Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

Gene Expression and DNA Methylation Profiling Suggest Potential Biomarkers for Azacitidine Resistance in Myelodysplastic Syndrome.

Myelodysplastic syndrome/neoplasm (MDS) comprises a group of heterogeneous hematopoietic disorders that present with genetic mutations and/or cytogenetic changes and, in the advanced stage, exhibit wide-ranging gene hypermethylation. Patients with higher-risk MDS are typically treated with repeated cycles of hypomethylating agents, such as azacitidine. However, some patients fail to respond to this therapy, and fewer than 50% show hematologic improvement. In this context, we focused on the potential use of epigenetic data in clinical management to aid in diagnostic and therapeutic decision-making. First, we used the F-36P MDS cell line to establish an azacitidine-resistant F-36P cell line. We performed expression profiling of azacitidine-resistant and parental F-36P cells and used biological and bioinformatics approaches to analyze candidate azacitidine-resistance-related genes and pathways. Eighty candidate genes were identified and found to encode proteins previously linked to cancer, chronic myeloid leukemia, and transcriptional misregulation in cancer. Interestingly, 24 of the candidate genes had promoter methylation patterns that were inversely correlated with azacitidine resistance, suggesting that DNA methylation status may contribute to azacitidine resistance. In particular, the DNA methylation status and/or mRNA expression levels of the four genes (AMER1, HSPA2, NCX1, and TNFRSF10C) may contribute to the clinical effects of azacitidine in MDS. Our study provides information on azacitidine resistance diagnostic genes in MDS patients, which can be of great help in monitoring the effectiveness of treatment in progressing azacitidine treatment for newly diagnosed MDS patients.

Acute Myeloid Leukemia with Myelodysplasia - Related Changes after Isolated Myeloid Sarcoma.

BACKGROUND: As a tumor mass, a myeloid sarcoma consists of myeloid blasts and presents at an anatomical site other than the bone marrow. In about one quarter of cases, myeloid sarcoma happens without an underlying acute myeloid leukemia or other myeloid neoplasm, and it may precede or coincide with AML or form acute blastic transformation of MDSs, MPNs, or MDS/MPNs. METHODS: Herein, we described a rare case of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), with WT1 mutation and high expression of TP53 after isolated myeloid sarcoma of lymph nodes showing a higher proportion of blasts, dysplasia of both megakaryocytes and granulocytes. CONCLUSIONS: The case highlights the importance of a bone marrow examination, including morphology, immunophenotyping, cytogenetic, and molecular examination in all cases to exclude the possibility of myeloid sarcoma, especially the morphological feature of bone marrow dysplasia in the early stage before AML.

Caspase 8 deletion causes infection/inflammation-induced bone marrow failure and MDS-like disease in mice.

Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by cytopenia in peripheral blood due to ineffective hematopoiesis and normo- or hypercellularity and morphologic dysplasia in bone marrow (BM). An inflammatory BM microenvironment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) are thought to be the major causes of ineffective hematopoiesis in MDS. Pyroptosis, apoptosis and necroptosis (collectively, PANoptosis) are observed in BM tissues of MDS patients, suggesting an important role of PANoptosis in MDS pathogenesis. Caspase 8 (Casp8) is a master regulator of PANoptosis, which is downregulated in HSPCs from most MDS patients and abnormally spliced in HSPCs from MDS patients with SRSF2 mutation. To study the role of PANoptosis in hematopoiesis, we generated inducible Casp8 knockout mice (Casp8-/-). Mx1-Cre-Casp8-/- mice died of BM failure within 10 days of polyI:C injections due to depletion of HSPCs. Rosa-ERT2Cre-Casp8-/- mice are healthy without significant changes in BM hematopoiesis within the first 1.5 months after Casp8 deletion. Such mice developed BM failure upon infection or low dose polyI:C/LPS injections due to the hypersensitivity of Casp8-/- HSPCs to infection or inflammation-induced necroptosis which can be prevented by Ripk3 deletion. However, impaired self-renewal capacity of Casp8-/- HSPCs cannot be rescued by Ripk3 deletion due to activation of Ripk1-Tbk1 signaling. Most importantly, mice transplanted with Casp8-/- BM cells developed MDS-like disease within 4 months of transplantation as demonstrated by anemia, thrombocytopenia and myelodysplasia. Our study suggests an essential role for a balance in Casp8, Ripk3-Mlkl and Ripk1-Tbk1 activities in the regulation of survival and self-renewal of HSPCs, the disruption of which induces inflammation and BM failure, resulting in MDS-like disease.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

Anesthetic Management of a Patient with MIRAGE Syndrome: A Case Report.

MIRAGE syndrome consists of Myelodysplasia, Infection, Growth restriction, Adrenal hypoplasia, Genital phenotypes, and Enteropathy. We report the uneventful anesthesia management of a 6-year-old female patient with MIRAGE syndrome. We think it can guide anesthesiologists caring for patients with this syndrome to find the appropriate method for them.

[Efficacy and safety analysis of venetoclax combined with hypomethylating agents for the treatment of higher-risk myelodysplastic syndromes in the real world].

Objective: To investigate the efficacy and safety of combining venetoclax (VEN) with hypomethylated drugs (HMA) in the treatment of higher-risk (IPSS-R score >3.5) myelodysplastic syndromes (MDS) . Methods: From March 2021 to December 2022, forty-five MDS patients with intermediate and high risk were treated with VEN in combination with HMAs. Clinical data were collected and analyzed retrospectively, including gender, age, MDS subtype, IPSS-R score, treatment regimen, and efficacy, etc. Kaplan-Meier method and Cox regression model were used to analyze univariate and multivariate of survival prognosis. Results: ①Forty-five patients with MDS, including ninety-one percent were classified as high or very high risk. According to the 2023 consensus proposal for revised International Working Group response criteria for higher-risk MDS, the overall response rate (ORR) was 62.2% (28/45), with the complete response rate (CR) was 33.3% (15/45). For twenty-five naïve MDS, the ORR was 68% (17/25) and the CR rate was 32% (8/25). In nonfirst-line patients, the ORR and CR were 55% (11/20) and 35% (7/20) respectively. The median cycle to best response was 1 (1-4). ②With a median followup of 189 days, the median overall survival (OS) time was 499 (95% confidence interval, 287-711) days, and most patients died from disease progression. Responders had a significantly better median OS time than nonresponders (499 days vs 228 days, P<0.001). Multifactor analysis revealed that IPSS-R score and response to treatment were independent prognostic factors for OS; the presence of SETBP1 gene mutations was associated with a longer hospital stay (51.5 days vs 27 days, P=0.017) . Conclusions: There is clinical benefit of venetoclax in combination with hypomethylated agents in patients with higher-risk MDS, but adverse events such as severe hypocytopenia during treatment should be avoided.

[Study of a patient with Myelodysplastic/myeloproliferative neoplasm with co-morbid neutrophilia and a novel NCOR1::GLYR1 fusion gene].

OBJECTIVE: To explore the genetic background for a patient with refractory myelodysplastic/myeloproliferative neoplasm (MDS/MPN) with co-morbid neutrophilia patient. METHODS: A MDS/MPN patient who was admitted to the First Affiliated Hospital of Nanjing Medical University in May 2021 was selected as the study subject. RNA sequencing was carried out to identify fusion genes in his peripheral blood mononuclear cells. Fusion gene sequence was searched through transcriptome-wide analysis with a STAR-fusion procedure. The novel fusion genes were verified by quantitative real-time PCR and Sanger sequencing. RESULTS: The patient, a 67-year-old male, had progressive thrombocytopenia. Based on the morphological and molecular examinations, he was diagnosed as MDS/MPN with co-morbid neutropenia, and was treated with demethylating agents and Bcl-2 inhibitors. Seventeen months after the diagnosis, he had progressed to AML. A novel fusion gene NCOR1::GLYR1 was identified by RNA-sequencing in his peripheral blood sample, which was verified by quantitative real-time PCR and Sanger sequencing. The patient had attained morphological remission after a DCAG regimen (a combinatory chemotherapy of decitabine, cytarabine, aclarubicin and granulocyte colony-stimulating factors) plus Chidamide treatment. A significant decrease in the NCOR1::GLYR1 expression was revealed by quantitative real-time PCR at post-chemotherapy evaluation. CONCLUSION: NCOR1::GLYR1 gene is considered as the pathogenic factor for the MDS/MPN patient with neutropenia.

[Pathogenesis of myelodysplastic syndromes by excessive mitochondrial fragmentation].

Myelodysplastic syndromes (MDS) are a group of heterogenous hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis in which clonal progenitor expansion occurs alongside impaired myelopoiesis. Inflammatory signaling activation due to dysregulated innate immunity is also a hallmark of MDS pathogenesis. We recently established a useful preclinical tool that recapitulates bona fide MDS phenotypes and gene expression profiles based on previously unreported co-mutations discovered during our clinical surveillance of mutations in patients with MDS. Notably, we focused unbiased transcriptome analysis on determining the distinct underlying mediators of MDS etiology, and identified excessive mitochondrial fission-mediated fragmentation in mutant HSCs and progenitors (HSC/Ps). We confirmed excessive mitochondrial fragmentation in HSC/Ps obtained from patients with MDS regardless of the mutational profile. Importantly, in vivo pharmacological inhibition of mitochondrial fission significantly attenuated inflammatory signaling activation, dysplasia formation and ineffective hematopoiesis phenotype, and prolonged survival of MDS mice, suggesting that excessive mitochondrial fragmentation could be a fundamental trigger of MDS pathogenesis. These findings provide new insights into the mechanistic basis of ineffective hematopoiesis, and a clue for targeting bone marrow failure caused by ineffective hematopoiesis in MDS.

[Clinical and genetic features of MDS associated with VEXAS syndrome].

VEXAS syndrome is a new disease entity characterized by the presence of cytoplasmic vacuoles in blood cells, X-linked autoinflammatory symptoms, and somatic variants in UBA1, which encodes an E1 ubiquitin-activating enzyme. Around 30-50% of VEXAS syndrome patients have concurrent MDS. We and others have recently analyzed clinical and genetic features of MDS associated with VEXAS syndrome and found that most of these cases are categorized in the low-risk subgroup with low bone marrow blast percentages. MDS associated with VEXAS syndrome tended to involve a smaller number of genes and lower-risk genetic alterations than classical MDS. In addition, anemia in MDS associated with VEXAS syndrome with active inflammation before treatment tended to respond well to steroids. In this review, we will present our recent findings together with others, focusing on the new disease entity and pathophysiology of VEXAS syndrome and clinical/genetic features of associated MDS.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

Combination therapy with venetoclax and azacitidine for the treatment of myelodysplastic syndromes with DDX41 mutations.

Myelodysplastic syndromes (MDS) patients with DEAD-box helicase 41 (DDX41) mutations have been reported to be treated effectively with lenalidomide; however, there are no randomized studies to prove it. Venetoclax and azacitidine are safe and effective in high-risk MDS/AML. In this study, we evaluated the efficacy of venetoclax and azacitidine combination therapy in eight consecutive MDS patients with DDX41 mutations at our centre from March 2021 to November 2023. We retrospectively analyzed the genetic features and clinical characteristics of these patients. Our findings suggest that MDS patients with DDX41 mutation may benefit from the therapy, for six subjects received this regimen as initial therapy and five of the six subjects achieved complete remission.

Construction and experimental validation of a novel ferroptosis-related gene signature for myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by morphological abnormalities and peripheral blood cytopenias, carrying a risk of progression to acute myeloid leukemia. Although ferroptosis is a promising target for MDS treatment, the specific roles of ferroptosis-related genes (FRGs) in MDS diagnosis have not been elucidated. METHODS: MDS-related microarray data were obtained from the Gene Expression Omnibus database. A comprehensive analysis of FRG expression levels in patients with MDS and controls was conducted, followed by the use of multiple machine learning methods to establish prediction models. The predictive ability of the optimal model was evaluated using nomogram analysis and an external data set. Functional analysis was applied to explore the underlying mechanisms. The mRNA levels of the model genes were verified in MDS clinical samples by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The extreme gradient boosting model demonstrated the best performance, leading to the identification of a panel of six signature genes: SREBF1, PTPN6, PARP9, MAP3K11, MDM4, and EZH2. Receiver operating characteristic curves indicated that the model exhibited high accuracy in predicting MDS diagnosis, with area under the curve values of 0.989 and 0.962 for the training and validation cohorts, respectively. Functional analysis revealed significant associations between these genes and the infiltrating immune cells. The expression levels of these genes were successfully verified in MDS clinical samples. CONCLUSION: Our study is the first to identify a novel model using FRGs to predict the risk of developing MDS. FRGs may be implicated in MDS pathogenesis through immune-related pathways. These findings highlight the intricate correlation between ferroptosis and MDS, offering insights that may aid in identifying potential therapeutic targets for this debilitating disorder.

Comparison of the 2022 world health organization classification and international consensus classification in myelodysplastic syndromes/neoplasms.

In 2022, two novel classification systems for myelodysplastic syndromes/neoplasms (MDS) have been proposed: the International Consensus Classification (ICC) and the 2022 World Health Organization (WHO-2022) classification. These two contemporary systems exhibit numerous shared features but also diverge significantly in terminology and the definition of new entities. Thus, we retrospectively validated the ICC and WHO-2022 classification and found that both systems promoted efficient segregation of this heterogeneous disease. After examining the distinction between the two systems, we showed that a peripheral blood blast percentage ≥ 5% indicates adverse survival. Identifying MDS/acute myeloid leukemia with MDS-related gene mutations or cytogenetic abnormalities helps differentiate survival outcomes. In MDS, not otherwise specified patients, those diagnosed with hypoplastic MDS and single lineage dysplasia displayed a trend of superior survival compared to other low-risk MDS patients. Furthermore, the impact of bone marrow fibrosis on survival was less pronounced within the ICC framework. Allogeneic transplantation appears to improve outcomes for patients diagnosed with MDS with excess blasts in the ICC. Therefore, we proposed an integrated system that may lead to the accurate diagnosis and advancement of future research for MDS. Prospective studies are warranted to validate this refined classification.

Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Myelodysplastic Neoplasms (MDS): The Current and Future Treatment Landscape.

Myelodysplastic neoplasms (MDS) are a heterogenous clonal disorder of hemopoietic stem cells characterized by cytomorphologic dysplasia, ineffective hematopoiesis, peripheral cytopenias and risk of progression to acute myeloid leukemia (AML). Our understanding of this disease has continued to evolve over the last century. More recently, prognostication and treatment have been determined by cytogenetic and molecular data. Specific genetic abnormalities, such as deletion of the long arm of chromosome 5 (del(5q)), TP53 inactivation and SF3B1 mutation, are increasingly associated with disease phenotype and outcome, as reflected in the recently updated fifth edition of the World Health Organization Classification of Hematolymphoid Tumors (WHO5) and the International Consensus Classification 2022 (ICC 2022) classification systems. Treatment of lower-risk MDS is primarily symptom directed to ameliorate cytopenias. Higher-risk disease warrants disease-directed therapy at diagnosis; however, the only possible cure is an allogenic bone marrow transplant. Novel treatments aimed at rational molecular and cellular pathway targets have yielded a number of candidate drugs over recent years; however few new approvals have been granted. With ongoing research, we hope to increasingly offer our MDS patients tailored therapeutic approaches, ultimately decreasing morbidity and mortality.

Germline MYOF1::WNK4 and VPS25::MYOF1 Chimeras Generated by the Constitutional Translocation t(17;19)(q21;p13) in Two Siblings With Myelodysplastic Syndrome.

BACKGROUND/AIM: Constitutional chromosomal aberrations are rare in hematologic malignancies and their pathogenetic role is mostly poorly understood. We present a comprehensive molecular characterization of a novel constitutional chromosomal translocation found in two siblings - sisters - diagnosed with myelodysplastic syndrome (MDS). MATERIALS AND METHODS: Bone marrow and blood cells from the two patients were examined using G-banding, RNA sequencing, PCR, and Sanger sequencing. RESULTS: We identified a balanced t(17;19)(q21;p13) translocation in both siblings' bone marrow, blood cells, and phytohemagglutinin-stimulated lymphocytes. The translocation generated a MYO1F::WNK4 chimera on the der(19)t(17;19), encoding a chimeric serine/threonine kinase, and a VPS25::MYO1F on the der(17), potentially resulting in an aberrant VPS25 protein. CONCLUSION: The t(17;19)(q21;p13) translocation found in the two sisters probably predisposed them to myelodysplasia. How the MYO1F::WNK4 and/or VPS25::MYO1F chimeras, perhaps especially MYO1F::WNK4 that encodes a chimeric serine/threonine kinase, played a role in MDS pathogenesis, remains incompletely understood.

Gene expression patterns of Sirtuin family members (SIRT1 TO SIRT7): Insights into pathogenesis and prognostic of Myelodysplastic neoplasm.

To assess and validate the gene expression profile of SIRTs (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) in relation to the pathogenesis and prognostic progression of Myelodysplastic neoplasm (MDS). Eighty bone marrow samples of patients with de novo MDS were diagnosed according to WHO 2022 and IPSS-R criteria. Ten bone marrow samples were obtained from elderly healthy volunteers and used as control samples. Gene expression levels of all SIRTs were assessed using RT-qPCR assays. Downregulation of SIRT2 (p = 0.009), SIRT3 (p = 0.048), SIRT4 (p = 0.049), SIRT5 (p = 0.046), SIRT6 (p = 0.043), and SIRT7 (p = 0.047) was identified in MDS patients compared to control individuals. Also, we identified that while SIRT2-7 genes are typically down-regulated in MDS patients compared to normal controls, there are relative expression variations among MDS patient subgroups. Specifically, SIRT4 (p = 0.029) showed increased expression in patients aged 60 or above, and both SIRT2 (p = 0.016) and SIRT3 (p = 0.036) were upregulated in patients with hemoglobin levels below 8 g/dL. SIRT2 (p = 0.045) and SIRT3 (p = 0.033) were highly expressed in patients with chromosomal abnormalities. Different SIRTs exhibited altered expression patterns concerning specific MDS clinical and prognostic characteristics. The downregulation in SIRTs genes (e.g., SIRT2 to SIRT7) expression in Brazilian MDS patients highlights their role in the disease's development. The upregulation of SIRT2 and SIRT3 in severe anemia patients suggests a potential link to manage iron overload-related complications in transfusion-dependent patients. Moreover, the association of SIRT2/SIRT3 with genomic instability and their role in MDS progression signify promising areas for future research and therapeutic targets. These findings underscore the importance of SIRT family in understanding and addressing MDS, offering novel clinical, prognostic, and therapeutic insights for patients with this condition.

Measurable residual disease monitoring by ddPCR in the early posttransplant period complements the traditional MFC method to predict relapse after HSCT in AML/MDS: a multicenter retrospective study.

BACKGROUND: Droplet digital PCR (ddPCR) is widely applied to monitor measurable residual disease (MRD). However, there are limited studies on the feasibility of ddPCR-MRD monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially targeting multiple molecular markers simultaneously. METHODS: Our study collected samples from patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) in complete remission after allo-HSCT between January 2018 and August 2021 to evaluate whether posttransplant ddPCR-MRD monitoring can identify patients at high risk of relapse. RESULTS: Of 152 patients, 58 (38.2%) were MRD positive by ddPCR within 4 months posttransplant, with a median variant allele frequency of 0.198%. The detectable DTA mutations (DNMT3A, TET2, and ASXL1 mutations) after allo-HSCT were not associated with an increased risk of relapse. After excluding DTA mutations, patients with ddPCR-MRD positivity had a significantly higher cumulative incidence of relapse (CIR, 38.7% vs. 9.7%, P < 0.001) and lower rates of relapse-free survival (RFS, 55.5% vs. 83.7%, P < 0.001) and overall survival (OS, 60.5% vs. 90.5%, P < 0.001). In multivariate analysis, ddPCR-MRD positivity of non-DTA genes was an independent adverse predictor for CIR (hazard ratio [HR], 4.02; P < 0.001), RFS (HR, 2.92; P = 0.002) and OS (HR, 3.12; P = 0.007). Moreover, the combination of ddPCR with multiparameter flow cytometry (MFC) can further accurately identify patients at high risk of relapse (F+/M+, HR, 22.44; P < 0.001, F+/M-, HR, 12.46; P < 0.001 and F-/M+, HR, 4.51; P = 0.003). CONCLUSION: ddPCR-MRD is a feasible approach to predict relapse after allo-HSCT in AML/MDS patients with non-DTA genes and is more accurate when combined with MFC. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT06000306. Registered 17 August 2023 -Retrospectively registered ( https://clinicaltrials.gov/study/NCT06000306?term=NCT06000306&rank=1 ).