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.

A Brief Overview of the Molecular Landscape of Myelodysplastic Neoplasms.

Myelodysplastic neoplasm (MDS) is a heterogeneous group of clonal hematological disorders that originate from the hematopoietic and progenitor cells and present with cytopenias and morphologic dysplasia with a propensity to progress to bone marrow failure or acute myeloid leukemia (AML). Genetic evolution plays a critical role in the pathogenesis, progression, and clinical outcomes of MDS. This process involves the acquisition of genetic mutations in stem cells that confer a selective growth advantage, leading to clonal expansion and the eventual development of MDS. With the advent of next-generation sequencing (NGS) assays, an increasing number of molecular aberrations have been discovered in recent years. The knowledge of molecular events in MDS has led to an improved understanding of the disease process, including the evolution of the disease and prognosis, and has paved the way for targeted therapy. The 2022 World Health Organization (WHO) Classification and the International Consensus Classification (ICC) have incorporated the molecular signature into the classification system for MDS. In addition, specific germline mutations are associated with MDS development, especially in pediatrics and young adults. This article reviews the genetic abnormalities of MDS in adults with a brief review of germline predisposition syndromes.

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.

TP53 Mutations in Acute Leukemias and Myelodysplastic Syndromes: Insights and Treatment Updates.

TP53 mutations are found in 5%-10% of de novo myelodysplastic syndrome (MDS) and AML cases. By contrast, in therapy related MDS and AML, mutations in TP53 are found in up to 30%-40% of patients. The majority of inactivating mutations observed in MDS and AML are missense mutations localized in a few prevalent hotspots. TP53 missense mutations together with truncating mutations or chromosomal loss of TP53 determine a loss-of-function effect on normal p53 function. Clonal expansion of TP53-mutant clones is observed under the selection pressure of chemotherapy or MDM2 inhibitor therapy. TP53-mutant clones are resistant to current chemotherapy, and when responses to treatment have been observed, they have correlated poorly with overall survival. The most heavily investigated and targeted agent for patients with TP53-mutant MDS and AML has been APR-246 (eprenetapopt) a p53 reactivator, in combination with azacitidine, but also in triplets with venetoclax. Despite positive results in phase II trials, a phase III trial did not confirm superior response or improved survival. Other agents, like magrolimab (anti-CD47 antibody), failed to demonstrate improved activity in TP53-mutant MDS and AML. Agents whose activity is not dependent on a functional apoptosis system like anti-CD123 antibodies or cellular therapies are in development and may hold promises. Delivering prognostic information in a dismal disease like TP53-mutated MDS and AML is particularly challenging. The physician should balance hope and realism, describing the trajectory of possible treatments and at the same time indicating the poor outcome, together with promoting adaptive coping in patients and elaborating on the nature of the disease.

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.

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.

[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.

Adding the epigenomic signature to the prognostic jigsaw of myelodysplastic neoplasm?

Defining mechanisms of resistance to hypomethylating agents (HMAs) and biomarkers predictive of treatment response remains challenging in myelodysplastic neoplasm (MDS). Currently available prognostic tools that predict overall survival and transformation to acute myeloid leukaemia have not been powered to predict responses to HMAs. Noguera-Castells et al. comprehensively characterized the epigenomic profile in patients with MDS treated with azacitidine and described a methylation signature-based prognostic tool in predicting responses to azacitidine. Commentary on: Noguera-Castells et al. DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: a multicentre retrospective study. Br J Haematol 2024;204:1838-1843.

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.

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.

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.

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.

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 ).

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.

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.

[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.