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Acute myeloid leukemia (AML) is a common type of acute leukemia (AL), belonging to malignant tumors of the hematopoietic system with the characteristics of rapid disease development, control with extreme difficulties, easy recurrence, poor prognosis, and incidence rate increasing with age. The traditionally diagnostic standard of French American British (FAB), being based on the morphological examination with high human subjectivity, can no longer meet the demand of clinical diagnosis and treatment of AML. Requirements of objective accuracy and low-dose sample, have become the indispensable method for AML diagnosis and monitoring prognosis. Flow cytometry is a modern technology that can quickly and accurately detect the series, antigen distribution, differentiation stage of AML cells, minimal residual lesions after AML therapy, so as to provide the great significance in guiding clinical diagnosis, hierarchical treatment, and prognosis judgement. This article will systematically elaborate on the application of flow cytometry in the diagnosis and classification of AML, and the detection of minimal residual lesions, thereby providing reference significance for dynamic monitoring and prognostic observation of AML with different immune subtypes of FAB.

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Disease relapse remains the first cause of mortality of hematological malignancies after allogeneic hematopoietic stem cell transplantation (allo-HCT) for acute myeloid and lymphoid leukemia (AML and ALL) and for myelodysplastic syndroms (MDS). More and more patients are eligible for allo-HCT over the years and for many of them, only reduced intensity conditioning is possible, which is associated with a higher risk of relapse. Knowledge and biotechnology allow us to better identify diseases at very high risk of relapse and to measure residual disease before allo-HCT. Planning post-transplant maintenance treatment as part of a prophylaxis strategy is now feasible. Monitoring biomarkers of residual disease and post-transplant chimerism after allo-HCT allows a preemptive strategy. Within the frame of the 14th annual workshops of the Francophone Society for Bone Marrow Transplantation and Cell Therapy, the working group reviewed the literature and discussed novel strategies and therapies used to prevent relapse post-allo-HCT. Innovative drugs have been developed recently. Their toxicity profile allows their use post-allo-HCT, albeit with precaution. We reviewed the use of FLT3 inhibitors for AML, BCR::ABL inhibitors for Philadelphia chromosome for ALL, hypomethylating agents and Bcl-2 inhibitors for AML and MDS. The indications of immunomodulation and infusion of donor lymphocytes have been reviewed. Finally, we outlined methods of follow-up and support for patients receiving these prophylactic treatments.

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Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However, these transplants are complicated by a high rate of relapse in very high cytogenetic risk or refractory diseases. The benefit of this therapeutic strategy for these serious malignant hemopathies could therefore be reassessed. As part of the 14th workshop for the harmonization of allograft practices organized by the francophone society of bone marrow transplantation and cellular therapy (SFGM-TC) (SFGM-TC) in Lille in September 2023, the role of allograft for very high risk or refractory AML and MDS was challenged after analysis of published studies.

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The prognosis of patients with high-risk acute myeloid leukemia (AML) is dismal even after allogeneic stem cell transplantation (allo-HSCT), with relapse remaining the leading cause of treatment failure. Here, we investigated whether ruxolitinib and decitabine plus modified busulfan-cyclophosphamide (mBu/Cy) conditioning could reduce relapse in high-risk AML after allo-HSCT. This prospective, single-arm, phase II trial enrolled 37 patients who received allo-HSCT between September 2020 and March 2022 at the First Medical Center of Chinese People's Liberation Army (PLA) General Hospital. Eligible patients (10-62 years) had relapsed/refractory, positive measurable residual disease (MRD) prior to conditioning or adverse genetic abnormalities. Ruxolitinib (35 mg twice daily, days - 15 to - 10) and decitabine (20 mg/m2/day, days - 15 to - 10) were administered followed by mBu/Cy conditioning. All patients achieved engraftment. The cumulative incidences (CIs) of acute graft-versus-host disease (GVHD) grades II-IV and III-IV were 35.0% and 10.5%, respectively. The 1-year cumulative incidence of chronic GVHD was 8.1%. The 1-year CI of relapse was 29.7% among all patients, 0% in patients who achieved the first complete remission (CR1) prior to conditioning, and 0% in those with MRD-negative prior to conditioning. The 1-year non-relapse mortality was 5.4%. The 1-year probabilities of overall survival, disease-free survival, and GVHD-free relapse-free survival were 70.3%, 62.2%, and 54.1%, respectively. In conclusion, the novel conditioning showed primary efficacy in terms of a reduction in relapse in high-risk patients with AML after allo-HSCT, especially in those who achieved CR1 and MRD-negative prior to conditioning. Also, the new conditioning regimen may help reduce the incidence of chronic GVHD. ClinicalTrials.gov identifier: NCT04582604.

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Homeodomain-only protein homeobox (HOPX) mainly exerts its transcriptional repression by physically sequestering the serum co-repressor and recruiting histone deacetylase (HDAC), possessing important potential as a prognostic gene in acute myeloid leukemia (AML). HDACs play crucial roles in cell growth, gene regulation, and metabolism, and they are also important factors in promoting AML progression. Therefore, this project attempts to investigate whether HOPX affects AML progression by interacting with HDAC2 protein. Bioinformatics analysis was employed to identify potential prognostic genes in AML. Flow cytometry and MTT assays were performed to analyze the cellular biological functions of the AML prognostic marker HOPX. The interaction network of HOPX was analyzed using the Search Tool for the Retrieval of Interacting Genes database, and the interaction between HOPX and HDAC2 was observed using endogenous and exogenous immunoprecipitation. HOPX is highly expressed in AML cells. Further research uncovered that low expression of HOPX can repress the proliferation activity, anti-apoptotic ability, and differentiation blockage of AML cells. Moreover, mechanistically, HOPX induced AML differentiation blockage and malignant progression through interaction with HDAC. HOPX can serve as a prognostic marker for AML and can interact with HDAC2 to induce AML differentiation blockage and malignant progression.

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OBJECTIVES: Acute myeloid leukemia (AML) with mast cell (MC) differentiation was recently described as an aggressive subgroup of AML cases. The objectives of this study were to assess the flow cytometric immunophenotypic features of AML-MC cases. METHODS: We characterized the immunophenotypic features of 21 AML-MC cases by flow cytometry and compared them to 20 reactive/regenerating bone marrow specimens. RESULTS: The number of MCs detected by flow cytometry in AML-MC cases ranged from 0.4% to 21.1%, with a median of 3.5%, significantly higher than that of normal/reactive bone marrow (BM) (median, 0.01%; range, 0.000%-0.396%; P < .0001). Immunophenotypically, MCs in AML-MC cases demonstrated immaturity, differing from MCs in normal/reactive BMs, including dimmer CD45 (100% vs 0%), lower side scatter (100% vs 0%), more frequent CD34 (81% vs 20%), and CD123 (100% vs 10%) positivity, and more frequent uniform/increased CD38 expression (95% vs 20%) (all P ≤ .0001). CD2 (0/5) and CD25 (2/6, 1 uniform and 1 partial) were assessed in a subset of cases. The myeloblasts in AML-MC were typically CD34+CD117+HLA-DR+ with unusually frequent expression of CD56 (57%, all partial) and CD25 (63%, mostly partial), increased CD117 (62%), and decreased CD38 (86%). The MC percentage determined by flow cytometry correlated well with MCs detected by tryptase immunohistochemistry (r = 0.76, P < .001). CONCLUSIONS: The MCs in AML-MC cases are characterized by dim CD45, low side scatter, CD34 and CD123 positivity, and uniform and increased CD38 expression. Flow cytometry is an excellent tool for identifying AML-MC cases.

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We focus on characterizing cell lines from young and aged-healthy and -AML (acute myeloid leukemia) cell lines, and our goal is to identify the key markers associated with the progression of AML. To characterize the age-related phenotypes in AML cell lines, we consider eigenCell analysis that effectively encapsulates the primary expression level patterns across the cell lines. However, earlier investigations utilizing eigenGenes and eigenCells analysis were based on linear combination of all features, leading to the disturbance from noise features. Moreover, the analysis based on a fully dense loading matrix makes it challenging to interpret the results of eigenCells analysis. In order to address these challenges, we develop a novel computational approach termed network-constrained eigenCells profile estimation, which employs a sparse learning strategy. The proposed method estimates eigenCell based on not only the lasso but also network constrained penalization. The use of the network-constrained penalization enables us to simultaneously select neighborhood genes. Furthermore, the hub genes and their regulator/target genes are easily selected as crucial markers for eigenCells estimation. That is, our method can incorporate insights from network biology into the process of sparse loading estimation. Through our methodology, we estimate sparse eigenCells profiles, where only critical markers exhibit expression levels. This allows us to identify the key markers associated with a specific phenotype. Monte Carlo simulations demonstrate the efficacy of our method in reconstructing the sparse structure of eigenCells profiles. We employed our approach to unveil the regulatory system of immunogenes in both young/aged-healthy and -AML cell lines. The markers we have identified for the age-related phenotype in both healthy and AML cell lines have garnered strong support from previous studies. Specifically, our findings, in conjunction with the existing literature, indicate that the activities within this subnetwork of CD79A could be pivotal in elucidating the mechanism driving AML progression, particularly noting the significant role played by the diminished activities in the CD79A subnetwork. We expect that the proposed method will be a useful tool for characterizing disease-related subsets of cell lines, encompassing phenotypes and clones.

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INTRODUCTION: The substantial economic burden of acute myeloid leukemia (AML) could be reduced with post-remission maintenance therapies that delay relapse. Real-world healthcare resource utilization (HCRU) data and costs among patients with AML receiving oral azacitidine (Oral-AZA) maintenance therapy or no maintenance are not well understood. We characterize HCRU and costs among these patients in clinical practice in the USA. METHODS: Data from IQVIA PharMetrics® Plus (January 1, 2016-June 30, 2022) were used. Patients ≥ 18 years who were newly diagnosed with AML, received first-line systemic induction therapy, and attained disease remission were eligible. Patients receiving Oral-AZA maintenance and those receiving no maintenance ("watch and wait" [W&W]) were matched 1:3 on baseline characteristics using propensity score matching (PSM) and followed until hematopoietic stem cell transplantation or end of continuous insurance enrollment, whichever occurred first. Outcomes included treatment patterns, inpatient and outpatient visits, and costs. RESULTS: After PSM, the Oral-AZA cohort included 43 patients and the W&W cohort 129. Of the 43 patients receiving Oral-AZA, 88.4% started at the recommended dose of 300 mg and 11.6% at 200 mg. The Oral-AZA cohort had significantly (p = 0.0025) longer median (95% CI) time to relapse from the index maintenance date (median not reached [NR; 9.0 months-NR] vs 3.3 months [0.8 months-NR]), and fewer per person per month (PPPM) hospitalizations (0.23 vs 0.61; p = 0.0005) and overall outpatient visits (5.77 vs 7.58; p = 0.0391) than the W&W cohort. Despite higher AML drug costs PPPM in the Oral-AZA cohort ($16,401 vs $10,651 for W&W), total healthcare costs PPPM were lower ($25,786 vs $38,530 for W&W; p < 0.0001). CONCLUSIONS: Patients with newly diagnosed AML treated with Oral-AZA maintenance in clinical practice had prolonged remission and lower HCRU and costs than patients receiving no maintenance therapy. These findings underscore the clinical and economic value of Oral-AZA in clinical practice.

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An 18-y-old American Saddlebred mare was admitted with fever and acute onset of neurologic signs including grade 3 of 5 ataxia, difficulty in prehension, and dull mentation. Because of financial restraints, desired testing could not be performed; the horse's condition declined despite supportive treatment, and euthanasia was elected. Postmortem examination revealed petechiae and ecchymoses in the meninges and neuroparenchyma of the encephalon. Blast-like neoplastic round cells were identified within the vasculature and areas of hemorrhage in the neuroparenchyma, the intestinal submucosa, and other organs, including the liver, kidney, lung, and mesenteric lymph node. Necrotizing enterocolitis and acute fibrinonecrotizing bacterial pneumonia were also noted. Of the atypical round cells in the encephalon, >70% expressed ionized calcium-binding adapter molecule 1 (Iba1), 10-20% expressed myeloperoxidase (MPO), and <10% expressed PAX5, CD3, CD20, CD79a, or MUM1. The bone marrow was diffusely effaced by neoplastic round cells expressing Iba1, and ~70% of these cells expressed MPO with no expression of CD3 or CD20. CD172a also immunolabeled a portion of the neoplastic cells. These findings were consistent with the diagnosis of acute myeloid leukemia-M1 with an unusual neurologic presentation.

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Venetoclax (VEN), a B-cell lymphoma 2 (BCL-2) selective inhibitor, is widely used for treating acute myeloid leukemia (AML) with promising results. However, the anti-leukemic effect of VEN in relapsed/refractory (R/R)- AML requires improvement. In this study, we observed that combining homoharringtonine (HHT) with VEN plus azacitidine resulted in a significantly higher response and better survival than VA alone in patients with R/R-AML. Basic research indicates that HHT combined with VEN has a highly synergistic effect against both resistant AML cells and primary cells with/without mesenchymal stem cell (MSC) co-culture in vivo, inhibiting proliferation and colony-forming capacity of AML cells associated with concomitant cell cycle arrest. Mechanistically, HHT sensitizes AML cells to VEN by downregulating the anti-apoptotic proteins MCL-1/BCL-xL, activating reactive oxygen species (ROS), leading to mitochondrial membrane potential loss, and attenuating fatty acid (FA) uptake. These findings adding HHT to VEN-based regimens may enhance outcomes in R/R-AML patients.

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Background/Aim: Rash is a common adverse event (AE) observed during cytarabine and idarubicin induction therapy in patients with acute myeloid leukemia (AML). Previous studies have highlighted the challenge in predicting the onset and duration of rash. This study aimed to determine the factors that affect the onset of rash in patients receiving induction therapy for AML. Patients and Methods: This retrospective study involved 97 patients with AML who received induction chemotherapy with cytarabine and idarubicin at the Department of Hematology, Kyushu University Hospital between January 2008 and June 2022. The factors associated with rash were identified through a multivariate stepwise logistic regression analysis. Subsequently, the patient's characteristics were compared between those with risk factors and those without risk factors using a matched pair analysis. Results: Pre-existing leukopenia [odds ratio (OR)=3.294; 95% confidence interval (CI)=1.272-8.531] and good performance status (PS=0) (OR=2.717; 95%CI=1.087-6.792) were significant risk factors for rash development. Conversely, the matched pair analysis indicated that patients with pre-existing leukopenia, excluding those with a PS score of 0, exhibited a significantly (p=0.015) higher incidence of rash than those without it. Conclusion: Both multivariate logistic regression analysis and matched pair analysis identified pre-existing leukopenia as a primary risk factor for rash development associated with cytarabine and idarubicin chemotherapy.

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Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR T cells, CD33PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR T cells further toward possible clinical application.

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BACKGROUND: Previous studies have shown that macrophage-mediated efferocytosis is involved in immunosuppression in acute myeloid leukemia (AML). However, the regulatory role of efferocytosis in AML remains unclear and needs further elucidation. METHODS: We first identified the key efferocytosis-related genes (ERGs) based on the expression matrix. Efferocytosis-related molecular subtypes were obtained by consensus clustering algorithm. Differences in immune landscape and biological processes among molecular subtypes were further evaluated. The efferocytosis score model was constructed to quantify molecular subtypes and evaluate its value in prognosis prediction and treatment decision-making in AML. RESULTS: Three distinct efferocytosis-related molecular subtypes were identified and divided into immune activation, immune desert, and immunosuppression subtypes based on the characteristics of the immune landscape. We evaluated the differences in clinical and biological features among different molecular subtypes, and the construction of an efferocytosis score model can effectively quantify the subtypes. A low efferocytosis score is associated with immune activation and reduced mutation frequency, and patients have a better prognosis. A high efferocytosis score reflects immune exhaustion, increased activity of tumor marker pathways, and poor prognosis. The prognostic predictive value of the efferocytosis score model was confirmed in six AML cohorts. Patients exhibiting high efferocytosis scores may derive therapeutic benefits from anti-PD-1 immunotherapy, whereas those with low efferocytosis scores tend to exhibit greater sensitivity towards chemotherapy. Analysis of treatment data in ex vivo AML cells revealed a group of drugs with significant differences in sensitivity between different efferocytosis score groups. Finally, we validated model gene expression in a clinical cohort. CONCLUSIONS: This study reveals that efferocytosis plays a non-negligible role in shaping the diversity and complexity of the AML immune microenvironment. Assessing the individual efferocytosis-related molecular subtype in individuals will help to enhance our understanding of the characterization of the AML immune landscape and guide the establishment of more effective clinical treatment strategies.

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Acute myeloid leukemia (AML) develops when hematopoietic stem cells acquire chromosomal and genetic abnormalities, transforming into leukemia stem cells (LSCs) and further gaining driver mutations. Advances in genomic analysis have identified numerous new gene mutations involved in AML development. Recent research has shown that individuals with germline mutations in genes like DDX41 and CEBPA develop AML upon acquiring additional somatic mutations, and the latest WHO classification separates AML with such mutations into distinct disease groups. LSCs are regulated by different metabolic processes than normal stem cells, contributing to drug resistance and relapse. LSCs rely on oxidative phosphorylation (OXPHOS) metabolism for energy production, and venetoclax inhibits this process, affecting LSCs. Resistant LSCs show enhanced glycolysis, which suggests that targeting both OXPHOS and glycolysis is crucial. While targeted therapies like FLT3, BCL-2, and IDH inhibitors have shown efficacy, resistance remains an issue, highlighting the need for new treatment strategies. CAR-T cell therapy is an emerging immunotherapy that shows particular promise for targeting CD123 and CLL-1, with acceptable toxicity. Future developments in CAR-T cell therapy and other immunotherapies are anticipated to improve AML treatment outcomes.

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Hematopoietic stem cells (HSCs) maintain homeostasis in the hematopoietic ecosystem, which is tightly regulated at multiple layers. Acute myeloid leukemia (AML) is a severe hematologic malignancy driven by genetic and epigenetic changes that lead to the transformation of leukemia stem cells (LSCs). Since somatic mutations in DNA methylation-related genes frequently occur in AML, DNA methylation is widely altered and functions as a starting engine for initiating AML. Additionally, RNA modifications, especially N6-methyladenosine (m6A), also play an important role in the generation and maintenance of the hematopoietic ecosystem, and AML development requires reprogramming of m6A modifications to facilitate cells with hallmarks of cancer. Given the complex pathogenesis and poor prognosis of AML, it is important to fully understand its pathogenesis. Here, we mainly focus on DNA methylation and RNA m6A modification in hematopoiesis and AML and summarize recent advances in this field.

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Measurable residual disease (MRD) in acute myeloid leukemia (AML) refers to the quantity of residual leukemic cells in a patient after treatment.According to the latest agreements, MRD in AML offering essential prognostic insights. However, there is ongoing debate regarding MRD-based monitoring and treatment strategies. There are multiple platforms for detecting MRD, each varying in sensitivity and suitability for different patients. MRD not only predicts treatment outcomes but also serves as an indicator of treatment effectiveness and a prognostic biomarker. In AML, most retrospective studies indicate that patients who are MRD-positive or show increasing MRD levels at specific time points during remission have significantly higher risks of relapse and mortality compared to MRD-negative patients. Although achieving MRD-negative status can improve patient prognosis, the possibility of relapse remains. Despite the correlation between MRD and clinical outcomes, MRD assessment methods are not yet standardized, leading to discrepancies in results across different techniques. To provide reliable MRD results, it is essential to optimize and standardize MRD detection methods. Methods for assessing MRD include multiparameter flow cytometry (MFC) and molecular assays, chosen based on disease characteristics. This review focuses on currently available MRD detection methods and discusses how the prognostic value of MRD test results informs personalized treatment strategies for AML patients.

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Acute myeloid leukemia (AML), as the most common malignancy of the hematopoietic system, poses challenges in treatment efficacy, relapse, and drug resistance. In this study, we have utilized 151 RNA sequencing datasets, 194 DNA methylation datasets, and 200 somatic mutation datasets from the AML cohort in the TCGA database to develop a multi-omics stratification model. This model enables comparison of prognosis, clinical features, gene mutations, immune microenvironment and drug sensitivity across subgroups. External validation datasets have been sourced from the GEO database, which includes 562 mRNA datasets and 136 miRNA datasets from 984 adult AML patients. Through multi-omics-based stratification model, we classified 126 AML patients into 4 clusters (CS). CS4 had the best prognosis, with the youngest age, highest M3 subtype proportion, fewest copy number alterations, and common mutations in WT1, FLT3, and KIT genes. It showed sensitivity to HDAC inhibitors and BCL-2 inhibitors. Both the M3 subtype and CS4 were identified as independent protective factors for survival. Conversely, CS3 had the worst prognosis due to older age, high copy number alterations, and frequent mutations in RUNX1, DNMT3A, and TP53 genes. Additionally, it showed higher proportions of cytotoxic cells and Tregs, suggesting potential sensitivity to mTOR inhibitors. CS1 had a better prognosis than CS2, with more copy number alterations, while CS2 had higher monocyte proportions. CS1 showed good sensitivity to cytarabine, while CS2 was sensitive to RXR agonists. Both CS1 and CS2, which predominantly featured mutations in FLT3, NPM1, and DNMT3A genes, benefited from FLT3 inhibitors. Using the Kappa test, our stratification model underwent robust validation in the miRNA and mRNA external validation datasets. With advancements in sequencing technology and machine learning algorithms, AML is poised to transition towards multi-omics precision medicine in the future. We aspire for our study to offer new perspectives on multi-drug combination clinical trials and multi-targeted precision medicine for AML.

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Acute myeloid leukemia (AML), a highly heterogeneous myeloid malignancy, remains a challenge in terms of proper risk stratification. In this study, we developed a novel pyroptosis prognostic model based on pyroptosis-related gene pairs, which exhibited excellent prognostic performance across multiple cohorts (N = 1506) and accurately predicted both adult and pediatric AML prognosis. Additionally, we integrated the pyroptosis risk model with other clinical risk factors to construct a highly operational nomogram. Moreover, our findings indicate a significant correlation between elevated pyroptosis risk scores and increased stemness of AML. Using CIBERSORT immune analysis, we found a decreased proportion of resting NK cells and activated mast cells in the high-risk group. Through analyzing the correlation between chemotherapy drug response sensitivity and risk scores, we found that AZD1332 and BPD-0008900 were extremely sensitive in the high-risk group.

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Early post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) relapse in patients with acute myeloid leukemia (AML) has an almost invariably dismal prognosis. Recent studies have demonstrated that FLT3 inhibition enhances the graft-versus-leukemia effect in vitro and in vivo. Thus, FLT-3 inhibitors may be viable treatment options in this setting. Here, we report three patients with FLT3 and NPM1 mutated AML who relapsed early after allo-HSCT and were treated with gilteritinib (associated with donor lymphocyte Infusion in two patients) to achieve long-term remission without a second transplantation.

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Leukemia is a malignant tumor with high heterogeneity and a complex evolutionary process. It is difficult to resolve the heterogeneity and clonal evolution of leukemia cells by applying traditional bulk sequencing techniques, thus preventing a deep understanding of the mechanisms of leukemia development and the identification of potential therapeutic targets. However, with the development and application of single-cell sequencing technology, it is now possible to investigate the gene expression profile, mutations, and epigenetic features of leukemia at the single-cell level, thus providing a new perspective for leukemia research. In this article, we review the recent applications and advances of single-cell sequencing technology in leukemia research, discuss its potential for enhancing our understanding of the mechanisms of leukemia development, discovering therapeutic targets and personalized treatment, and provide reference guidelines for the significance of this technology in clinical research.