FLT3 Internal Tandem Duplication Mutations Research Articles

Quizartinib with donor lymphocyte infusion for post-transplant relapse of FLT3-ITD-positive acute myeloid leukemia.

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-positive acute myeloid leukemia (AML) has a poor prognosis, particularly with DNMT3A and NPM1 mutations. Quizartinib, a FLT3 inhibitor showing clinical benefit in FLT3-ITD-positive AML, has unclear safety and efficacy when combined with donor lymphocyte infusion (DLI). We report a case of FLT3-ITD-positive AML with DNMT3A and NPM1 mutations that relapsed after allogeneic hematopoietic stem cell transplantation (allo-HCT) and was treated with quizartinib and DLI. A 49-year-old man was diagnosed with AML. Target-sequencing analysis of the bone marrow revealed FLT3-ITD, DNMT3A R882, and NPM1 mutations. Although the patient achieved complete remission (CR) through induction therapy and received allo-HCT, he relapsed on day 71. Quizartinib was initiated on day 79, and the patient achieved CR with incomplete recovery on day 106. He did not desire a second allo-HCT and continued quizartinib in combination with DLI, which was started on day 156 and administered eight times every 2 to 3months. The patient achieved hematological CR on day 163 and remained in molecular CR 3years after allo-HCT without adverse effects. Quizartinib combined with DLI may be a feasible treatment for early relapse of FLT3-ITD-positive AML after allo-HCT, even with concurrent DNMT3A and NPM1 mutations.

Concurrent versus sequential or no triazole anti-fungal therapy in patients undergoing 7 + 3 plus midostaurin induction for FLT-3 acute myelogenous leukemia.

Midostaurin is a multikinase inhibitor approved for the treatment of adult patients with newly diagnosed FMS-like tyrosine kinase 3 mutated (FLT3m) acute myeloid leukemia (AML). Azole antifungal medications are commonly used in AML and are known to interact with anti-cancer drugs such as midostaurin through the CYP3A pathway. However, there are no midostaurin related dose modifications recommended with strong CYP3A inhibitors. We retrospectively reviewed 40 patients between 2017-2022 and compared efficacy and safety outcomes in patients who received azole antifungals concurrently to those who did not receive an azole or received it sequentially to midostaurin for treatment of FLT3m AML. Median age of both groups was approximately 55 years and 70% of patients harbored FLT-3 internal tandem duplication mutations. Most patients in the concurrent arm were on either posaconazole (33%) or isavuconazole (50%) for antifungal prophylaxis and micafungin (72%) for the sequential/no azole arm. Overall CR/CRi rate with concurrent versus sequential/no azole were 72% and 77%, and non-hematologic grade 3 toxicities were 22% and 40% (p = 0.21), respectively. Rates of dose reductions (6% vs. 0%, p = 0.26) and held doses (17% vs. 14%, p = 0.79) were not different between concurrent and sequential/no azole. There were no differences in the rates of new fungal infection during induction between the two groups. Azoles given concurrently or sequentially with midostaurin were found to be equally safe and effective in the treatment of newly diagnosed FLT3 AML. Additional confirmatory studies are needed due to our limited sample size.

Genetic risk stratification and outcomes among treatment-naive patients with AML treated with venetoclax and azacitidine

AbstractThe European LeukemiaNet (ELN) acute myeloid leukemia (AML) genetic risk classification systems are based on response to intensive chemotherapy; their ability to discriminate outcomes in older patients treated with venetoclax-azacitidine may be suboptimal. This pooled analysis of the phase 3 VIALE-A trial (NCT02993523) and phase 1b study (NCT02203773) examined prognostic stratification according to the 2017 and 2022 ELN risk classifications and derived new molecular signatures differentiating venetoclax-azacitidine–treated patients based on overall survival (OS). Overall, 279 patients treated with venetoclax-azacitidine and 113 patients treated with placebo-azacitidine were analyzed. The ELN 2017 or 2022 prognostic criteria classified most patients as adverse-risk AML (60.2% and 72.8% for venetoclax-azacitidine and 65.5% and 75.2% for placebo-azacitidine, respectively). Although outcomes with venetoclax-azacitidine improved across all ELN risk groups compared with placebo-azacitidine, ELN classification systems poorly discriminated venetoclax-azacitidine outcomes. By applying a bioinformatic algorithm, new molecular signatures were derived differentiating OS outcomes with venetoclax-azacitidine. The mutational status of TP53, FLT3 internal tandem duplication (FLT3-ITD), NRAS, and KRAS categorized patients into higher-, intermediate-, and lower-benefit groups (52%, 25%, and 23% of patients, respectively), each associated with a distinct median OS (26.5 months [95% confidence interval (CI), 20.2-32.7]; 12.1 months [95% CI, 7.3-15.2]; and 5.5 months [95% CI, 2.8-7.6], respectively). ELN prognostic classifiers did not provide clinically meaningful risk stratification of OS outcomes in patients treated with venetoclax-azacitidine. TP53, FLT3-ITD, NRAS, and KRAS mutation status allows the classification of these patients into 3 risk groups with distinct differences in median OS. These trials were registered at www.clinicaltrials.gov as #NCT02993523 and #NCT02203773.

Intensive induction chemotherapy vs hypomethylating agents in combination with venetoclax in NPM1-mutant AML

AbstractAlthough intensive induction chemotherapy (IC) remains the standard of care for younger patients with acute myeloid leukemia (AML), hypomethylating agents + venetoclax (HMA/VEN) can lead to durable remission among older patients with nucleophosmin 1 (NPM1) mutations. Whether IC or HMA/VEN is superior in patients aged ≥60 years with NPM1-mutant AML is unknown. We performed an international, multicenter retrospective cohort study of 221 patients (147 IC and 74 HMA/VEN) with previously untreated NPM1-mutant AML. Composite complete remission (cCR) (defined as CR + CR with incomplete count recovery) rate was similar for IC and HMA/VEN (cCR, 85% vs 74%; P = .067). Although overall survival (OS) was favorable with IC in unselected patients compared with HMA/VEN (24-month OS, 59% [95% confidence interval (CI), 52-69%] vs 38% [95% CI, 27-55%]; P = .013), it was not statistically different among patients aged 60-75 years (60% [95% CI, 52-70%] vs 44% [95% CI, 29-66%]; P = .069) and patients who received an allogeneic stem cell transplant (70% [95% CI, 58-85%] vs 66% [95% CI, 44-100%]; P = .56). Subgroup analyses suggested that patients with normal cytogenetics (24-month OS, 65% [95% CI, 56-74%] with IC vs 40% [95% CI, 26-60%] with HMA/VEN; P = .009) and without FLT3 internal tandem duplication mutations might benefit from IC compared with HMA/VEN (24-month OS, 68% [95% CI, 59-79%] vs 43% [95% CI, 29-63%]; P = .008). In multivariable analysis, OS was not statistically different between patients treated with IC and HMA/VEN (hazard ratio for death with HMA/VEN vs IC, 0.71; 95% CI, 0.40-1.27; P = .25).

Combining the novel FLT3 and MERTK dual inhibitor MRX-2843 with venetoclax results in promising antileukemic activity against FLT3-ITD AML

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately one third of acute myeloid leukemia (AML) patients. FLT3-Internal tandem duplication (FLT3-ITD) mutations are the most common FLT3 mutations and are associated with a poor prognosis. Gilteritinib is a FLT3 inhibitor that is US FDA approved for treating adult patients with relapsed/refractory AML and a FLT3 mutation. While gilteritinib monotherapy has improved patient outcome, few patients achieve durable responses. Combining gilteritinib with venetoclax (VEN) appears to make further improvements, though early results suggest that patients with prior exposure to VEN fair much worse than those without prior exposure. MRX-2843 is a promising inhibitor of FLT3 and MERTK. We recently demonstrated that MRX-2843 is equally potent as gilteritinib in FLT3-ITD AML cell lines in vitro and primary patient samples ex vivo. In this study, we investigated the combination of VEN and MRX-2843 against FLT3-ITD AML cells. We found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-mutated AML cell lines and primary patient samples. Importantly, we found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-ITD AML cells with acquired resistance to cytarabine (AraC) or VEN+AraC. VEN and MRX-2843 significantly reduce colony-forming capacity of FLT3-ITD primary AML cells. Mechanistic studies show that MRX-2843 decreases Mcl-1 and c-Myc protein levels via transcriptional regulation and combined MRX-2843 and VEN significantly decreases oxidative phosphorylation in FLT3-ITD AML cells. Our findings highlight a promising combination therapy against FLT3-ITD AML, supporting further in vitro and in vivo testing.

Molecular, clinical, and therapeutic determinants of outcome in NPM1-mutated AML

AbstractAlthough NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcomes; however, only FLT3-internal tandem duplication (ITD) mutation and adverse karyotype are currently used for risk stratification because of inconsistent results and uncertainty about how other factors should influence treatment, particularly given the strong prognostic effect of postinduction measurable residual disease (MRD). Here, we analyzed a large group of patients with NPM1 mutations (NPM1mut) AML enrolled in prospective trials (National Cancer Research Institute [NCRI] AML17 and AML19, n = 1357) to delineate the impact of baseline molecular and clinical features, postinduction MRD status, and treatment intensity on the outcome. FLT3-ITD (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.01-1.63), DNMT3A (HR, 1.65; 95% CI, 1.32-2.05), WT1 (HR, 1.74; 95% CI, 1.27-2.38), and non-ABD NPM1mut (HR, 1.64; 95% CI, 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients who achieved MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD-negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin) regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the high-risk molecular subgroups.

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FMS-like tyrosine kinase 3 (FLT3) is a viable and important therapeutic target for acute myeloid leukemia (AML). FLT3 internal tandem duplication (FLT3-ITD) mutations have been identified in approximately 30% of AML patients, and are associated with unfavorable prognosis, higher risk of relapse, drug resistance, and poor clinical outcome. Even FLT3 inhibitors have demonstrated promising efficacy, they cannot cure AML or even significantly extend the lives of patients with FLT3-ITD mutations. This is partly because of poor water solubility, insufficient membrane penetration and short half-life of small molecule inhibitors. Besides, the presence of enzymes like CYP3A4 in bone marrow accelerate the elimination and metabolism of FLT3 inhibitors, resulting in low plasma concentrations and side effects. Here we report the erythrocyte membrane-camouflaged FLT3 inhibitor nanoparticles to enhance FLT3-ITD AML treatment. Briefly, we physically coextruded red blood cell (RBC) membrane vesicles with nanoparticles derived from FLT3 inhibitor F30 to obtain F30@RBC-M, which exhibited comparable potent FLT3-ITD inhibitory effects compared to free F30in vitro, while displaying a higher potent antitumor efficacy in xenograft models due to the prolonged circulation properties. Furthermore, administration of F30@RBC-M significantly extended the survival of mice in a transplanted mouse model than F30 free drug. These findings suggest that RBC membrane-coated nanoparticles derived from FLT3 inhibitors hold promise as a tool to enhance the therapeutic efficacy to treat FLT3-ITD AML.

Azacitidine, Venetoclax, and Gilteritinib in Newly Diagnosed and Relapsed or Refractory FLT3-Mutated AML.

Azacitidine plus venetoclax is a standard of care for patients with newly diagnosed AML who are unfit for intensive chemotherapy. However, FLT3 mutations are a common mechanism of resistance to this regimen. The addition of gilteritinib, an oral FLT3 inhibitor, to azacitidine and venetoclax may improve outcomes in patients with FLT3-mutated AML. This phase I/II study evaluated azacitidine, venetoclax, and gilteritinib in two cohorts: patients with (1) newly diagnosed FLT3-mutated AML who were unfit for intensive chemotherapy or (2) relapsed/refractory FLT3-mutated AML (ClinicalTrials.gov identifier: NCT04140487). The primary end points were the maximum tolerated dose of gilteritinib (phase I) and the combined complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate (phase II). Fifty-two patients were enrolled (frontline [n = 30]; relapsed/refractory [n = 22]). The recommended phase II dose was gilteritinib 80 mg once daily in combination with azacitidine and venetoclax. In the frontline cohort, the median age was 71 years and 73% of patients had an FLT3-internal tandem duplication (ITD) mutation. The CR/CRi rate was 96% (CR, 90%; CRi, 6%). Sixty-five percent of evaluable patients achieved FLT3-ITD measurable residual disease <5 × 10-5 within four cycles. With a median follow-up of 19.3 months, the median relapse-free survival (RFS) and overall survival (OS) have not been reached and the 18-month RFS and OS rates are 71% and 72%, respectively. In the relapsed/refractory cohort, the CR/CRi rate was 27%; nine additional patients (41%) achieved a morphologic leukemia-free state. The most common grade 3 or higher nonhematologic adverse events were infection (62%) and febrile neutropenia (38%), which were more frequent in the relapsed/refractory cohort. The combination of azacitidine, venetoclax, and gilteritinib resulted in high rates of CR/CRi, deep FLT3 molecular responses, and encouraging survival in newly diagnosed FLT3-mutated AML. Myelosuppression was manageable with mitigative dosing strategies.

CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.

Acute myeloid leukemia (AML)patients with FLT3 internal tandem duplication (FLT3-ITD)and DNA methyltransferase 3A (DNMT3A)R882 double mutations had a worse prognosis compared withAML with FLT3-ITDor DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL)in AML with FLT3-ITDand DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITDand DNMT3A-R882double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP).Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-Ccells. An in vivo AML animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosisand repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITDand DNMT3A-R882 double-mutated AML.

Prognostic risk signature in patients with acute myeloid leukemia treated with hypomethylating agents and venetoclax

AbstractHypomethylating agents (HMAs) and venetoclax (Ven) represent the standard of care for patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. However, the European LeukemiaNet (ELN) risk classifications have been validated for patients treated with intensive therapy. In this study, we validate a recently proposed new molecular prognostic risk signature (mPRS) for patients with AML treated with HMAs and Ven. This classification allocated patients to favorable, intermediate (N/KRAS or FLT3–internal tandem duplication mutations), and lower (TP53 mutations) benefit groups. We retrospectively analyzed 159 patients treated with HMA and Ven. The mPRS classification allocated 74 (47%), 31 (19%), and 54 (34%) patients to the higher, intermediate, and lower-benefit groups, respectively. The overall response rate was 71% (86%, 54%, and 59% in the higher, intermediate, and lower-benefit groups, respectively). The median overall survival (OS) and event-free survival (EFS) times were 30 and 19 months, respectively, in the higher-benefit group; 12 and 8 months in the intermediate-benefit group; and 5 and 4 months in the lower-benefit group (P < .001). The C-index for OS and EFS was higher when stratifying patients according to mPRS classification than with the ELN 2022 classification. The 2-year cumulative incidence of relapse was 35%, 70%, and 60% in the higher, intermediate, and lower-benefit groups, respectively (P = .005). The mPRS classification accurately segregated groups of patients with AML treated with HMA plus Ven. In these patients, N/KRAS and TP53 mutations appear to negatively affect outcomes; therefore, new treatment approaches are warranted.

C-Kit and Flt3 mutation status in acute myeloid leukaemia with cytogenetic correlation and prognosis: A series of 75 cases

Recent WHO classification (5 edition) has included Acute Myeloid Leukaemia (AML) with mutations in NPM1, CEBPA as separate entities along with AML with cytogenetic abnormalities in AML with recurrent genetic abnormalities. Even though C-kit and FLT3 mutations in AML have no diagnostic importance, prognostic significance in different subtypes of AML for the presence of these mutations are not the same.To assess the correlation between French American British (FAB) AML classification, specific cytogenetic abnormalities with C-kit, and FLT3 mutation in AML and to evaluate the prognosis and survival of AML patients with respect to cytogenetic abnormalities and C-kit and FLT3 mutation status. Retrospectively all AML cases in which C-kit and FLT3 mutation status was assessed were retrieved; C-kit D816V mutation status had been assessed by Real time PCR; For FLT3 mutation, both Internal tandem duplication (ITD) and D835V mutation status had been assessed using PCR and gel electrophoresis; The data regarding morphological with immunophenotypic diagnosis, conventional karyotyping, FISH for translocation 8;21 (t (8;21)) and inversion16 / translocation16;16 (t (16;16) were also retrieved in all these cases along with follow-up from hospital records. Total 75 cases were included; Male/ Female ratio was 1.21:1 (41/34); Median age was 31 (Range: 2 - 64); 18 cases had translocation 8;21 (t (8;21)); 3 cases showed inversion16 / translocation16;16 (t (16;16); Out of the 18 cases which showed t (8;21), 10 cases had associated loss of sex chromosome. Eight cases had C-kit D816V mutation; three of which had t (8;21) while two had inversion 16. 12 cases had FLT3 mutations among which nine were ITD while three had D835V mutation. On karyotyping, one of these cases showed hyperdiploidy while the majority had normal karyotype. A single case had both C-kit D816V mutation and FLT3 D835V mutation with inversion 16 on karyotyping; Most common type of AML in both cases with FLT3 mutation and C-kit mutation was AML-M2 (FAB); Commonest karyotyping abnormality for cases with C-kit mutation was t(8;21); while for FLT3 mutation, the majority had normal karyotype; The single case which had both C-kit D816V mutation and FLT3 D835V mutation was alive event-free at three-year follow-up. Both FLT3 ITD and TKD mutations had a worse prognosis in our study. However, AML cases with C-kit mutation had a similar prognosis comparable to C kit negative cases. A large-scale study is required to elucidate the significance of this.

The Clinical Utility of FLT3 Mutation Testing in Acute Leukemia: A Canadian Consensus.

FMS-like tyrosine kinase 3 (FLT3) mutations are detected in approximately 20-30% of patients with acute myeloid leukemia (AML), with the presence of a FLT3 internal tandem duplication (FLT3-ITD) mutation being associated with an inferior outcome. Assessment of FLT3 mutational status is now essential to define optimal upfront treatment in both newly diagnosed and relapsed AML, to support post-induction allogeneic hematopoietic stem cell transplantation (alloSCT) decision-making, and to evaluate treatment response via measurable (minimal) residual disease (MRD) evaluation. In view of its importance in AML diagnosis and management, the Canadian Leukemia Study Group/Groupe canadien d'étude sur la leucémie (CLSG/GCEL) undertook the development of a consensus statement on the clinical utility of FLT3 mutation testing, as members reported considerable inter-center variability across Canada with respect to testing availability and timing of use, methodology, and interpretation. The CLSG/GCEL panel identified key clinical and hematopathological questions, including: (1) which patients should be tested for FLT3 mutations, and when?; (2) which is the preferred method for FLT3 mutation testing?; (3) what is the clinical relevance of FLT3-ITD size, insertion site, and number of distinct FLT3-ITDs?; (4) is there a role for FLT3 analysis in MRD assessment?; (5) what is the clinical relevance of the FLT3-ITD allelic burden?; and (6) how should results of FLT3 mutation testing be reported? The panel followed an evidence-based approach, taken together with Canadian clinical and laboratory experience and expertise, to create a consensus document to facilitate a more uniform approach to AML diagnosis and treatment across Canada.

A study of the synthetic route and resistance of gilteritinib

Gilteritinib is used to treat acute myeloid leukemia (AML) carrying FMS-like tyrosine kinase 3 (FLT3) mutations. As it is a new drug, there are still many aspects that need improvement. This article will be written according to the following three points: Analysis of its mechanism of action with the receptor; Analysis of the three main synthetic routes of gilteritinib and reasonable suggestions are given; Causes and solutions to the emergence of drug resistance. The following 4 points have been found that:(1) FLT3-internal tandem duplication (ITD) mutation and FLT3-tyrosine kinase domain (TKD) mutation, leukemic cells' ability to proliferate, differentiate, and survive aberrant changes due to both kinds of mutations. (2) Gilteritinib combines with FLT3's ATP pocket, and the compound shaped from FLT3 to gilteritinib was synthesized by the juxtamembrane (JM) domain. (3) Synthesis route 3 in this paper is most suitable for industrial mass production. (4) Resistance to gilteritinib is divided into primary resistance and secondary resistance, research in the future should focus on finding new therapeutic targets.

Interaction between Presenting Features, Co-Occurring Mutations, MRD and Induction Treatment Influences Outcome in Adults with NPM1 Mutated AML - an Analysis of 1357 Patients in the UK NCRI AML17 and AML19 Studies

Background NPM1 mutated (mut) AML is considered favourable risk unless associated with FLT3 internal tandem duplication (ITD) or adverse karyotype. However many other genetic and clinical risk factors have been identified, including high white cell count (WCC), secondary disease, co-mutations and presence of a “triple hit” genotype ( NPM1mut, FLT3 ITD and DNMT3Amut). Although alternative classification systems have been proposed, few studies have been large enough to robustly evaluate clinical and molecular subgroups within NPM1mut, and none have incorporated the effects of measurable residual disease (MRD) status or treatment intensity. Therefore, clinical decision making remains challenging and inconsistent. Here, we performed comprehensive genotyping and molecular MRD assessment in two consecutive prospective randomised studies to clarify these issues. Methods NCRI AML17 (2009-2014) and AML19 (2015-2020) were large randomised trials for younger adults with newly diagnosed AML. Induction regimens included DA +/- etoposide (ADE) and FLAG-IDA, with or without Gemtuzumab. Patients underwent sequential MRD assessment by RT-qPCR, and diagnostic DNA samples were analysed by targeted NGS. Overall survival (OS) was calculated with the Kaplan-Meier method, while cumulative incidence of relapse (CIR) considered death as a competing risk. Relapse events included MRD relapse. All reported hazard/odds ratios are from multivariable analyses including age, prior haematological malignancy, prior chemo/radiotherapy, WCC (log transformed), cytogenetic risk and mutation profile ( FLT3 ITD, DNMT3A, PTPN11, N/KRAS, TET2, IDH1, IDH2, WT1, TP53 MDS-related genes). Results 1357 patients with NPM1mutAML were identified, 888 in AML17 and 469 in AML19. 1100 patients had DNA available for panel sequencing, and 981 underwent MRD monitoring of whom 737 had an evaluable post course 2 (PC2) peripheral blood (PB) sample. Morphological remission (CR+CRi) was achieved in 1264/1357 patients (93%) and PC2 PB MRD negativity in 594/737 (81%). We confirmed the adverse impact of PB PC2 MRD positivity: 3y CIR was 65% vs 29% in those MRD- (p&amp;lt;0.001), and 3yr OS was 40% vs 79% (p&amp;lt;0.001). For PB PC2 MRD+ patients, CIR was &amp;gt;50% in all subgroups. There was a lower rate of PC2 PB MRD negativity in patients with high presenting WCC (OR 1.14, p=0.05), FLT3 ITD (OR 2.58, p&amp;lt;0.001), DNMT3Amut (OR 2.2, p&amp;lt;0.001) and WT1mut (HR 2.04, p=0.02). There was no effect of FLT3 allelic ratio, while patients with both FLT3 ITD and DNMT3A mutations had the lowest rate of MRD- (64%, Figure A). Of the 594 PC2 MRD- patients, we observed higher 3y CIR in patients with high WCC (HR 1.14, p=0.01), DNMT3Amut (HR 1.83, p=0.001), WT1mut(HR 1.96, p=0.01) and IDH1mut (HR 1.9, p=0.002), and lower CIR in patients with N/KRASmut(HR 0.53, p=0.01). Although presence of FLT3 ITD alone was not associated with CIR (HR 0.88, p=0.7), patients with both FLT3 ITD and DNMT3A mutations had 3y CIR from MRD- of 40% (HR 1.74, p=0.03). MDS-related gene mutations had no impact on achieving MRD-, nor relapse from MRD-. Considering all patients, OS was poorer with increasing age (HR 1.03, p&amp;lt;0.001), WCC (HR 1.07, p&amp;lt;0.001), adverse karyotype (HR 2.48, p=0.01), FLT3 ITD (HR 1.34, p=0.01), DNMT3Amut (HR 1.56, p&amp;lt;0.001) and WT1mut(HR 1.71, p&amp;lt;0.001). 3y OS was 52% in patients with both FLT3 ITD and DNMT3Amut, 63% with FLT3 ITD only, 67% with DNMT3A only and 75% with neither. Considering MRD- patients, only age (HR 1.02, p=0.04), DNMT3Amut(HR 1.75, p=0.007) and WT1mut (HR 1.8, p=0.048) remained associated with OS. There was no benefit to CR1 allogeneic transplant in MRD- patients (HR 0.83, 95%CI 0.51-1.34, p=0.4), including in any subgroups (heterogeneity p=0.9). Compared with other treatments, FLAG-Ida increased MRD- (87% vs 79%, p=0.009), reduced relapse from MRD- (16% vs 34%, p&amp;lt;0.001) and improved 3y OS (79% vs 63%, HR 0.52, p&amp;lt;0.001). The OS benefit of FLAG-Ida was greatest in patients with at least one of the features associated with failure to achieve MRD- and relapse from MRD- (high WCC, triple hit or WT1mut, HR 0.39 95%CI 0.26-0.58, heterogeneity p&amp;lt;0.01). Conclusion The “triple hit” ( NPM1, DNMT3A and FLT3 ITD) genotype, WT1mut and high WCC were associated with poor outcomes, due to a lower probability of achieving MRD-, and a higher relapse risk from MRD- remission. Survival was improved for patients receiving intensified induction with FLAG-Ida including patients in these high-risk groups.

Stag2-Cohesin Loss Attenuates Flt3 ITD Myeloid Blast Expansion Yet Preserves Mutant HSC

FLT3 is mutated through an internal tandem duplication (ITD) in 20-25% of acute myeloid leukemia (AML), driving aberrant STAT5/AKT signaling and leukemogenesis. Murine modelling of ITD showed marked myeloid progenitor expansion and hematopoietic stem cell (HSC) exhaustion in various co-mutant settings. Here we present a Stag2 Δ Flt3 ITD model with stark reversal of these two aforementioned features. STAG2, a cohesin complex member, maintains the integrity of the 3D genome partitioning structure known as topologically structural domains. Loss of Stag2 impairs the access and engagement of key hematopoietic transcription factors such as PU.1 to their target genes. sAML is diagnosed when patients present leukemia with known history of hematological malignancies or chemotherapy/radiotherapy treatment. Comparing to de novo AML, sAML often arises in older patients and harbors a poor prognosis with a 5-year overall survival rate of &amp;lt;30%. Advances in genomic studies found various epigenetic mutations, such as STAG2, are often associated with sAML and AML-myelodysplastic related changes (MRC) subtype. STAG2 mutations are found in 14-20% of sAML cases and is suggested to reside within a dominant clone during the pre-leukemia phase, MDS to sAML transformation, such as with FLT3 ITD acquisition and persists during remission. To understand the mechanistic contribution of STAG2-cohesin loss with FLT3 ITD, we generated sequential mutagenesis murine models where the order of Stag2 and Flt3 ITD mutation is set as either ITD 1st Stag2 2nd ( de novo like) or Stag2 1stITD 2nd (sAML like) using tamoxifen-inducible Cre/Flpo recombinase or pIpC-inducible Mx1Cre. In the de novo like model, ITD is constitutively active then Stag2 is deleted when mice reach 6-8 weeks of age. Surprisingly, loss of Stag2 attenuates LSK to MP transition at 4 weeks post deletion, while MPP3 remains elevated, suggesting aberrant remodeling of myeloid differentiation. In the sAML like model, Stag2 is deleted via Mx1Cre and waited for 4 months to mimic the MDS phase, which is then followed by activation of ITD mutation via Flpo, which represents the MDS to sAML transformation. After activating both mutations, mice were followed for another 4 months before analyzing the hematopoietic stem and progenitor compartment. In contrast to the de novo like model, sequential Stag2 1stITD 2nd preserves the HSC population defined by either immunophenotyping or transcriptome via scRNAseq ( Figure 1). The mutant HSC is more quiescent but retains the capacity to reconstitute lethally irradiated recipients in the short term. Similar to the de novo like model, sequential Stag2 1stITD 2nd mice also exhibits a blocked myeloid differentiation. Comparing to ITD mutant, Stag2 1stITD 2nd LSK cells have decreased expression of Socs2 and Cish. While functionally determining the role of mutant HSC, we are performing RNA-seq during at early timepoints post ITD activation to determine how preceding Stag2 mutation could have altered the stem cell fate decision. Targeted therapy with inhibitors of FLT3 have had an overall survival benefit in FLT3-mutant AML, though the magnitude of effect has been modest. STAG2 mutations are more likely to be identified in poor responders to FLT3 inhibition as both reported by us with Pexidartinib treatment, as well as in the setting of Crenolanib treatment where expansion of the STAG2-mutant clone was observed during treatment. Thus, remodeling of the chromatin landscape though altered CTCF binding or cohesin function might impact leukemia identity in FLT3-mutant AML. Our data highlights an important regulatory role of Stag2-cohesin in Flt3 ITD mediated leukemogenesis, while generating a model that mimics the genetic evolution of sAML. This model will not only shed light on the sAML pathogenesis but also with creates a pre-clinical testing platform with potential therapeutic relevance.

A Phase I/II Trial of the FLT3 Kinase Inhibitor XY0206 in Patients with Relapsed/Refractory Acute Myeloid Leukemia

Abstract Background: The FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) patients, which are associated with worse survival and higher relapse risk. XY0206 is a novel oral FLT3 inhibitor with preclinical activity against FLT3 internal tandem duplication mutations (FLT3-ITD). The Phase Ⅰ/Ⅱ clinical trail with XY0206 (NCT04471064) is currently underway to explore the initial efficacy, safety and pharmacokinetic (PK) of XY0206 in patients with relapsed/refractory AML (R/R AML). Methods: This open-label, multicenter, dose-escalation and dose-expansion Phase Ⅰ/Ⅱ trail enrolled subjects from August 2020 to September 2022 who were aged ≥18 years with R/R AML. Six dose-escalation or dose-expansion cohorts (12.5, 25, 37.5, 50, 62.5 mg per day, or 25 mg twice a day) were set up to explore the safety/tolerability and antileukemic activity of XY0206, and to determine the maximal tolerated dose (MTD) of XY0206. Safety and tolerability were assessed by monitoring dose-limiting toxicities (DLT), treament-emergent adverse events (TEAE) and adverse drug reaction (ADR). Cohort expansion was based on safety and antileukemic activity. Results: As of December 14, 2022, a total of 61 subjects were enrolled (37 with FLT3 mutation-positive). Demographics and baseline characteristics of patients are presented in Table 1. MTD of XY0206 was established as 50 mg when two of the three patients enrolled in the 62.5 mg dose-escalation cohort could not tolerate continuous medication. The 37.5 mg, 50 mg and 25 mg BID dose cohorts were further expanded. Antileukemic activity was assessed in the Full Analysis Set (FAS, n=61), with overall response rate (ORR) was 34.4% (n=21/61), composite complete remission (CRc) was 32.8% (n=20/61), complete remission (CR) was 6.6% (n=4/61) and CR with partial hematologic recovery (CRh) was 9.8% ( n=6/61 ). Among the patients with FLT3 mutation-positive (FLT3 mut+, n= 37), ORR was 48.6%(n=18/37), CRc was 45.9% ( n=17/37), CR was 2.7% (n=1/37) and CRh was 16.2% (n=6/37). Among the patients with FLT3-ITD mutation (n=30), ORR was 56.7% (n=17/30), CRc was 53.3% (n=16/30), CR was 3.3% (n=1/30) and CRh was 20%( n=6/30). The 37.5 mg dose cohort as the target dose was further expanded to include FLT3 mut+ patients only. CRc in FLT3 mut+ patients ( n= 19 ) and FLT3-ITD mutation patients (n=16) was 57.9% (n=11/19) and 68.8% (n=11/16 ), CR was 5.3% (n=1/19) and 6.3% (n=1/16), CRh was 26.3% (n=5/19 ) and 31.3% (n=5/16), respectively. In terms of safety, the overall incidence and severity of TEAE/ADR in 61 subjects did not correlate with dose significantly. The most common grade 3 and above adverse events (≥ 50% ) were leukopenia (65.6% ), thromobocytopenia (63.9%), neutropenia (62.3%), animia (52.5%) and hypokalemia (50.8%). The fatal adverse event was disease progression, which was mostly related to AML. Conclusion: XY0206 is a novel FLT3 inhibitor that showed a good safety profile, a potent anti-leukemic activity and improved efficacy in patients with FLT3 mut+ R/R AML.

Measurable Residual FLT3-ITD before Allogeneic Transplant for Acute Myeloid Leukemia

Background: In the past year, multiple studies have demonstrated the drastically increased risk of relapse and death in patients with Acute Myeloid Leukemia (AML) who have persistent FLT3 internal tandem duplication (ITD) mutations detectable in clinical remission by next generation sequencing (NGS). Translating this evidence into a change in clinical practice requires both widely available testing and a clear understanding of the clinical implications of FLT3-ITD detection at defined threshold levels. In the largest of the recent studies (Pre-MEASURE, PMID: 36881031), we performed FLT3-ITD MRD detection in blood DNA collected from 608 patients at the time of first remission (CR1) prior to allogeneic hematopoietic cell transplant (alloHCT). To evaluate cross-assay comparability and the impact of residual disease burden levels, we performed analysis on this cohort using a commercially available FLT3-ITD MRD NGS assay. Methods: Of the 608 patients aged 18 or older from the Pre-MEASURE study who received an alloHCT for FLT3-ITD mutated AML during CR1 at a CIBMTR reporting site in the USA between the years of 2013-2019, patients not experiencing non-relapse mortality and with sufficient DNA from pretransplant blood available were included in this analysis. A commercially available testing kit (IVS, Invivoscribe, San Diego, CA), which can detect FLT3-ITD MRD by NGS for up to 21 patients on one Illumina MiSeq flow cell, was used to establish a workflow following clinical testing standards and validated to detect ITDs down to a variant allele fraction (VAF) of at least 0.005%. Results were compared to those previously reported using an anchored multiplex PCR-based (AMP) targeted NGS assay. Overall survival (OS) and cumulative incidence of relapse were estimated with the day of transplant as time 0 using Kaplan-Meier estimation (log-rank tests) and Cox proportional hazards models. Results: A total of 451 patients were included in this study, of which 173 (38%) experienced relapse (median: 5.2 months; range 0.7-52.1) and 133 (29%) died (median: 10.7 months; range 2-53.8) post-alloHCT. The IVS assay identified 232 FLT3-ITD variants with a median VAF of 0.005% (range 0.0002-44%) and length of 51bp (range 5-228). IVS positive patients had a median of 1 FLT3-ITD variant (range 1-15) and median maximum VAF of 0.01% (range 0.0003-44%). Utilizing the same VAF cutoff of 0.01% for MRD positivity for the IVS test as used in the Pre-MEASURE study, patients testing positive prior to alloHCT exhibited higher rates of relapse (79% vs 31%, p&amp;lt;0.0001) and decreased survival (29% vs 77%, p&amp;lt;0.0001) at 3 years compared to testing negative. These results closely align with those previously reported using the AMP assay (Figure A). The IVS test has a superior limit of detection compared with the AMP test, allowing investigation of alternative VAF thresholds. In total, 151 patients (33.5%) were FLT3-ITD MRD positive by IVS of which only 75 (17%) had VAF ≥0.01%. A threshold-free approach for the IVS test improved the ability to identify patients at risk of relapse, predicting 63% of relapses within 6 months of transplant compared to 43% with the 0.01% cutoff, but resulted in decreased specificity as 42% of all positive patients relapsed within 6 months vs. 57% with the 0.01% VAF cutoff. In multivariable analysis, IVS FLT3-ITD MRD status was associated with both relapse and overall survival (Figure B). Additionally, stratifying patients by their level of residual FLT3-ITD disease burden prior to alloHCT revealed a dose-dependent increase in risk for death (Figure B) and relapse. Any detectable level of residual FLT3-ITD over 0.001% VAF exhibited increased risk, with VAFs ≥0.01% being the highest. Conclusions: In this analysis of patients with AML from the Pre-MEASURE study, we show cross-assay evidence that the detection of residual FLT3-ITD variants in pre-transplant blood during CR1 is associated with significantly increased risk of relapse and death after alloHCT. The highly sensitive assay used in this study could identify ~2/3 of patients who relapsed within 6 months and demonstrated that a dose-dependent correlation between residual FLT3-ITD level and adverse clinical outcomes exists, with VAF ≥0.01% representing the highest risk. This evidence provides strong justification for NGS-based AML MRD clinical testing for persistent FLT3-ITD prior to alloHCT to identify patients at high risk of post-transplant relapse and death.

Discovery of LWY713 as a potent and selective FLT3 PROTAC degrader with in vivo activity against acute myeloid leukemia

Fms-like tyrosine kinase 3 (FLT3) has been validated as a therapeutic target for acute myeloid leukemia (AML). While a number of FLT3 kinase inhibitors have been approved for AML treatment, the clinical data revealed that they cannot achieve complete and sustained suppression of FLT3 signaling at the tolerated dose. Here we report a series of new, potent and selective FLT3 proteolysis targeting chimera degraders. The optimal compound LWY713 potently induced the degradation of FLT3 with a DC50 value of 0.64 nM and a Dmax value of 94.8% in AML MV4-11 cells with FLT3-internal tandem duplication (ITD) mutation. Mechanistic studies demonstrated that LWY713 selectively induced FLT3 degradation in a cereblon- and proteasome-dependent manner. LWY713 potently inhibited FLT3 signaling, suppressed cell proliferation, and induced cell G0/G1-phase arrest and apoptosis in MV4-11 cells. Importantly, LWY713 displayed potent in vivo antitumor activity in MV4-11 xenograft models.

Real-World Data for FLT3-Mutated Acute Myeloid Leukemia Patients Treated in Resource-Constrained Settings

Background: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a well-established prognostic marker in acute myeloid leukemia (AML), whereas the impact of mutations in the Tirosine-Kinase Domain (TKD) is controversial. The association of TK inhibitors (TKIs) with intensive chemotherapy improved the overall survival (OS) of FLT3-mutated AML patients in randomized clinical trials and is recommended by the European LeukemiaNet panel of experts. Nevertheless, TKIs are not available for most patients treated in Low- and Middle-Income Countries (LMIC) and there is a scarcity of data concerning the outcome of treatment in these resource-constrained settings nowadays. Methods: This is a real-world study encompassing newly diagnosed adults with FLT3-mutated AML intensively treated at public hospitals in Brazil between January 2015 and December 2020, who were registered in the International Consortium on Acute Leukemias (ICAL) ICAML2015 Study. No patient received TKIs as part of the treatment. FLT3 mutation was tested in diagnostic samples by fragment analysis method. We used the Adapted Genetic Risk (AGR) score reported by Silveira et al. (2020), which is an adaptation of the European Leukemia Net (ELN) 2017 classification. The primary endpoints were event-free survival (EFS) and OS, calculated by Kaplan-Meier method. Relapse rate was calculated through competing risk analysis. Results: Overall, 350 pts (median age: 51y; 52.8% females) were included. FLT3-ITD and -TKD mutations were detected in 17.5% and 7.2% of the patients, respectively. Ninety patients (35.1%) were classified in the favorable risk group, 110 (42.9%) in the intermediate, and 56 (21.9%) in the adverse risk group, according to the AGR. The most frequent co-mutation was in the NPM1 gene. The remaining features are summarized in Table 1. Seventy-eight patients underwent allogeneic stem-cell transplantation (HSCT) in first complete remission (22.2%), of whom 27 had FLT3-ITD (i.e. 43.9% of patients harboring FLT3-ITD underwent HSCT). With a median follow-up period of 3.5 years, the median 4-year OS rate was 24.2% (95% CI 19.8-30.1), and the 4-y EFS 22.9% (95% CI 18.6-28.2). When only patients with FLT3-ITD mutations were analyzed, the 4-y OS rate was 14.4% which was inferior to that of patients with wild-type FLT3 (27.5%; p=0.057) (Figure 1). The cumulative incidence of relapse at four years was 39.4% for patients with FLT3-ITD and 43.9% for those with wildtype FLT3 (p=0.5), while the non-relapse mortality (NRM) at four years was 28.1% and 32.4%, respectively. The four-year OS rate for patients with FLT3-ITD mutations who underwent HSCT was 49.1% (95% CI 38.9-62.1). Patients with FLT3-ITD were further subdivided according to the allelic ratio (AR) in those with low (&amp;lt;0.5) and high (³ 0.5), and no significant difference in OS was detected between the two groups. Of note that patients harboring FLT3-TKD mutations showed a better overall survival rate compared to those with wild-type FLT3 (HR 0.49, 95% CI 0.26-0.93, p=0.029), but this result needs to be interpreted cautiously due the small size of the cohort. Conclusion: our results show that the frequency of FLT3-ITD mutations and their association with worse prognosis was similar to those described in high-income countries previously to the introduction of TKI as the standard of care. Therefore, allogeneic HSCT remains a valid consolidation strategy for FLT3-mutated patients. However, less than half of the patients for whom HSCT was indicated were actually transplanted. Additionally, the allelic ratio of FLT3-ITD should no longer be considered a prognostic marker for intensively treated patients not receiving FLT3 inhibitors.

Idarubicin Versus Daunorubicin Versus Mitoxantrone for Induction Chemotherapy in Acute Myeloid Leukemia: Patient Registration Study of Turkish Society of Hematology-Acute Myeloid Leukemia Working Group

Introduction: Standard induction chemotherapy in newly diagnosed acute myeloid leukemia (AML) includes a combination of standard-dose cytarabine and an anthracycline (idarubicin [IDA], daunorubicin [DNR], or mitoxantrone [MXR]). Anthracycline selection is still not standardized, and the impact of anthracycline selection on survival and response in patients with adverse genetic risk is of interest. The study aimed to retrospectively compare the effectiveness of induction regimens with respect to anthracycline preference. Methods: The study population was recruited from the Turkish AML Registry project database based on documentation of newly diagnosed AML and induction chemotherapy with IDA, DAU, or MXR in combination with cytarabine. Patients diagnosed before 31 August 2022 were received typical “7 + 3” induction chemotherapy containing 3 days of IDA, DAU, or MXR. Results: The study included 317 newly diagnosed AML patients (median age 51; range, 18 - 79; 55.2% male). Of the patients, IDA was administered in 266 (83.9%), DNR in 42 (13.2%), and MXR in 9 (2.8%). The three anthracycline treatment groups were similar between age, gender, type of AML by the World Health Organization (WHO), and risk classification by 2017 European LeukemiaNet (ELN) when evaluated according to the baseline disease characteristics. At the time of diagnosis, although the median white blood cell levels were 19 x 10 9/L (0.4 - 355) in the IDA, 7.8 x 10 9/L (0.7 - 357.2) in the DAU, and 5.5 x 10 9/L (0.6 - 34) in the MXR therapy group (p = 0.019); the hemoglobin and platelet levels and bone marrow blast percentage were similar between the treatment groups. FMS-like tyrosine kinase-3 internal tandem duplication ( FLT3-ITD) (p = 0.561) and Nucleophosmin 1 gene ( NPM1) (p = 0.430) mutation frequencies were also similar between treatment groups. Morphological complete remission (CR) was documented in 65% of all evaluable patients. Among the patient groups, CR was 67.7% for IDA, 50% for DNR, and 55.6% for MXR (p = 0.081). In univariate analysis (Table 1), significant predictors of overall survival (OS) were age ≥60 years (p &amp;lt;0.001), acute leukemias of ambiguous lineage (p &amp;lt;0.001), presence of disseminated intravascular coagulation (DIC) (p = 0.012), and FLT3-ITD mutation (p = 0.018); no significant interaction was noted between NPM1 mutation and OS (p = 0.059). Additionally, when idarubicin was taken as a reference, there was no meaningful relationship between anthracycline type and OS (p values for daunorubicin and mitoxantrone were 0.145 and 0.356, respectively). In multivariate analysis (Table 1), age ≥60 years (p = 0.001), acute leukemias of ambiguous lineage (p = 0.007), and the presence of DIC (p = 0.040) remained significant. After a median follow-up of 29.8 months, 32.5% relapse cases were documented and similarly distributed among IDA (33.1%), DNR (31%), and MXR (22.2%) therapy groups (p = 0.771). During the same period, allogeneic hematopoietic stem cell transplantation (AHSCT) was documented in 10.9% and 26.2% of patients treated for IDA and DNR, respectively. No patient progressed to AHSCT in the MXR arm (p &amp;lt;0.001). In the study, 142 (44.8%) deaths were reported, and there was a 48.5%, 26.2%, and 22.2% mortality rate between the IDA, DNR, and MXR treatment groups, respectively. There was no significant difference in OS by anthracycline type in both the study cohort and age group &amp;lt;60 years (Figures 1A and 1B). There was also no significant difference in OS in patients with the adverse genetic risk group and &amp;lt;60 years age group with adverse genetic risk, according to 2017ELN (Figures 1C and 1D). The results may have been influenced by the number of patients in the treatment groups of the retrospective study and by differences in fitness levels that needed to be measured systematically. Conclusions: We are encouraged that our findings mostly agree with previously published studies and that IDA, DNR, and MXR offer comparable survival values in the research and the younger patient groups. In the future, the ever-changing treatment environment in patient groups with targetable mutations makes choosing the optimal anthracycline for induction chemotherapy in AML much more difficult.