with FLT3‐ITD

To determine the long‐term efficacy of FLT3 inhibitors (FLT3i) in the salvage setting for relapsed and refractory (rel/ref) acute myeloid leukemia (AML) with FLT3 internal tandem duplication (AML FLT3‐ITD), we conducted a retrospective study of 120 patients with rel/ref AML FLT3‐ITD who received salvage therapy with either FLT3i‐containing regimen (FLT3i group, N = 45) or conventional cytotoxic regimen (conventional group, N = 75). The median overall survival (OS) after the first salvage in the FLT3i group was 6·9 vs. 4·6 months in the conventional group (P = 0·17). The OS was better in the FLT3i group among patients with initial complete remission (CR) duration ≤12 months or with primary refractory disease (6·9 vs. 3·7 months; P < 0·01). The OS was better when FLT3i was combined with cytotoxic agents versus monotherapy (17 vs. 4·8 months; P = 0·017). Multivariate analysis revealed that the use of FLT3i was an independent predictor of OS (hazard ratio 0·58; 95% confidence interval, 0·38–0·88). Incorporating FLT3i into salvage strategies may improve long‐term outcome of patients with AML FLT3‐ITD. Prospective studies to validate this conclusion are warranted.