FLT3 Internal Tandem Duplication Research Articles

Bmf-500: An Orally Bioavailable Covalent Inhibitor of FLT3 with High Selectivity and Potent Antileukemic Activity in FLT3-Mutated AML

Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) are the most frequent genetic alteration in AML and are associated with poor prognosis. Though several FLT3 inhibitors have entered clinical trials and reached commercialization, adverse events and dose-limiting toxicities often restrict the therapeutic window and limit their long-term use. Such limitations impact the ability to achieve long-lasting response in patients and ultimately result in therapy-induced resistance. Exquisite potency combined with high selectivity and improved safety profile is expected to help improve tolerance and overall treatment outcomes of FLT3-targeted therapy. BMF-500 was designed as a highly potent and selective, covalent, small molecule inhibitor of FLT3, that binds irreversibly to a reactive cysteine in the kinase active site. BMF-500 is a picomolar inhibitor with markedly improved potency over gilteritinib, a reversible inhibitor of FLT3. BMF-500 selectively killed AML cells harboring FLT3 activating mutations, including MV4-11 and MOLM-13, and engineered cells expressing FLT3 internal tandem duplications (FLT3-ITD) and/or FLT3 tyrosine kinase domain (TKD) mutations. In ex vivo cultures, BMF-500 as a single agent induced potent growth inhibition of patient-derived AML cells harboring either FLT3-ITD or FLT3 non-ITD mutations. The potent covalent inhibition of FLT3 by BMF-500 manifested in effective and durable cellular response that was improved over gilteritinib. For example, a 3-hour exposure followed by wash-out of BMF-500 outperformed 4 days of continuous exposure to gilteritinib, at all concentrations tested. In cells harboring FLT3 activating mutations, BMF-500 induced dose-dependent inhibition of FLT3 phosphorylation and downstream signaling, including phospho-STAT5 and phospho-ERK. A 1-hour pulse treatment with BMF-500 was sufficient to achieve deep and durable target inhibition for greater than 24 hours, an effect not observed with gilteritinib under similar conditions. Profiling BMF-500 across a broad panel of kinases and key cell-surface receptors revealed high selectivity for FLT3 mutants and selectivity against cKit. Potent FLT3 inhibition and high selectivity of BMF-500 translated to sustained tumor regression and improved survival in both subcutaneous and disseminated xenograft models of mutant FLT3-driven AML. Orally administered BMF-500 was well tolerated over 4 weeks of dosing. Study results including efficacy and PD response will be presented. Collectively these data demonstrate BMF-500 to be a novel FLT3 inhibitor with best-in-class potential, given its efficacy, durability, and selectivity in comparison to existing FLT3 inhibitors.

V-FAST Master Trial: Subgroup Analysis of Outcomes with CPX-351 Plus Midostaurin in Adults with Newly Diagnosed Acute Myeloid Leukemia By FLT3 Mutation Type

CPX-351 (United States: Vyxeos®; European Union/United Kingdom: Vyxeos® liposomal), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in patients aged ≥1 year in the United States and in adults in the European Union/United Kingdom. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7+3 chemotherapy, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin (MID). V-FAST (Vyxeos - First Phase Assessment with Targeted Agents) is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial (NCT04075747) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (MID, venetoclax, enasidenib). Eligible adults in the CPX-351 + MID cohort were aged 18 to 75 years, had newly diagnosed AML (de novo AML or secondary AML) with a FLT3 mutation, were fit for intensive chemotherapy, and had an ECOG performance status of 0 to 2. The dose-exploration phase (3+3 design) determined a recommended phase 2 dose of CPX-351 100 units/m2 (daunorubicin 44 mg/m2 + cytarabine 100 mg/m2) on Days 1, 3, and 5 + MID 50 mg twice daily on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose. Herein, we report outcomes for a subgroup analysis based on the presence of a FLT3 internal tandem duplication (ITD) and/or tyrosine kinase domain (TKD) mutation within the cohort of adults primarily with de novo AML who were treated with CPX-351 + MID in the V-FAST trial. A total of 23 patients had sufficient data available to be included in the general safety and efficacy analyses in this ongoing trial at the time of the data cutoff on March 22, 2022. Of the patients included in this analysis, 18 patients had a FLT3 ITD mutation and 6 patients had a FLT3 TKD mutation; 1 patient had both FLT3 mutation types and was included in both subgroups. Patient baseline characteristics are shown in the Table. A diagnosis of de novo AML was reported for 78% of patients with FLT3 ITD mutation and all (100%) patients with a FLT3 TKD mutation. Treatment-emergent adverse events (TEAE) occurring in >50% of patients with a FLT3 ITD or TKD mutation, respectively, included febrile neutropenia (78%; 83%), nausea (67%; 67%), alanine aminotransferase increased (61%; 33%), leukopenia (61%; 50%), and thrombocytopenia (56%; 67%). All patients experienced a ≥3 grade TEAE, primarily hematologic events and infections. Serious TEAEs were reported for 6 (33%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation; no serious TEAE was reported more than once. Four (22%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation died during the study; there were no deaths on or before study Day 60. Complete remission (CR) was achieved by 14/17 (82%) patients with a FLT3 ITD mutation and by 5/6 (83%) patients with a FLT3 TKD mutation (Figure). Among patients in CR and with a measurable residual disease (MRD) assessment by multicolor flow cytometry, MRD negativity was achieved by 5/10 (50%) patients with a FLT3 ITD mutation and 1/3 (33%) patients with a FLT3 TKD mutation, all after the first induction cycle. Median (interquartile range) recovery times to an absolute neutrophil count ≥500/μL and platelet count ≥50,000/μL, respectively, were 35 (34, 43) and 34 (28, 36) days for patients in CR with a FLT3 ITD mutation and 30 (29, 35) and 30 (29, 40) days for those in CR with a FLT3 TKD mutation. A total of 10/18 (56%) patients with a FLT3 ITD mutation and 2/6 (33%) patients with a FLT3 TKD mutation have proceeded to hematopoietic cell transplantation to date. In conclusion, preliminary results from the V-FAST trial suggest the combination of CPX-351 + MID is feasible with a manageable safety profile and promising remission rates in adults with newly diagnosed, FLT3-mutated AML. Although conclusions are limited by the small numbers of patients in each subgroup, results are generally consistent for patients with FLT3 ITD and TKD mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes of Patients with NPM1+ Acute Myeloid Leukemia (AML) Enrolled in the CONNECT® Myeloid Disease Registry

Background Mutations in NPM1 (NPM1+) occur in ∼30% of patients with AML. NPM1+ has been associated with favorable outcomes following intensive chemotherapy (IC) and demonstrated positive prognostic value in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) mutations. Recently, several studies have reported differences in outcomes in patients with NPM1 mutations based on age and treatment. In this analysis we evaluated treatment patterns and outcomes in AML based on NPM1 mutational status and primary induction therapy in patients who were enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011). Methods Patients aged 55 years or older with AML were grouped by known NPM1 status (NPM1+ vs NPM1‒) and induction therapy (non-IC vs IC). Overall survival (OS) and event-free survival (EFS) were evaluated using the Cox model adjusted for age. For the EFS analyses, an event was defined as death, disease progression, or refractory to therapy. Results A total of 483 patients with known NPM1 status were assessed (NPM1+, n = 123; NPM1‒, n = 360). Most patients were male (NPM1+, 61.5%; NPM1‒, 60.7%) and white (NPM1+, 78.0%; NPM1‒, 85.6%; Table). Median age, Eastern Cooperative Oncology Group performance status (ECOG PS), and frailty group were similar in the NPM1+ and NPM1‒ groups. Favorable and adverse European LeukemiaNet (ELN) risk scores at baseline were different in the NPM1+ and NPM1‒ groups. The majority of patients received non-IC regimens as induction therapy (NPM1+, 69.1%; NPM1-, 75.0%). Overall, patients with NPM1+ AML had significantly longer median OS (26 mo) compared with patients with NPM1- AML (15 mo) (HR [95% CI]: NPM1+ vs NPM1- 0.58 [0.44-0.76]; P < 0.01). When analyzed by type of induction regimen (non-IC vs IC), although OS remained numerically longer for patients in the NPM1+ groups, the differences were not statistically significant. Among patients who received non-IC regimens, median OS was 19 and 14 mo in the NPM1+ and NPM1- groups, respectively (Figure). Among patients who received IC regimens, median OS was 24 and 16 mo in the NPM1+ and NPM1- groups, respectively. There was a significant improvement in EFS for patients in the NPM1+ group vs the NPM1- group (20 mo vs 11 mo; hazard ratio [HR] [95% CI], NPM1+ vs NPM1- 0.61 [0.47-0.80]; P < 0.01). This was also true for patients treated with non-IC regimens (15 vs 11 mo in the NPM1+ and NPM1- groups, respectively; HR [95% CI], NPM1+ vs NPM1- 0.67 [0.46-1.00]; P = 0.048). In contrast with the non-IC treated regimens, in patients treated with IC regimens, NPM1+ status was not associated with significantly longer EFS compared to NPM1- status. Patients with known NPM1 status were further evaluated for additional mutations and the following differences were noted: the frequency of mutations in tumor protein p53 (TP53) (1.6% vs 18.6%), RUNX1 (0.8% vs 14.8%), ASXL1 (2.4% vs 17.6%) IDH2 (8.9% vs 15.7%), and NRAS/KRAS (8.9% vs 14.3%) was lower in patients with NPM1+ vs NPM1- AML, respectively, whereas the frequency of mutations in FLT3-ITD (35.8% vs 17.1%), FLT3- tyrosine kinase domain (TKD) (14.6% vs 6.2%), DNMT3A (23.6% vs 15.7%), and IDH1 (14.6% vs 8.6%) was higher in patients with NPM1+ vs NPM1- AML, respectively. Conclusions Overall, patients with NPM1+ AML had longer OS and EFS than patients with NPM1‒ AML. This was observed among patients with NPM1+ AML for both non-IC and IC induction regimens. Patterns of co-mutations differed between the NPM1+ and NPM1- groups and is a likely contributing factor to differences between outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Risk Stratification Model for Cytogenetically Normal Acute Myeloid Leukemia with FLT3-ITD Mutation

Introduction Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are observed in about 30 % of patients with newly diagnosed cytogenetically normal acute myeloid leukemia (CN-AML) and has now been acknowledged to be associated with a poor survival. However, long-term follow-up revealed a high degree of heterogeneity in the clinical prognosis of these patients. Further risk stratification to identify high-risk patients is urgently needed. Methods A total of 106 patients with CN-AML and FLT3-ITD mutations who visited the People's Hospital of Peking University from 2007-2019 were included, and target region sequencing was performed by 266 gene panel. Patients were randomly divided into training and validation cohort according to the ratio of 7:3. Molecular and clinical data in training cohort were incorporated to construct a nomogram model. Cumulative incidence of relapse (CIR), Relapse-free survival (RFS) and survival by allogenic hematopoietic stem cell transplantation (allo-HSCT) and consolidation chemotherapy were compared. Results Clinical variables, genes, and genetic groups with mutations occurring in more than 10 patients were enrolled for univariate analysis of RFS (allo-HSCT was recorded as a censored event). And to avoid bias patients with RFS ≤ 4.57 months (median interval from complete remission to allo-HSCT) would be excluded from the subsequent analyses unless receiving an allo-HSCT was treated as a censored event. Three variables were eventually entered into the nomogram analysis: DNMT3A mutation (+ vs. -), minimal residual disease detected by multiparameter flow cytometry (MPFC-MRD) at the end of the 2nd course of consolidation chemotherapy (CON2) (+ vs. -, represents ≥ vs. ＜0.01%), typical ITDs (+ vs. -, represents ITDs have insertion sequences that exactly match wild-type FLT3 gene vs. ITDs have insertion sequences containing other exogenous nucleotides). And the model demonstrated good discrimination in both the training and validation cohort with C-index value of 0.82 (95% confidence interval, 0.7-0.86), 0.86 (95% confidence interval, 0.76-0.95), respectively. The Nomogram model was constructed as: Score = 1.44×DNMT3A mutation+2.16×MFPC-MRD-0.94×typical ITDs (Figure 1, A). According to the variables in nomogram model, patients with risk scores ≤ 0 were assigned to the low-risk subgroup (N = 39) and the rest were assigned to the high-risk subgroup (N = 67). The 5-year RFS was significantly higher in the low-risk group than in the high-risk group (training cohort, HR 3.68 [1.16-11.66], P = 0.002; validation group, HR 4.45 [1.52-13.00], P = 0.03) (Figure 1, B-C). We then interrogated the effect of consolidation chemotherapy and allo-HSCT as post-remission therapies in the two risk subgroups. Allo-HSCT did not further improve the prognosis of patients in the low-risk group (allo-HSCT vs. chemotherapy, 5-year CIR, HR 0.46 [0.11-1.93], P = 0.29; 5-year RFS, HR 0.41 [0.09-1.86], P = 0.24; 5-year survival, HR 0.91 [0.18-4.68], P = 0.91) (Figure 2, A-D). In contrast, in the high-risk group of patients, transplantation showed a significant survival benefit (5-year CIR, HR 0.21 [0.06-0.74], P = 0.01; 5-year RFS, HR 0.23 [0.06-0.80], P = 0.01; 5-year survival, HR 0.25 [0.06-1.11], P = 0.05) (Figure 2, E-H). Conclusion Our study suggests that risk stratification by our novel nomogram scoring system can identify patients at high risk of relapse in CN-AML with FLT3-ITD mutations and allo-HSCT further benefits their prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antileukemic Activity of Ypn-005, a CDK7 Inhibitor, Inducing Apoptosis through c-MYC and FLT3 Suppression in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is an aggressive blood cancer with a high rate of relapse associated with adverse survival outcomes, especially in elderly patients. An aberrant expression of cyclin-dependent kinase 7 (CDK7) is associated with poor outcomes and CDK7 inhibition has showed antitumor activities in various cancers. In this study, we explored the potential efficacy of YPN-005, a highly selective CDK7 inhibitor, and evaluated a possible mechanism of the antileukemic effect of YPN-005 in vitro, in vivo, and ex vivo AML models. Methods We investigated the efficacy of YPN-005 in AML cell lines, xenograft mouse model, and primary AML cells. The anti-proliferative effect of YPN-005 was measured by cell viability assay using CellTiter-Glo assay, and the apoptotic level was evaluated using Annexin V/PI staining. The phosphorylation of RNA polymerase II, c-MYC, MCL1, and phosphorylated FLT3/STAT5 protein expression was measured using an immunoblotting assay. To reveal the anti-leukemic effect of YPN-005 on AML cells in vivo, we observed tumor load using the IVIS spectrum in vivo imaging system (PerkinElmer) in an orthotopic mouse model using pGFP-transduced MOLM-13 cell line. Results YPN-005 effectively inhibited the proliferation of AML cells by inducing apoptosis and reducing phosphorylation of RNA polymerase II. The c-MYC protein expression decreased with the treatment of YPN-005 (Figure 1.), and the effect of YPN-005 was negatively correlated with c-myc expression. YPN-005 also showed antileukemic activities in primary AML cells, especially those harboring FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation and in in vivo mouse model (Figure 2.). Phosphorylated FLT3/STAT5 was decreased and FLT3/STAT5 was downregulated with YPN-005 treatment. Conclusion In conclusion, our data suggest that YPN-005, a CDK7 inhibitor has a potential role in treating AML by inducing apoptosis and suppressing c-MYC and FLT3/STAT5. The antileukemic property of YPN-005 needs to be further evaluated in FLT3-mutated AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Non-Canonical FLT3 Mutations (FLT3m) in Patients (Pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) - Outcomes and Prognosis after FLT3 Inhibitor (FLT3i) and Venetoclax Based Regimens

Non-Canonical FLT3 Mutations (FLT3m) in Patients (Pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) - Outcomes and Prognosis after FLT3 Inhibitor (FLT3i) and Venetoclax Based Regimens

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Throughput Screening to Elucidate In Vivo Oncogenicity of Loss-of-Function Alterations in Lymphoid Malignancies

Large-scale genetic studies utilizing next-generation sequencing have identified many recurrently altered genes and revealed their overall picture in lymphoid malignancies. Although limited genes mutated at high frequency have been extensively investigated using genetically engineered mouse models, less attention has been directed to hundreds of rarely mutated genes (so-called long tail driver genes). Here we performed in vivo CRISPR loss-of-function screening to examine oncogenicity of somatic alterations, cooperability between them, and association between genotype and lineage in lymphoid malignancies. As many driver candidates mutated with various frequencies have been reported in diffuse large B-cell lymphomas (DLBCL), we constructed a custom lentiviral CRISPR library targeting 86 genes (12 sgRNAs each) in which loss-of-function alterations (mutations and deletions) are recurrently (> 5%) observed in DLBCL. These sgRNA-targeted genes also covered the majority of driver candidates of other precursor and mature lymphoid neoplasms, such as 71% and 87% of those observed in B-acute lymphoblastic leukemia (B-ALL/LBL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Transplantation of the CRISPR library-transduced murine hematopoietic stem/progenitor (Lin-c-Kit+Sca-1+) cells caused a variety of hematologic malignancies in 180 (90%) out of 199 recipient mice, resulting in earlier death (with a median of 228 [range, 37‒476] days) of transplanted mice than control mice. Hematologic, immunophenotypic, and pathologic analyses of 132 tumors revealed that these malignancies consisted of B- and T-cell lymphomas (17% and 20%), B- and T-ALL/LBL (14% and 15%) and myeloid malignancy (14%) according to Bethesda criteria, although the diagnosis of 22 (17%) tumors could not be determined. sgRNA sequencing of 109 tumors showed that at least 1 sgRNA-targeted genes were detected in almost all (>99%) tumors, with a median of 3 (range, 1‒10) genes per tumor. Enrichment analysis of sgRNA-targeted genes demonstrated significant overrepresentation of 13 genes, such as Trp53 and Irf2bp2, pointing to stronger oncogenic capacity of these driver genes. These sgRNA-targeted genes showed lineage and tissue specificity, such as Pax5, Kmt2d, Atm, and S1pr2 for B-cell lineage and Zc3h12a and Socs1 for T-cell lineage, while only weak enrichment was observed in myeloid neoplasms. These tumors showed characteristic disease phenotype according to sgRNA-targeted genes: Pax5-targeted tumors were usually B-ALL/LBL, whereas Kmt2d-targeted tumors developed as B-cell lymphomas with earlier onset than those harboring other targeted genes. All of Zc3h12a-targeted tumors were T-cell lymphoma with CD4+CD8- immunophenotype and low clonality, while Socs1-targeted tumors primarily developed as T-ALL/LBL with CD4+CD8+ immunophenotype and frequent thymus involvement. Whole-exome sequencing (n = 108) not only confirmed targeted disruption by sgRNAs but also identified additional driver alterations in 76% and 40% of B-cell and T-cell neoplasms, respectively, suggesting the necessity of second-hit alterations in these tumors. These additional driver alterations were associated with a specific lineage. Frequent C-terminal truncating mutations of Notch1 and loss-of-function mutations of Ikzf1 were found in T-cell malignancies, while Jak1 and Jak3 hotspot missense mutations and Flt3 internal tandem duplications were common in B-cell malignancies, suggesting cooperative relationship between specific alterations and their association with lineage. In addition, hierarchical clustering of RNA-sequencing data (n = 105) identified several clusters associated with disease phenotypes and sgRNA-targeted genes. These clusters showed distinct expression signatures, including germinal center B-cell-like signatures in Kmt2d-targeted tumors, MYC target signatures in Pax5-targeted tumors, and inflammation-related signature in Zc3h12a-targeted tumors. In summary, our in vivo CRISPR loss-of-function screening enables high-throughput evaluation of the tumorigenic potential of many genetic abnormalities, their cooperability and association with lineage in lymphoid malignancies. Such approach accelerates understanding of genotype-phenotype association and discovery of potential therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Prognostic Impact of FLT3 in NPM1-Mutated AML: Co-Occurrence of FLT3-ITD and FLT3-TKD Confers Poor Outcomes

Background:NPM1 and FLT3 are commonly mutated in patients with acute myeloid leukemia (AML). While FLT3 internal tandem duplication (ITD) is known to confer worse prognosis even in the setting of NPM1 according to the recent European LeukemiaNet (ELN) 2022 criteria, the prognostic impact of FLT3 tyrosine kinase domain (TKD) in this population remains unclear. One recent study suggests FLT3-TKD and NPM1 co-mutation defines a highly favorable prognostic group (Boddu et al.). Furthermore, NPM1-mutated AML with both FLT3-ITD and FLT-TKD mutations are rare and not well described. Objective: In this study, we explored the prognostic impact of FLT3-ITD and/or FLT3-TKD in patients with NPM1-mutated AML. Methods: This was a multicenter, retrospective study (Moffitt Cancer Center, Weill Cornell, and Memorial Healthcare System) of NPM1-mutated AML patients who were diagnosed and treated from 2013 to June 2022. Inclusion was restricted to NPM1-mutated AML patients who also harbored FLT3-ITD, FLT-TKD, or both (n=107). All patients have next generation sequencing (NGS) performed at diagnosis. Kaplan-Meier, univariate (log-rank), and multivariate (Cox regression) analyses were performed. Results: Among 107 patients (47M/60F, median age 62 at diagnosis, range 23-79), 85% (91/107) had de novo AML. By ELN 2017 criteria, 37.4% (40/107) had favorable risk, 50.5% (54/107) had intermediate risk, and 9.3% (10/107) had adverse risk disease. Over 90% had intermediate risk cytogenetics at the time of diagnosis. Common co-mutations included DNMT3A (53.3%), TET2 (24.3%), WT1 (12.1%), IDH2 (8.4%), and IDH1 (6.5%) (Table 1). There were 66.4% (71/107) of patients with NPM1+FLT3-ITD, 23.4% (25/107) with NPM1+FLT3-TKD, and 10.3% (11/107) with NPM1 and both FLT3-ITD and FLT-TKD (NPM1+FLT3). Patients with NPM1+FLT3-ITD achieved CR/CRi in 84.5% (60/71) similar to patients with NPM1+FLT3-TKD, 88% (22/25), p=1.0. Most patients in both groups received intensive frontline therapy (80.3% vs. 72%, p=0.408). Forty-two (59.2%, 42/71) NPM1+FLT3-ITD patients received allogeneic hematopoietic cell transplant (HCT) compared to 36% (9/25) of NPM1+FLT3-TKD patients, p=0.193. The median overall survival (mOS) was not significantly different between NPM1+FLT3-ITD (42.4 months) and NPM1+FLT3-TKD (not reach), p=0.51. The NPM1+FLT3 cohort had dismal mOS of 8.4 months, which is significantly worse compared to those with NPM1+FLT3-ITD (p<0.005) and NPM1+FLT3-TKD (p=0.01) (Figure 1). Univariate analysis showed co-occurrence of FLT3-ITD and FLT-TKD significantly impacted OS (HR 2.94, 95% CI: 1.42-6.08, p=0.004). Multivariate analysis using covariates including age over 60, AML type, mutation burden (4 or more), and co-occurrence of FLT3-ITD and FLT-TKD confirmed its prognostic significance on survival (HR 2.96, 95% CI: 1.41-6.20, p=0.004). Conclusions: Our findings suggest very poor outcomes in patients with NPM1+FLT3 and no significant difference observed in survival between NPM1+FLT3-ITD and NPM1+FLT3-TKD. The prognosis of NPM1-mutated AML is increasingly being refined as more knowledge is gained in the molecular level. More studies are needed to understand the different gene-gene interactions that underlie this unique subset. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognosis of Pediatric Acute Myeloid Leukemia with KMT2A-MLLT3 According to DNA Methylation Patterns: Jccg JPLSG AML-05 Study

Introduction Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by cytogenetic abnormalities, including fusion genes, mutations, epigenetic modifications, and dysregulated gene expression. DNA methylation patterns have recently been appeared to be associated with molecular subtypes, chromosomal abnormalities, gene fusion, and AML prognosis. DNA methylation pattern of adult AML has also been proposed as a novel biomarker for AML prognosis. However, few studies have discussed the influence of DNA methylation status on the clinical features of pediatric AML. We analyzed genome-wide DNA methylation in 127 pediatric patients with AML to determine its association with clinical features, genetic alterations, and prognostic impact. Methods Between 2006 and 2010, 443 pediatric patients with de novo AML (0-17 years) participated in the Japanese AML-05 trial of the Japanese Pediatric Leukemia/Lymphoma Study Group. Of these patients, 127 patients were enrolled in this study. Their cytogenetic features were as follows: normal karyotype, 26 patients; RUNX1-RUNX1T1, 11; CBFB-MYH11, 5; KMT2A rearrangement, 47 (22 of 47 had KMT2A-MLLT3); NUP98-NSD1, 8; NUP98-KDM5A, 5; CBFA2T3-GLIS2, 4; FUS-ERG, 4; and other cytogenetics, 18. This cohort included 35 patients with FLT3-internal tandem duplication (ITD), 9 with CEBPA biallelic mutations, 15 with high MECOM (also known as EVI1) expression, and 29 with high PRDM16 (also known as MEL1) expression. In all patients, genome-wide DNA methylation analysis using Infinium MethylationEPIC BeadChip (Illumina) was performed. Results and Discussion Based on unsupervised hierarchical clustering, patients were divided into six clusters (cluster 1-6) associated with genetic alterations. Cluster 1 (n = 28) was characterized by the presence of KMT2A rearrangement with low MECOM expression. CBFA2T3-GLIS2 and NUP98-KDM5A considered as adverse prognostic factors were included in cluster 2 (n = 15). Cluster 3 (n = 16) comprised 11 RUNX1-RUNX1T1 and 5 CBFB-MYH11. No CBF-AML was included in other clusters. All nine patients with biallelic CEBPA mutations, which indicated a favorable outcome, were classified under cluster 4 (n = 18). Interestingly, all four patients with FUS-ERG were also classified under cluster 4. Clusters 5 (n = 15) and 6 (n = 35) comprised patients with molecular features related to adverse outcomes, such as FLT3-ITD, KMT2A-MLLT3 with high MECOM expression, and high PRDM16 expression. All seven patients with NUP98-NSD1 were classified under cluster 6. Patients with AML with KMT2A rearrangement showed a significant difference in DNA methylation levels at 500 CpG sites compared with those without. Patients were categorized into four clusters (clusters A-D) based on the methylation values of the 500 CpG sites that were significantly different between patients with AML with or without KMT2A rearrangement, and 96% (45 of 47) of patients with AML with KMT2A rearrangement were classified under clusters A (n = 32) or B (n = 13). The remaining two patients were classified under clusters C (n = 1) and D (n = 1). Interestingly, all 32 patients with AML with KMT2A rearrangement classified under cluster A showed low MECOM expression. Moreover, 22 patients with AML with KMT2A-MLLT3 were classified under clusters A (n = 14) and B (n = 8). Patients with KMT2A-MLLT3 in cluster B had a significantly worse prognosis than those in cluster A (5-year overall survival, 30% vs. 100%; P = 0.001, and 5-year event-free survival, 0% vs. 100%; P < 0.001). All eight patients with KMT2A-MLLT3 in cluster B had high MECOM expression, whereas all 14 patients in cluster A had low MECOM expression. These results indicate that the methylation pattern corresponded to MECOM expression. Our findings suggest that DNA methylation levels at specific CpG sites are useful biomarkers for the prognosis of pediatric patients with AML.

Impact of Allogeneic Hematopoietic Cell Transplantation in First Complete Remission in Addition to FLT3 Inhibition with Quizartinib in Acute Myeloid Leukemia with FLT3-Internal Tandem Duplication: Results from the Quantum-First Trial

Background Patients with acute myeloid leukemia (AML) exhibiting an FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD+), face particularly poor outcomes, even after allogeneic hematopoietic cell transplantation (allo-HCT), which appears to only partially mitigate the poor prognosis associated with this mutation. (Döhner H et al., Blood 2017, Stone RM et al., NEJM 2017; Larson RA et al., Leukemia 2021). QuANTUM-First (NCT02668653) is a phase 3, global, randomized, double-blind, placebo-controlled trial evaluating the novel, highly potent, and selective type II FLT3 inhibitor quizartinib (Quiz) for patients with newly diagnosed FLT3-ITD+ AML who were treated with curative intent. This pivotal trial demonstrated that the addition of Quiz to intensive induction, consolidation including allo-HCT in CR1, followed by single-agent continuation therapy for up to 3 years resulted in a significant improvement in overall survival (OS) for this population. The objectives of our analyses were to evaluate the impact of allo-HCT in CR1 and the interrelationship with Quiz on clinical outcomes of QuANTUM-First and on the feasibility of continuation therapy with Quiz after allo-HCT. Methods Patients aged 18-75 years with newly diagnosed AML were centrally screened for FLT3-ITD before initiation of standard induction therapy. Patients were randomized to Quiz (40 mg/d, on days 8-21) or placebo (PBO) and stratified by region (North/South America, Europe, and Asia/other regions), age (<60 years, ≥60 years), and white blood cell count (WBC; <40 × 109/L, ≥40 × 109/L) at diagnosis. Patients who achieved complete remission (CR) or CR with incomplete hematologic recovery (CRi) received up to 4 cycles of high-dose cytarabine plus Quiz (40 mg/d) or PBO and/or allo-HCT followed by up to 3 years of continuation therapy with Quiz (30-60 mg/d) or PBO. Patients who underwent allo-HCT could start continuation therapy 30 to 180 days after allo-HCT. The primary endpoint was OS. The impact of allo-HCT in CR1 on OS was assessed as a time-dependent covariable in univariable and multivariable regression analyses. Results In QuANTUM-First, 539 patients with FLT3-ITD+ AML were randomized to Quiz (n=268) or PBO (n=271). There were 294 (54.5%) females and 245 (45.5%) males. Of the 539 randomized patients (median age, 56 years; range, 20-75 years), 297 (55.1% [297/539]; Quiz, 54.9% [147/268]; PBO, 55.4% [150/271]) achieved CR and 71 (13.2% [71/539]; Quiz, 16.8% [45/268]; PBO, 9.6% [26/271]) achieved CRi after 1 to 2 courses of induction. Allo-HCT was performed in CR1 in 157 patients (52.9% of CR patients [157/297]; Quiz, 57.1% [84/147]; PBO, 48.7% [73/150]) predominantly with grafts from unrelated donors (49.7%), followed by siblings (32.5%) and other related donors (17.8%). Patients proceeding toward allo-HCT in CR1 were younger (median age, 51 years) and had a slightly higher proportion of WBC ≥40 × 109/L at diagnosis compared with patients who did not undergo allo-HCT in CR1. After completion of allo-HCT, 61 patients in the Quiz arm (72.6%) and 36 patients in the PBO arm (49.3%) started 3 years of continuation therapy. An additional 115 allo-HCTs were performed within the trial outside CR1 (Quiz, n=60; PBO, n=55). Multivariable regression analysis was stratified by region, age, and WBC, including allo-HCT in CR1 as time dependent and adjusted for FLT3-ITD variant allelic frequency, as well for sex. The analysis revealed Quiz treatment (hazard ratio [HR], 0.770; 95% confidence interval (CI), 0.609-0.973; P = 0.0284) and allo-HCT in CR1 (HR, 0.424; 95% CI, 0.301-0.597; P < 0.0001) as favorable factors with respect to OS. Based on this model, at any given time, the HR was 0.326 (95% CI, 0.216-0.493) for the patients randomized to Quiz and proceeding to allo-HCT in CR1 compared with patients randomized to PBO who had not yet received allo-HCT in CR1 by that time. Conclusions In patients who underwent allo-HCT in CR1, longer survival was observed in those treated with quizartinib versus placebo. Furthermore, irrespective of allo-HCT performed in CR1, quizartinib when combined with standard induction and consolidation therapy and continued for up to 3 years as a single agent improves survival over placebo in patients with newly diagnosed FLT3-ITD+ AML.

Phase 1/2, Open-Label, Dose Escalation, Dose Expansion Study of Menin Inhibitor DSP-5336 in Adult Patients with Acute Leukemia with and without Mixed-Lineage Leukemia (MLL)-Rearrangement (R) or Nucleophosmin 1 (NPM1) Mutation (m)

Patients who relapse after initial acute leukemia (AL) treatment have median survivals <6 mo for AML and <1 y for ALL, especially AML or ALL with MLL rearrangements (MLLr) or relapsed AML with NPM1 mutations. Translocations of the MLL gene (5% to 10% of adult patients) produce an aggressive subtype, with a median overall survival (OS) of approximately 9 months. The NPM1 mutation (NPM1m) occurs in 30-35% of adult de novo AML and is associated with favorable prognosis. However, 40-50% of these patients have concurrent FLT3-internal tandem duplication mutations negating this favorability. DSP-5336, a novel oral small-molecule, was developed to prevent menin protein binding to MLL fusion proteins that drive leukemogenesis. Nonclinical studies of DSP-5336 showed selective growth inhibition against human AL cell lines with MLLr or NPM1m. Evidence of antitumor activity and survival advantage was demonstrated in 3 AML xenograft models with MLLr and/or NPM1m. Treatment with DSP-5336 may reduce leukemic cells and benefit patients with relapsed or refractory (R/R) MLLr or NPM1m AML or pts with R/R MLLr ALL. An open-label, single-arm, phase 1/2 study (NCT04988555) will evaluate the safety and efficacy of DSP-5336 and determine the recommended phase 2 dose (RP2D) in adults aged ≥18 y with relapsed/refractory AML or ALL after at least one line of therapy. Key inclusion criteria are ECOG performance status 0-2 and adequate organ function. Patients will be dosed twice daily in 28-day cycles. In phase 1, 21-30 pts will be enrolled into multiple dose cohorts (1-6 pts per dose level), with dose escalation determined using a two-parameter Bayesian logistic regression model. The RP2D will be informed by the maximum biologic effect or maximum tolerated dose, whichever is lower. The phase 2 study will include two arms (relapsed or refractory [R/R] AML with MLLr and R/R AML with NPM1m). Patients will be treated at the RP2D to evaluate clinical activity and safety, monitored using Bayesian posterior probability to optimize enrollment, with Bayesian stopping rules. Bayesian monitoring of responses begins after the first 10 enrolled patients are evaluable for efficacy. This study is recruiting in the United States and Japan. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Leukoreductive response to the combination of sorafenib and chemotherapy in hyperleukocytosis of FLT3-ITD mutated pediatric AML.

Twelve to 22% of pediatric acute myeloid leukemia (AML) patients present with hyperleukocytosis, which is one of the main risk factors of early death due to its clinical complications: leukostasis, causing pulmonary or central nervous system injuries, tumor lysis syndrome, and disseminated intravascular coagulation. Sorafenib is a multi-kinase inhibitor that blocks the Fms-Related Tyrosine Kinase 3 receptor (FLT3) in AML patients with a FLT3-internal tandem duplication (FLT3-ITD), leading to a reduction of proliferation. Here we report four de novo diagnosed or relapsed pediatric FLT3-ITD-positive AML patients with hyperleukocytosis, which were treated with sorafenib in combination with cytoreductive chemotherapy prior to the start of the induction phase. We observed a fast reduction of white blood cells in peripheral blood and bone marrow. This resulted in a rapid clinical stabilization of the patients. Adverse side effects-such as dermatologic toxicity, elevation of transaminases and hypertension-occurred but were mild and inductive chemotherapy could be started in parallel or subsequently. This implies sorafenib as a safe and effective treatment option in combination with chemotherapy during cytoreductive prephase for children with this life-threatening condition.

Evaluating the clinical significance of FLT3 mutation status in Syrian newly diagnosed acute myeloid leukemia patients with normal karyotype

The FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) is one of the most prevalent mutations, affecting between 20 and 30 percent of cases in patients with acute myeloid leukemia (AML). The Patients with a FLT3-ITD mutation have a poor prognosis. In the present study, we investigated the FLT3 (ITD-TKD) mutations in 100 newly adult Syrian patients with AML-Normal karyotype (NK). Our results revealed that prevalence of FLT3-ITD mutation was 24%. Interestingly, 20 patients had a typical duplication mutation and four patients had different mutations. From those four mentioned patients, two of them carried a 39 base pair (bp) duplication in different location: (c.1838_1877dup39, p.591–603dup) and (c.1836_1874 dup 39, p.591–603dup), the third patient, showed FLT3-ITD duplication and a newly insertion together, this insertion was not demonstrated before: (c.1842_1865dup24, c.1865_1866insGAA). Finally, the fourth patient exhibited a duplication of 21bp (c.1855_1875dup21, p.597–603dup). In addition, statistically significant differences were observed for the relation between the presence of FLT3-ITD mutation and lactate dehydrogenase (LDH) level, overall survival (OS), relapse, and event free survival (EFS). We demonstrated that our patients with FLT3-ITD mutation had a poor prognosis. Also, the frequency of FLT3-TKD mutation was low 2% and no compound between the two mutations was found, as individuals showed to carry the two mutations were not detected. These findings are likely useful for a better understanding of molecular leukemogenetic steps in AML-NK patients and may be beneficial for clinical relevance for risk grouping, study design and choice of therapy in Syrian population.

Clinical characteristics and prognostic analysis of acute myeloid leukemia patients with PTPN11 mutations

ABSTRACT Objectives Little is known about the clinical impact of germline/somatic mutations of PTPN11 in acute leukemia. The aim of this study was to investigate the clinical characteristics and prognostic impact of PTPN11 mutations in patients with acute myeloid leukemia (AML). Methods: Seventy-four patients with PTPN11 mutation-positive AML treated at our institution were enrolled in this study. The prevalence of PTPN11 mutations was examined using targeted next-generation sequencing technology, and patients with AML and PTPN11 mutations were screened. Clinical characteristics, prognostic impact, and association between PTPN11 mutations and other mutations were analyzed retrospectively. Results: PTPN11 mutations co-occurred more commonly with DNMT3A, NPM1, and FLT3 internal tandem duplication mutations. Compared with PTPN11 wild-type (WT) patients, PTPN11 mutation-positive AML patients presented with higher white blood cell (WBC) and platelet (PLT) counts. In 74 PTPN11 positive AML patients, PTPN11 mutations had an adverse effect on overall survival (OS) (62.5%) and a negative prognostic effect on event-free survival (EFS) (50%). Allo-hematopoietic stem cell transplantation (HSCT) abrogated the negative effect of mutations in PTPN11; the OS and EFS of AML patients with PTPN11 mutations who received transplantation were longer than those of AML patients with PTPN11 mutations who did not undergo allo-HSCT (P = 0.001, EFS; P < 0.001, OS). Discussion: Newly diagnosed PTPN11 mutation-positive AML patients with high WBC and PLT counts or presenting no remission after first induction chemotherapy suffer from high mortality rates. Conclusion: Given the lack of targeted therapies for PTPN11 mutations, timely HSCT is necessary for patients.

Prognostic Value of FLT3-Internal Tandem Duplication Residual Disease in Acute Myeloid Leukemia.

The applicability of FLT3-internal tandem duplications (FLT3-ITD) for assessing measurable residual disease (MRD) in acute myeloid leukemia (AML) in complete remission (CR) has been hampered by patient-specific duplications and potential instability of FLT3-ITD during relapse. Here, we comprehensively investigated the impact of next-generation sequencing (NGS)-based FLT3-ITD MRD detection on treatment outcome in a cohort of patients with newly diagnosed AML in relation to established prognostic factors at diagnosis and other MRD measurements, ie, mutant NPM1 and multiparameter flow cytometry. In 161 patients with de novo FLT3-ITD AML, NGS was performed at diagnosis and in CR after intensive remission induction treatment. FLT3-ITD MRD status was correlated with the cumulative incidence of relapse and overall survival (OS). NGS-based FLT3-ITD MRD was present in 47 of 161 (29%) patients with AML. Presence of FLT3-ITD MRD was associated with increased risk of relapse (4-year cumulative incidence of relapse, 75% FLT3-ITD MRD v 33% no FLT3-ITD MRD; P < .001) and inferior OS (4-year OS, 31% FLT3-ITD MRD v 57% no FLT3-ITD MRD; P < .001). In multivariate analysis, detection of FLT3-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 3.55; P < .001) and OS (hazard ratio 2.51; P = .002). Strikingly, FLT3-ITD MRD exceeds the prognostic value of most generally accepted clinical and molecular prognostic factors, including the FLT3-ITD allelic ratio at diagnosis and MRD assessment by NGS-based mutant NPM1 detection or multiparameter flow cytometry. NGS-based detection of FLT3-ITD MRD in CR identifies patients with AML with profound risk of relapse and death that outcompetes the significance of most established prognostic factors at diagnosis and during therapy, and furnishes support for FLT3-ITD as a clinically relevant biomarker for dynamic disease risk assessment in AML.

The Translational Landscape Revealed the Sequential Treatment Containing ATRA plus PI3K/AKT Inhibitors as an Efficient Strategy for AML Therapy.

The present study aimed to better understand the possibility of utilizing all-trans retinoic acids (ATRA) in acute myeloid leukemia (AML). We found that ATRA significantly suppressed global translation and protein synthesis in AML cells. The efficacy of ATRA in treating AML required its translational regulatory functions, as shown by the fact that the decrease in the universal eukaryotic initiation factor 4E (eIF4E) was essential to maintain the induction of cell growth arrest and differentiation by ATRA. By establishing a specific translational landscape, we suggested that transcripts with simple 5'UTR gained a translational advantage in AML cells during ATRA stress. Based on that, the genes translationally regulated by ATRA were mainly enriched in phosphatidylinositol-3-kinase/Akt (PI3K/AKT) signaling; we subsequently revealed that PI3K/AKT activation was required for ATRA to effectively induce AML cell differentiation. However, PI3K/AKT has been reported to promote the stemness of AML cells. As such, we further suggested that sequential treatment including ATRA and PI3K/AKT inhibitor induced robust apoptosis, extremely inhibited the clonality of AML cells, and suppressed the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-driven transformation of CD34+ hematopoietic stem/progenitor cells. Future clinical studies are warranted to further support the clinical application of the sequential strategy for the effective treatment of AML.

Frugal alignment-free identification of FLT3-internal tandem duplications with FiLT3r

BackgroundInternal tandem duplications in the FLT3 gene, termed FLT3-ITDs, are useful molecular markers in acute myeloid leukemia (AML) for patient risk stratification and follow-up. FLT3-ITDs are increasingly screened through high-throughput sequencing (HTS) raising the need for robust and efficient algorithms. We developed a new algorithm, which performs no alignment and uses little resources, to identify and quantify FLT3-ITDs in HTS data.ResultsOur algorithm (FiLT3r) focuses on the k-mers from reads covering FLT3 exons 14 and 15. We show that those k-mers bring enough information to accurately detect, determine the length and quantify FLT3-ITD duplications. We compare the performances of FiLT3r to state-of-the-art alternatives and to fragment analysis, the gold standard method, on a cohort of 185 AML patients sequenced with capture-based HTS. On this dataset FiLT3r is more precise (no false positive nor false negative) than the other software evaluated. We also assess the software on public RNA-Seq data, which confirms the previous results and shows that FiLT3r requires little resources compared to other software.ConclusionFiLT3r is a free software available at https://gitlab.univ-lille.fr/filt3r/filt3r. The repository also contains a Snakefile to reproduce our experiments. We show that FiLT3r detects FLT3-ITDs better than other software while using less memory and time.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Molecular profiling of patients with cytogenetically normal acute myeloid leukemia and hyperleukocytosis.

Acute myeloid leukemia (AML) with initial hyperleukocytosis is associated with high early mortality and a poor prognosis. The aims of this study were to delineate the underlying molecular landscape in the largest cytogenetic risk group, cytogenetically normal acute myeloid leukemia (CN-AML), and to assess the prognostic relevance of recurrent mutations in the context of hyperleukocytosis and clinical risk factors. The authors performed a targeted sequencing of 49 recurrently mutated genes in 56 patients with newly diagnosed CN-AML and initial hyperleukocytosis of ≥100G/L treated in the AMLCG99 study. The median number of mutated genes per patient was 5. The most common mutations occurred in FLT3 (73%), NPM1 (75%), and TET2 (45%). The predominant pathways affected by mutations were signaling (84% of patients), epigenetic modifiers (75% of patients), and nuclear transport (NPM1; 75%) of patients. AML with hyperleukocytosis was enriched for molecular subtypes that negatively affected the prognosis, including a high percentage of patients presenting with co-occurring mutations in signaling and epigenetic modifiers such as FLT3 internal tandem duplications and TET2 mutations. Despite these unique molecular features, clinical risk factors, including high white blood count, hemoglobin level, and lactate dehydrogenase level at baseline, remained the predictors for overall survival and relapse-free survival in hyperleukocytotic CN-AML.