Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury.

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

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.

Reporting bone marrow biopsies for myelodysplastic neoplasms and acute myeloid leukaemia incorporating WHO 5th edition and ICC 2022 classification systems: ALLG/RCPA joint committee consensus recommendations.

The classification of myeloid neoplasms continues to evolve along with advances in molecular diagnosis, risk stratification and treatment of disease. An approach for disease classification has been grounded in international consensus that has facilitated understanding, identification and management of molecularly heterogeneous entities, as well as enabled consistent patient stratification into clinical trials and clinical registries over time. The new World Health Organization (WHO) and International Consensus Classification (ICC) Clinical Advisory Committee releasing separate classification systems for myeloid neoplasms in 2022 precipitated some concern amongst haematopathology colleagues both locally and internationally. While both classifications emphasise molecular disease classification over the historical use of morphology, flow cytometry and cytogenetic based diagnostic methods, notable differences exist in how morphological, molecular and cytogenetic criteria are applied for defining myelodysplastic neoplasms (MDS) and acute myeloid leukaemias (AML). Here we review the conceptual advances, diagnostic nuances, and molecular platforms required for the diagnosis of MDS and AML using the new WHO and ICC 2022 classifications. We provide consensus recommendations for reporting bone marrow biopsies. Additionally, we address the logistical challenges encountered implementing these changes into routine laboratory practice in alignment with the National Pathology Accreditation Advisory Council reporting requirements for Australia and New Zealand.

A new therapeutic perspective: Erastin inhibits tumor progression by driving ferroptosis in myelodysplastic syndromes.

Ferroptosis is a recently identified and evolutionarily conserved form of programmed cell death. This process is initiated by an imbalance in iron metabolism, leading to an overload of ferrous ions. These ions promote lipid peroxidation in the cell membrane through the Fenton reaction. As the cell's antioxidant defenses become overwhelmed, a fatal buildup of reactive oxygen species (ROS) occurs, resulting in the rupture of the plasma membrane. Ferroptosis is implicated in conditions such as ischemia-reperfusion injuries and a range of cancers. In our research, we explored ferroptosis in myelodysplastic syndromes (MDS) by measuring iron levels, transferrin receptor expression, and glutathione peroxidase 4 (GPX4) mRNA. Our findings revealed that MDS patients had significantly higher Fe2+ levels in CD33+ cells and increased transferrin receptor mRNA compared to healthy individuals. GPX4 expression was also higher in MDS but not statistically significant. To investigate potential treatments for myeloid hematological diseases through ferroptosis induction, we treated the myelodysplastic syndrome cell line (SKM-1) and two myeloid leukemia cell lines (KG-1 and K562) with erastin, an iron transfer inducer. We observed that erastin treatment led to glutathione depletion, reduced GPX4 activity, and increased ROS, culminating in cell death by ferroptosis. Furthermore, combining erastin with azacitidine demonstrated a synergistic effect on MDS and leukemia cell lines, suggesting a promising approach for treating these hematological conditions with this drug combination. Our experiments confirm erastin's ability to induce ferroptosis in MDS and highlight its potential synergistic use with azacitidine for treatment.

[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.

Prevalence of massively diluted bone marrow cell samples aspirated from patients with myelodysplastic syndromes (MDS) or suspected of MDS: A retrospective analysis of nationwide samples in Japan.

Bone marrow (BM) examination is a key element in the diagnosis and prognostic grading of myelodysplastic syndromes (MDSs), and obtaining adequate BM cell samples is critical for accurate test results. Massive haemodilution of aspirated BM samples is a well-known problem; however, its incidence in patients with MDS has not been well studied. We report the first study to examine the incidence of massive haemodilution in nationwide BM samples aspirated from patients diagnosed with or suspected of MDS in Japan. Among 283 cases available for analysis, BM smears from 92 cases (32.5%) were hypospicular (massively haemodiluted) and, particularly, no BM particles were observed in 52 cases (18.4%). Regarding hypospicular cases, we examined how the doctors in charge interpreted the BM smears of their patients. In only 19 of 92 cases (20.7%), doctors realised that the BM smears were haemodiluted. Furthermore, the BM biopsy, which can help diagnose hypospicular cases, was oftentimes not performed when the haemodilution was overlooked by doctors (not performed in 50 of 73 such cases). These real-world data highlight that not only researchers who are working to improve diagnostic tests but also clinicians who perform and use diagnostic tests must realise this common and potentially critical problem.

Revisiting the most often used item in the haematological tool box-The extent of haemodilution in bone marrow aspirates.

In this issue, a nationwide retrospective Japanese study finds that, in a second opinion setting, one-third of bone marrow aspirates from patients suspected of myelodysplastic syndromes are heavily haemodiluted. Moreover, in four-fifths of such cases, the failure to obtain the correct material for diagnosis went undetected by the referring institution. These data are intriguing, but given their special set-up, caution should be exerted in transposing them to other countries. Commentary on: Ogata et al. Prevalence of massively diluted bone marrow cell samples aspirated from patients with myelodysplastic syndromes (MDS) or suspected MDS: A retrospective analysis of nationwide samples in Japan. Br J Haematol 2024;204:1856-1861.

Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

Rapid progression from MDS to AML with gastric submucosal tumour as an extramedullary infiltration.

We present a rare case of myeloid sarcoma in the stomach of an elderly woman initially diagnosed with anaemia. Myeloid sarcoma, an unusual extramedullary manifestation of acute myeloid leukaemia (AML), primarily affects lymph nodes, bones, spine and skin, with gastrointestinal involvement being infrequent. Despite normal results from the initial endoscopy, a follow-up examination after 4 months revealed multiple submucosal gastric tumours. These developments coincided with worsening of anaemia and an increase in peripheral myeloblasts. Pathological evaluation and immunohistochemical staining confirmed gastric extramedullary infiltration associated with AML. This case highlights the importance of comprehensive diagnostic processes when suspecting leukaemic transformations, especially in myelodysplastic syndrome (MDS). Due to financial constraints, additional critical studies such as cytogenetics and next-generation sequencing were not performed. Nonetheless, this rare case demonstrates the visual observation of rapid progression from MDS to AML and concurrent early myeloid sarcoma development in an elderly patient.

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.

Single-cell transcriptomics dissects the transcriptome alterations of hematopoietic stem cells in myelodysplastic neoplasms.

BACKGROUND: Myelodysplastic neoplasms (MDS) are myeloid neoplasms characterized by disordered differentiation of hematopoietic stem cells and a predisposition to acute myeloid leukemia (AML). The underline pathogenesis remains unclear. METHODS: In this study, the trajectory of differentiation and mechanisms of leukemic transformation were explored through bioinformatics analysis of single-cell RNA-Seq data from hematopoietic stem and progenitor cells (HSPCs) in MDS patients. RESULTS: Among the HSPC clusters, the proportion of common myeloid progenitor (CMP) was the main cell cluster in the patients with excess blasts (EB)/ secondary AML. Cell cycle analysis indicated the CMP of MDS patients were in an active proliferative state. The genes involved in the cell proliferation, such as MAML3 and PLCB1, were up-regulated in MDS CMP. Further validation analysis indicated that the expression levels of MAML3 and PLCB1 in patients with MDS-EB were significantly higher than those without EB. Patients with high expression of PLCB1 had a higher risk of transformation to AML. PLCB1 inhibitor can suppress proliferation, induce cell cycle arrest, and activate apoptosis of leukemic cells in vitro. CONCLUSION: This study revealed the transcriptomic change of HSPCs in MDS patients along the pseudotime and indicated that PLCB1 plays a key role in the transformation of MDS into leukemia.

Comparative analysis of feature-based ML and CNN for binucleated erythroblast quantification in myelodysplastic syndrome patients using imaging flow cytometry data.

Myelodysplastic syndrome is primarily characterized by dysplasia in the bone marrow (BM), presenting a challenge in consistent morphology interpretation. Accurate diagnosis through traditional slide-based analysis is difficult, necessitating a standardized objective technique. Over the past two decades, imaging flow cytometry (IFC) has proven effective in combining image-based morphometric analyses with high-parameter phenotyping. We have previously demonstrated the effectiveness of combining IFC with a feature-based machine learning algorithm to accurately identify and quantify rare binucleated erythroblasts (BNEs) in dyserythropoietic BM cells. However, a feature-based workflow poses challenges requiring software-specific expertise. Here we employ a Convolutional Neural Network (CNN) algorithm for BNE identification and differentiation from doublets and cells with irregular nuclear morphology in IFC data. We demonstrate that this simplified AI workflow, coupled with a powerful CNN algorithm, achieves comparable BNE quantification accuracy to manual and feature-based analysis with substantial time savings, eliminating workflow complexity. This streamlined approach holds significant clinical value, enhancing IFC accessibility for routine diagnostic purposes.

5G2 mutant mice model loss of a commonly deleted segment of chromosome 7q22 in myeloid malignancies.

Monosomy 7 and del(7q) are among the most common and poorly understood genetic alterations in myelodysplastic neoplasms and acute myeloid leukemia. Chromosome band 7q22 is a minimally deleted segment in myeloid malignancies with a del(7q). However, the rarity of "second hit" mutations supports the idea that del(7q22) represents a contiguous gene syndrome. We generated mice harboring a 1.5 Mb germline deletion of chromosome band 5G2 syntenic to human 7q22 that removes Cux1 and 27 additional genes. Hematopoiesis is perturbed in 5G2+/del mice but they do not spontaneously develop hematologic disease. Whereas alkylator exposure modestly accelerated tumor development, the 5G2 deletion did not cooperate with KrasG12D, NrasG12D, or the MOL4070LTR retrovirus in leukemogenesis. 5G2+/del mice are a novel platform for interrogating the role of hemopoietic stem cell attrition/stress, cooperating mutations, genotoxins, and inflammation in myeloid malignancies characterized by monosomy 7/del(7q).

HSPA9/mortalin inhibition disrupts erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and anemia. They are the most common myeloid blood cancers in American adults. The full complement of gene mutations that contribute to the phenotypes or clinical symptoms in MDS is not fully understood. Around 10%-25% of MDS patients harbor an interstitial heterozygous deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes, including HSPA9. The HSPA9 gene encodes for the protein mortalin, a highly conserved heat shock protein predominantly localized in mitochondria. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells. In this study, we explored the role of HSPA9 in regulating erythroid maturation using human CD34+ cells. We inhibited the expression of HSPA9 using gene knockdown and pharmacological inhibition and found that inhibition of HSPA9 disrupted erythroid maturation as well as increased expression of p53 in CD34+ cells. To test whether the molecular mechanism of HSPA9 regulating erythroid maturation is TP53-dependent, we knocked down HSPA9 and TP53 individually or in combination in human CD34+ cells. We found that the knockdown of TP53 partially rescued the erythroid maturation defect induced by HSPA9 knockdown, suggesting that the defect in cells with reduced HSPA9 expression is TP53-dependent. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to the anemia observed in del(5q)-associated MDS patients due to the activation of TP53.

Applications of Circulating Tumor DNA in Myelodysplastic Syndromes and Acute Myeloid Leukemia: Promises and Challenges.

The term 'liquid biopsy' has become widely used by clinicians with the development of non-invasive diagnostic and monitoring techniques for malignancies. Liquid biopsy can provide genetic information for early diagnosis, risk stratification, treatment selection and postoperative follow-up. In the era of personalized medicine, liquid biopsy is an important research direction. In recent years, research on circulating tumour DNA (ctDNA) in hematological malignancies has also made great progress. This review provides an overview of the current understanding of circulating tumour DNA in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Additionally, recent advancements in the monitoring of minimal/measurable residual disease (MRD) through ctDNA are discussed.