Myeloid Precursors Research Articles

Epigenetic Enzyme Mutations in Myeloid Malignancies Are Selected By Chromatin-Remodeling Requirements That Vary By Lineage- and Maturation-Stage

Introduction/Methods: Enzymes that modify histone H3 at lysine 27 (H3K27) to thereby regulate gene transcription (epigenetic enzymes) are recurrently inactivated by deletion and/or mutation in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemias (AML). Frustrating understanding of mechanisms is that writers and erasers of the same modification, e.g., methyltransferases that create, and demethylases that remove, H3K27 trimethylation (H3K27me3), a repression ('off') mark, are recurrently inactivated. Moreover, acetyltransferases that write H3K27 acetylation (H3K27ac), an 'on' mark mutually exclusive with H3K27me3, are also recurrently inactivated. One clue to underlying mechanisms is that MDS/MPN/AML present with diverse lineage and maturation phenotypes - perhaps these emerge from, or select for, the diverse epigenetic enzyme mutations. We therefore identified genes upregulated with specific myeloid lineage-commitment, maturation and function fates (~500 genes each) and then examined distributions of H3K27me3 and H3K27ac at these gene-loci in: (i) embryonic stem cells (ESC); (ii) hematopoietic stem and progenitor cells (HSPC); (iii) mature myeloid cells (monocyte [mono], pro-erythroblast, megakaryocyte [MK]); and (iv) AML cells.Results: Terminal-myeloid programs underwent substantial remodeling to gain H3K27ac ‘on’ mark from ESC/HSPC to mature myeloid (Fig.1A). Providing a mechanism for this, the H3K27 acetyltransferases EP300 and CREBBP were recruited into the RUNX1/SPI1 myeloid-lineage master transcription factor (MTF) hub by cooperation between their transcription activating domains. Mutated/translocated RUNX1, or mutated-NPM1 that cytoplasmically dislocated SPI1, disrupted this cooperation, and reverted hub content to default recruitment of histone deacetylases (HDAC) instead. Demonstrating cause-effect, inhibiting these HDAC renewed AML cell maturation to terminal lineage-fates. Meanwhile, MYC-target (proliferation) genes have high baseline H3K27ac in ESC and HSPC and do not require major remodeling during ontogeny (Fig.1A). An H3K27ac remodeling requirement for lineage-maturation but not proliferation/housekeeping explains selection pressure for inactivating mutations in EP300 or CREBBP, that we found in ~1.2% of MDS/MPN/AML in our (n=690) and other series. Consistent with pan-lineage-maturation needs for H3K27ac, the mutations were found in all lineage sub-types. H3K27me3 ‘off’ mark was mostly erased from myeloid programs in HSPC, but was greater at MK vs erythroid, and also at mono vs granulocyte genes (Fig.1B). This implied more need for H327me3 demethylase (KDM6A/UTX) for HSPC commitment into MK vs erythroid, or mono vs granulocyte, lineages. Accordingly, KDM6A was most upregulated in MK and mono-lineage cells (Fig.1C), and myeloid-conditional Kdm6a knockout decreased platelets and increased red cells in the spleen - reported by others: https://doi.org/10.1182/blood.V128.22.1467.1467. RUNX1-ETO has been shown to specifically impede granulocytic but not mono differentiation - https://www.nature.com/articles/2403396 and KDM6A inactivating mutations were significantly more likely to occur secondary to RUNX1-ETO (4-9% BEAT and AMLSG case series) vs other cytogenetics (0.005%). Selection for KDM6A secondary mutations could thus be to channel myeloid precursors toward lineages most efficiently impeded by primary mutations. Although H3K27me3 was substantially erased at all myeloid-commitment and terminal programs (except MK) in HSPC vs ESC, subsequent lineage-commitment and maturation entailed rewriting H3K27me3 at preceding HSPC and alternate lineage-fate programs, including at MTF genes for alternate fates (Fig.1B, D). Primary MDS/MPN/AML and AML cell lines with inactivating mutations/deletions in H3K27 methyltransferase EZH2 thus displayed aberrant co-expression of lineage MTFs and gene expression programs of normally mutually exclusive lineages (Fig.1E-G).Conclusion. Epigenetic remodeling requirements vary by myeloid lineage and maturation stage. Thus, epigenetic enzyme mutations are selected by, and cause, lineage-context of transformation. This knowledge can guide choice of specific epigenetic enzyme inhibitors to remedy the lineage-maturation defects that drive and define myeloid malignancies. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

ASXL1 Mutation Cooperating with RUNX1 Mutation Further Perturbs the Differentiation of Neutrophils

Acute myeloid leukemia(AML) is characterized by an accumulation of myeloid progenitors. This disease can be caused by various germline mutations, including runx1 and asxl1. Recently several reports showed that mutations of ASXL1 and RUNX1 tend to coexist in AML patients. However, the impact on hematopoiesis remained uncertain. Thus far, lack of an animal model was the main limitation to study the function of the double mutations. In this work, we studied the combination role of runx1 and asxl1 by establishing a novel in vivo zebrafish model. First, we identified the characterization of 6646 patients with myeloid maliganacies in the cbioprotal database. Second, we generated a zebrafish model with double mutations by crossing the ENU-induced runx1 mutant and the CRISPR-CAS9 edited asxl1 mutant. Our results showed that truncation was the most frequent mutation type in RUNX1-mutated patients(56.3%) and ASXL1-mutated patients(87.8%). Besides, the median survival time of patients carrying double mutations was shorter than those with single RUNX1 mutation(9.61 months vs. 16.06 months ) or single ASXL1 mutation(9.61 months vs. 14.47 months). In zebrafish models, we found that the pu.1(myeloid precursor marker) positive cells increased while the gata1(proerythrocyte marker) didn't change in double mutants . Furthermore, mpo and lyz (neutrophil marker) positive cells further reduced while mpeg1 and mfap4(monocyte marker) positive cells didn't show significant difference in double mutants.Overall, we demonstrated that ASXL1 mutation cooperating with RUNX1 mutation further impeded the differentiation of the myeloid progenitors into mature neutrophils and it possibly explained the cause for a worse prognosis of patients with double mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of Polymorphism in the Genes TLR3, TLR4 and TLR9 in Patients with Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a bone marrow malignancy characterized by uncontrolled proliferation of transformed myeloid precursors. It is the most common acute leukemia and is a highly heterogeneous disease. AML leads to impaired hematopoiesis, which may result in increased risk of infection. Toll-like receptors (TLRs) are major elements of the innate immune system which recognize pathogen-associated molecular patterns. They also have a role in autoimmune diseases and cancer through their recognition of endogenous danger-associated molecular patterns. Various TLRs were found to be expressed in AML cells, and their expression was found to affect growth, differentiation, and immunostimulatory capacity in AML.We retrospectively analysed 90 patients (48 females and 42 males) with diagnosis of AML, hospitalized in Department and Clinic of Hematology, Blood Neoplasms and Bone Marrow Transplantation of Wroclaw Medical University. The median age of patients was 62 (range:18-81). Infectious complications after induction chemotherapy occurred in 62 patients. In the present study, we detected the effect of seven single nucleotide polymorphisms (SNPs) located in the genes coding for TLR3 (rs5743305, rs3775296, 3775291), TLR4 (rs4986791, rs4986790) and TLR9 (rs187084, rs5743836). DNA was isolated from samples of peripheral blood taken on EDTA. Melting curve genotyping with the use of LightSNiP asays(TIB MOLBIOL, Germany) was employed to determining TLR3, TLR4 and TLR9 genotypes, and the reactions were performed LightCycler 480 device (Roche Applied Science, Germany).Statistical analyses were performer using Real Statistics Resource Pack for Microsoft Excel.We found that TLR3 rs5743305 AA homozygosity was more common in AML patients with infectious complications than in patients unaffected by infections (p=0.015). TLR4 rs4986791 C and TLR9 rs5743836 C were associated with lower levels of the inflammation marker CRP (p=0.015 and p=0.048, for rs4986791 and rs5743836, respectively), TLR4 rs4986791 C and was less frequently detected among patients with CRP>5 (p=0.028). We also found TLR9 rs187084 C to be more common in patients with adverse cytogenetic risk according to ELN criteria (0.012). Additionally, a trend for higher prevalence of extramedullary disease was observed for carriers of TLR3 rs3775296 T (p=0.054), TLR3 3775291 GG (p=0.068), TLR4 rs4986790 G (p=0.078), as well as TLR9 rs187084 TT (p=0.053).In conclusion, our results suggest that SNPs in the genes coding for TLR3, TLR4 and TLR9 may be implicated in clinical outcome of AML and is related with increased risk of infectious complications. DisclosuresWróbel: BMS: Honoraria; Roche: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BeiGene: Honoraria.

The Pan-Clk/Dyrk Inhibitor Cirtuvivint (SM08502) Exposes Mechanistic Underpinnings of Alternative Splicing As a Therapeutic Vulnerability in Heme Malignancies

Mutations in spliceosomal genes are one of the most common classes of somatic alterations in patients with Myelodysplastic Syndrome (MDS) and occur across the entire spectrum of myeloid malignancies, including 10‒25% of patients with acute myeloid leukemia (AML). These mutations occur in higher proportions in AML subjects greater than 60 years of age, or when AML has transformed from an antecedent MDS. Spliceosomal gene mutations are implicated in the production of pathological RNA splicing patterns that block cell differentiation and maintain a myeloid precursor phenotype. This suggests deranged pre-mRNA splicing is a mechanistic determinant of many heme malignancies and, as such, has provoked interest in therapeutic modulation of pre-mRNA splicing as a treatment paradigm. The CLK/DYRK family of protein kinases has been recognized as an integration hub for signal transduction-dependent modulation of alternative pre-mRNA splice junction selection through direct phosphorylation of the serine/arginine-rich splicing factor (SRSF) splice junction enhancer-binding proteins. Thus, these kinases potentially represent a druggable intervention point in alternative splicing-dependent cancers. The isoquinoline SM08502 (cirtuvivint) is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual specificity tyrosine phosphorylation-regulated kinases (DYRK1-4) with activity against only a minimal number of the remaining members of the CMGC-family kinases and the kinome as a whole. Here the consequence of pan-CLK/DYRK kinase inhibition on cell viability and tumorigenicity was evaluated across a panel of human tumor-derived AML, DLBCL, MCL, myeloma, T-ALL, and CML/CLL models. EC 50s in response to cirtuvivint ranged from 0.014 μM‒0.495 μM in 4-day in vitro cell viability assays, with low-dose responders enriched in subsets of AML, myeloma, DLBCL, MCL and T-ALL. Cell viability EC 50s were associated with induction of programed cell death at drug exposures that inhibited accumulation of phosphorylated SRSF proteins and the anti-apoptotic protein MCL-1. To directly evaluate the contribution of CLK/DYRK kinases to alternative splicing profiles, high-depth RNAseq analysis was performed across 4 cell lines (3 acute myeloid leukemia cell lines and 1 mantle cell lymphoma cell line) +/- a 6-hour exposure to 1µM cirtuvivint. Both baseline and drug-induced changes in alternative splicing events (ASEs) were measured using a multivariate analysis of splicing transcripts (rMATS). The frequency of cirtuvivint-induced ASEs was approximately 20% of total detected ASEs. Concordant drug-induced ASEs among the tested cell lines were in genes enriched in pathways known to drive oncogenesis in hematopoietic lineages, including the MAP kinase and mTOR signaling pathways. Tumor growth inhibition assays in immunocompromised mice showed a range of model-specific responses, including tumor stasis and partial to complete tumor regression at clinically relevant exposures. A cell-based synthetic-lethal screen of cirtuvivint across 36 small molecule inhibitors identified multiple BCL-2 inhibitors among the most prominent synergistic combinations. Consistent with this, combination of the BCL-2 inhibitor venetoclax with cirtuvivint was sufficient to induce tumor regressions in AML xenograft models (KG-1 and HL-60) that were resistant to either single-agent drug at the same concentrations. These observations support further evaluation of CLK/DYRK inhibitors as a therapeutic strategy for heme malignancies dependent upon alternative pre-mRNA splicing. DisclosuresBossard: Biosplice Therapeutics: Current Employment. McMillan: Prizer: Ended employment in the past 24 months. Beaupre: Pfizer: Ended employment in the past 24 months. White: Pfizer: Ended employment in the past 24 months.

NT-Probnp As Personalized Medicine Tool and New Biomarker Predicting Response to Chemotherapy and Survival in AML

NT-Probnp As Personalized Medicine Tool and New Biomarker Predicting Response to Chemotherapy and Survival in AML

Histamine Deficiency Promotes Myofibroblasts Transformation from HDC-Expressing CD11b+ Myeloid Cells in Injured Hearts Post Myocardial Infarction.

Myocardial infarction (MI) is a significant contributor to the development of heart failure. Histidine decarboxylase (HDC), the unique enzyme that converts L-histidine to histamine, is highly expressed in CD11b+ immature myeloid cells. However, the relationship between HDC-expressing macrophages and cardiac myofibroblasts remains to be explained. Here, we demonstrate that the GFP (green fluorescent protein)-labeled HDC+CD11b+ myeloid precursors and their descendants could differentiate into fibroblast-like cells in myocardial interstitium. Furthermore, we prove that CD11b+Ly6C+ monocytes/macrophages, but not CD11b+Ly6G+ granulocytes, are identified as the main cellular source for bone marrow-derived myofibroblast transformation, which could be regulated via histamine H1 and H2 receptor-dependent signaling pathways. Using HDC knockout mice, we find that histamine deficiency promotes myofibroblast transformation from Ly6C+ macrophages and cardiac fibrosis partly through upregulating the expression of Krüppel-like factor 5 (KLF5). Taken together, our data uncover a central role of HDC in regulating bone marrow-derived macrophage-to-myofibroblast transformation but also identify a histamine receptor (HR)-KLF5 related signaling pathway that mediates myocardial fibrosis post-MI. CD11b+Ly6C+ monocytes/macrophages are the main cellular source for bone marrow-derived myofibroblast transformation. Histamine inhibits myofibroblasts transformation via H1R and H2R-dependent signaling pathways, and ameliorates cardiac fibrosis partly through upregulating KLF5 expression.

A rare case of post COVID-19 hemophagocytic lymphohistiocytosis in a pediatric patient

Introduction/ObjectiveHemophagocytic Syndromes are a cluster of disorders related to cytotoxic dysfunction of T/NK-cells and are mainly subdivided into Primary (familial) and Secondary (acquired) forms, with the latter usually linked to patients with viral infections; including EBV, CMV among many others. A myriad of other causes have been associated with hemophagocytic lymphohistiocytosis (HLH), most notably systemic inflammatory conditions; especially Juvenile Rheumatoid Arthritis and hematolymphoid malignancies particularly T/NK-cell lymphomas.Methods/Case ReportA previously healthy 7-year-old boy, presented to the ER with fever and a skin rash over both lower limbs of 1 week duration. Two weeks prior he was tested for COVID-19 and was found to be positive. Physical examination further revealed slightly palpable liver and spleen. CBC was done and exhibited pancytopenia, further testing showed elevated LDH, hyperferritinemia and hypertriglyceridemia. However, serological testing for rheumatological conditions was unremarkable. Imaging studies were done and were noncontributory. Subsequently, a bone marrow aspirate and biopsy were done. The bone marrow aspirate showed afew histiocytes engulfing red blood cells and nuclear debris (hemophagocytic cells), complete trilineage maturation and normal M:E ratio of 3:1. Trephine biopsy was hypocellular for age and estimated at about 70%, composed of myeloid and erythroid precursors with various degrees of maturation. Megakaryocytes were adequate in number and showed normal morphology. Extensive histiocytic infiltration as highlighted by CD68 immunostain and focal phagocytosis were identified. CD34 highlighted <5% blasts, PAS special stain showed no fungal elements and no fibrosis was evident by Reticulin special stain. The background was devoid of lymphoid aggregates or granulomas. Stainable iron stores were depleted. No sideroblasts were identified. The patient was treated with corticosteroid and showed marked improvement and was discharged after 3 days.Results (if a Case Study enter NA)NAConclusionHemophagocytic lymphohistiocytosis can be a critical sequela of COVID-19 infection. Suggested mechanisms include impaired/delayed T-cell response and elevated levels of several inflammatory cytokines. Clinical suspesion is important in the diagnosis of these cases. Further study of this correlation is needed as we explore clinical sequelae of COVID-19 infection.

Glucocorticoids inhibit human hematopoietic stem cell differentiation toward a common ILC precursor

Innate lymphoid cells (ILCs) comprise cytotoxic natural killer (NK) cells and helper ILCs (hILCs). Human hILC development is less characterized as compared with that of NK cells, although all ILCs are developmentally related. It has been reported that the immunosuppressive drugs glucocorticoids (GCs) regulate ILC function, but whether they control ILC differentiation from hematopoietic stem cells (HSCs) is unknown. This study sought to analyze the effect of GCs on ILC development from HSCs. This study exploited an invitro system to generate and expand from peripheral blood HSCs a multipotent CD56+ ILC precursor able to differentiate into NK cells, ILC1s, and ILC3s. We also analyzed exvivo, at different time points, the peripheral blood of recipients of allogeneic HSC transplantation who were or were not treated with GCs and compared ILC subset reconstitution. Invitro, GCs favor the generation of NK cells from myeloid precursors, while they strongly impair lymphoid development. In support of these data, recipients of HSC transplantation who had been treated with GCs display a lower number of circulating hILCs, including the ILC precursor (ILCP) previously identified as a systemic substrate for tissue ILC differentiation. GCs impair the development of the CD117+ ILCP from CD34+ HSCs, while they do not affect the further steps of ILCP differentiation toward NK cells and hILC subsets. This reflects an association of GC treatment with a marked reduction of circulating hILCs in the recipients of HSC transplantation.

Large Granular Lymphocyte Expansion in Myeloid Diseases and Bone Marrow Failure Syndromes: Whoever Seeks Finds.

Large granular lymphocytes (LGL) are lymphoid cells characterized by either a T-cell or a natural killer phenotype whose expansion may be reactive to toxic, infectious, and neoplastic conditions, or result from clonal selection. Recently, the higher attention to LGL clones led to their detection in many clinical conditions including myeloid neoplasms and bone marrow failures. In these contexts, it is still unclear whether LGL cells actively contribute to anti-stem cell autoimmunity or are only a reaction to dysplastic/leukemic myelopoiesis. Moreover, some evidence exists about a common clonal origin of LGL and myeloid clones, including the detection of STAT3 mutations, typical of LGL, in myeloid precursors from myelodysplastic patients. In this article we reviewed available literature regarding the association of LGL clones with myeloid neoplasms (myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemias) and bone marrow failures (aplastic anemia and pure red cell aplasia, PRCA) focusing on evidence of pathogenic, clinical, and prognostic relevance. It emerged that LGL clones may be found in up to one third of patients, particularly those with PRCA, and are associated with a more cytopenic phenotype and good response to immunosuppression. Pathogenically, LGL clones seem to expand after myeloid therapies, whilst immunosuppression leading to LGL depletion may favor leukemic escape and thus requires caution.

OAB-056: A machine learning model based on tumor and immune biomarkers to predict undetectable measurable residual disease (MRD) in transplant-eligible multiple myeloma (MM)

<h3>Background</h3> There is expectation of using biomarkers to personalize treatment. Yet, a successful treatment selection cannot be confirmed before 5 or 10 years of progression-free survival (PFS). Treatment individualization based on the probability of an individual patient to achieve undetectable MRD with a singular regimen, could represent a new model towards personalized treatment with fast assessment of its success. This idea has not been investigated previously. <h3>Methods</h3> We sought to define a machine learning model to predict undetectable MRD in newly-diagnosed transplant-eligible MM patients (n=278). The training (n=152) and internal validation cohort (n=60) consisted of 212 active MM patients enrolled in the GEM2012MENOS65 clinical trial. The external validation cohort was defined by 66 high-risk smoldering MM patients enrolled in the GEM-CESAR clinical trial, which treatment differed only by the substitution of bortezomib by carfilzomib during induction and consolidation. Patients were included in the study based on data availability of 17 parameters (p≤.05) associated with MRD outcomes. <h3>Results</h3> We started by investigating patients' MRD status after VRD induction, HDT/ASCT and VRD consolidation (GEM2012MENOS65) according to their ISS and Revised-ISS, LDH levels, and cytogenetic alterations. High LDH levels and del(17p13), two features relatively infrequent at diagnosis, were the only parameters associated with lower rates of undetectable MRD. The ISS and R-ISS were not predictive. Therefore, we aimed to evaluate other disease features associated with MRD outcomes and develop more effective models based on machine learning logistic regression. The most effective one resulted from integrating cytogenetic [t(4;14) and/or del(17p13)], tumor burden (plasma cell clonality in bone marrow and circulating tumor cells in peripheral blood) and immune related (myeloid precursors, mature B cells, intermediate neutrophils, eosinophils, CD27negCD38pos T cells and CD56brightCD27neg NK cells) biomarkers. Data obtained for an individual patient can be substituted into our formula, which results in a numerical probability of achieving undetectable (>0.5) vs persistent (0.685 or <0.365 (observed in 102/212 patients), MRD outcomes are respectively predicted with higher confidence. Standard-confidence, high-confidence, and external validation predictions were accurate in 152/212 (71.7%), 85/102 (83.3%), and 48/66 (72.7%) patients respectively. Patients predicted to achieve undetectable MRD using standard and high-confidence values showed longer PFS and OS than those with probability of persistent MRD. <h3>Conclusions</h3> We demonstrated that selecting a regimen based on probable MRD outcomes, and confirming soon after if that probability was accurate, is a possible new approach towards individualized treatment in MM. The model is available at www.MRDpredictor.com to facilitate its use in clinical practice.

Looking beyond VEXAS: Coexistence of undifferentiated systemic autoinflammatory disease and myelodysplastic syndrome

It has been established that individuals with myelodysplastic syndromes (MDS) have a higher frequency of systemic inflammatory disorders. On the other hand, patients with autoimmune diseases are at increased risk of MDS development. Both diseases can be associated with various genetic lesions and share diverse pathogenetic mechanisms. Recently identified VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, associated with somatic mutations in UBA1, encompasses a range of inflammatory conditions involving multiple organs along with hematological pathologies, including MDS, as well as characteristic bone marrow vacuolization of myeloid and erythroid precursors. This novel syndrome drove further attention to complex associations between MDS and adult-onset inflammatory conditions. The present narrative literature review discusses the clinical presentation, pathophysiology, management of concurrent MDS and systemic inflammatory diseases in parallel to the clinical picture of VEXAS syndrome.

Characteristic bone marrow findings in patients with UBA1 somatic mutations and VEXAS syndrome

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a newly characterized syndrome with underlying somatic UBA1 mutations in myeloid cells linking hematologic disease with autoinflammatory rheumatologic disorders. Hematologic abnormalities, particularly peripheral blood cytopenia(s) may prompt bone marrow evaluation in patients with known or unrecognized VEXAS syndrome. This review highlights key findings and diagnostic considerations encountered during bone marrow examination in patients with this disorder. Frequently reported hematologic changes include macrocytic anemia, cytoplasmic vacuoles in myeloid and erythroid precursors, marrow hypercellularity, and varying degrees of dysplasia. Myelodysplastic syndrome and plasma cell neoplasms have been diagnosed in patients with VEXAS syndrome. Macrophage activation syndrome and/or hemophagocytic lymphohistiocytosis and monoclonal B-cell lymphocytosis have also been reported. The bone marrow is a target organ in VEXAS syndrome. Heightened awareness of the bone marrow features and hematologic complications may aid in identifying individuals with VEXAS who may benefit from increased disease surveillance or alternative therapeutic strategies.

Advanced Flow Cytometry Analysis Algorithms for Optimizing the Detection of "Different From Normal" Immunophenotypes in Acute Myeloid Blasts.

Acute myeloid leukemias (AMLs) are a group of hematologic malignancies that are heterogeneous in their molecular and immunophenotypic profiles. Identification of the immunophenotypic differences between AML blasts and normal myeloid hematopoietic precursors (myHPCs) is a prerequisite to achieving better performance in AML measurable residual disease follow-ups. In the present study, we applied high-dimensional analysis algorithms provided by the Infinicyt 2.0 and Cytobank software to evaluate the efficacy of antibody combinations of the EuroFlow AML/myelodysplastic syndrome panel to distinguish AML blasts with recurrent genetic abnormalities (n = 39 AML samples) from normal CD45low CD117+ myHPCs (n = 23 normal bone marrow samples). Two types of scores were established to evaluate the abilities of the various methods to identify the most useful parameters/markers for distinguishing between AML blasts and normal myHPCs, as well as to distinguish between different AML groups. The Infinicyt Compass database-guided analysis was found to be a more user-friendly tool than other analysis methods implemented in the Cytobank software. According to the developed scoring systems, the principal component analysis based algorithms resulted in better discrimination between AML blasts and myHPCs, as well as between blasts from different AML groups. The most informative markers for the discrimination between myHPCs and AML blasts were CD34, CD36, human leukocyte antigen-DR (HLA-DR), CD13, CD105, CD71, and SSC, which were highly rated by all evaluated analysis algorithms. The HLA-DR, CD34, CD13, CD64, CD33, CD117, CD71, CD36, CD11b, SSC, and FSC were found to be useful for the distinction between blasts from different AML groups associated with recurrent genetic abnormalities. This study identified both benefits and the drawbacks of integrating multiple high-dimensional algorithms to gain complementary insights into the flow-cytometry data.

Reticular dysgenesis exacerbated by hemophagocytic lymphohistiocytosis and the presence of unusual histiocyte-like cells in bone marrow

We report a rare case of agranulocytosis and lymphopenia complicated with hemophagocytic lymphohistiocytosis. Diagnosis of reticular dysgenesis was made by detection of a pathogenic stop gain variant in the AK2 gene on targeted next generation sequencing and confirmed by Sanger sequencing. Parents were found to be carriers for this variant. Bone marrow aspirate and biopsy was also performed with a clinical diagnosis of severe combined immunodeficiency with HLH. However, no hemophagocytosis was noted in the bone marrow aspirate or trephine biopsy. Instead, it showed aggregates of large histiocyte-like cells, scattered erythroid precursors and megakaryocytes. These cells were confused to be some form of storage cells, but did not resemble storage cells seen in Gaucher’s disease or Niemann Pick disease. Myeloid precursors were very few in number. Reticular dysgenesis was not suspected during admission due to a lack of awareness of this entity. Testing for sensorineural deafness in neonates with severe agranulocytosis and lymphopenia would facilitate an early diagnosis of reticular dysgenesis. To the best of our knowledge, hemophagocytic lymphohistiocytosis has not been previously reported in association with reticular dysgenesis.

Multiplex Screening for Interacting Compounds in Paediatric Acute Myeloid Leukaemia.

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by the malignant transformation of myeloid precursor cells with impaired differentiation. Standard therapy for paediatric AML has remained largely unchanged for over four decades and, combined with inadequate understanding of the biology of paediatric AML, has limited the progress of targeted therapies in this cohort. In recent years, the search for novel targets for the treatment of paediatric AML has accelerated in parallel with advanced genomic technologies which explore the mutational and transcriptional landscape of this disease. Exploiting the large combinatorial space of existing drugs provides an untapped resource for the identification of potential combination therapies for the treatment of paediatric AML. We have previously designed a multiplex screening strategy known as Multiplex Screening for Interacting Compounds in AML (MuSICAL); using an algorithm designed in-house, we screened all pairings of 384 FDA-approved compounds in less than 4000 wells by pooling drugs into 10 compounds per well. This approach maximised the probability of identifying new compound combinations with therapeutic potential while minimising cost, replication and redundancy. This screening strategy identified the triple combination of glimepiride, a sulfonylurea; pancuronium dibromide, a neuromuscular blocking agent; and vinblastine sulfate, a vinca alkaloid, as a potential therapy for paediatric AML. We envision that this approach can be used for a variety of disease-relevant screens allowing the efficient repurposing of drugs that can be rapidly moved into the clinic.

Clinical Heterogeneity of the VEXAS Syndrome: A Case Series

The objective of this study is to describe the clinical features and outcomes of patients with the newly defined vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome. Nine men with somatic mutations in the UBA1 gene were identified; the most frequent variant was p.Met41Thr (7 of 9, 78%). The median age at VEXAS diagnosis was 74 (67, 76.5) years, and patients had a median duration of symptoms for 4 years before diagnosis. Refractory constitutional symptoms (88%), ear and nose chondritis (55%), and inflammatory arthritis (55%) were common clinical features. Vasculitis was noted in 44%. All patients had significantly elevated inflammatory markers and macrocytic anemia. Thrombocytopenia was present in 66% at diagnosis of VEXAS. Eight patients had bone marrow biopsies performed. All bone marrows were hypercellular, and there was vacuolization of the erythroid (100%) or myeloid precursors (75%). Glucocorticoids attenuated symptoms at prednisone doses ≥20 mg per day, but no other immunosuppressive agent showed consistent long-term control of disease. One patient with coexisting plasma-cell myeloma received plasma-cell–directed therapy with improvement of the inflammatory response, which is a novel finding. In conclusion, VEXAS syndrome is a clinically heterogeneous, treatment-refractory inflammatory condition caused by somatic mutation of the UBA1 gene. Patients often present with overlapping rheumatologic manifestations and persistent hematologic abnormalities. As such, internists and subspecialists, including pathologists, should be aware of this condition to avert diagnostic delay, now that the etiology of this syndrome is known.

Epigenetics of Osteoporosis

AbstractFragile bone is the root cause of osteoporosis. For inherited or acquired reasons, the fragile bone does not provide sufficient fracture resistance to withstand the physical strains of a normal lifestyle. Accordingly, clinical characteristics consist of fragility fractures that occur during daily life activities or low energy trauma. Hip fractures and vertebral fractures are so called "major osteoporotic fractures”, that also cause the highest burden of disease. Although the clinical osteoporosis manifestations are relatively uniform, there is a vast spectrum of underlying molecular causes. Impaired bone formation, accelerated bone loss, and impaired lifetime adaptive regeneration according to physical impact characterize the cruder facets of osteoporosis. The signaling cascades that govern bone formation and metabolism may be altered by genetically or epigenetically inherited defects or acquired epigenetic changes due to tissue aging and/or underlying diseases. While molecular genetics and mechanisms and specific osteoporosis treatments have made impressive progress over the last three decades, there is still an urgent need to better understand the role of epigenetics in this disease.Epigenetic mechanisms such as covalent modifications of DNA, histones, or essential core factors like the osteogenic transcription factors (e. g., RUNX2) and inhibitory modulators of osteogenic WNT-signaling (e. g., Dickkopf-1 (DKK-1), sclerostin (SOST)) are all intricately implicated in developmental bone formation and adaptive regeneration and remodeling processes throughout adult life. These mechanisms are accompanied by chromatin architecture and gene expression changes of small (e. g., microRNAs (miRs)) and long, noncoding RNAs (lncRNAs). The timely execution of these mechanisms either facilitates or inhibits bone formation and remodeling. Together, epigenetic mechanisms controlling bone homeostasis widen the spectrum of potential dysregulations that can cause osteoporosis and open new avenues for therapeutic interventions.Apart from the core mechanisms of bone formation and regeneration, recent research revealed that tissue-resident cells of the immune system such as tissue-specific macrophages, myeloid precursors, and lymphocytes have surprisingly fundamental influence on tissue regeneration, including bone. Those tissue resident cells are also subject to epigenetic changes and may substantially contribute to the development of disease. Epigenetic constellations can be inherited, but the dynamic epigenetic mechanisms involved in physiological processes of tissue regeneration may also be affected by pathologies such as cellular aging and senescence. Recently, several studies aimed at identifying DNA methylation signatures in peripheral blood leukocytes from osteoporosis patients that reveal novel disease mechanisms and potential targets for diagnosis and treatment. Overall, these studies rendered, however, yet inconclusive results.By contrast, studies using bone marrow-derived skeletal progenitors identified transcriptome changes in osteoporosis patients, which could have epigenetic reasons in the absence of genetic causes. Respective changes may be related to the local milieu in bone and bone marrow as a kind of segmental attitude of a specific tissue acquired through tissue aging and/or supported by underlying aging-associated diseases such as arteriosclerosis or aging of cells of the immune system.In summary, there is cumulating evidence linking epigenetic factors to the pathogenesis of aging-associated osteoporosis. However, we are currently still limited in our knowledge with respect to the causal traits that are common, inherited, or acquired in a lifetime in the respective tissues and cells involved in bone formation and regeneration. During the following years, the field will most certainly learn more about molecular processes and factors that can be targeted therapeutically and/or used as biomarkers for risk assessment.

Granulocytic and Monocytic Myeloid-Derived Suppressor Cells are Functionally and Prognostically Different in Patients with Chronic Myeloid Leukemia.

Myeloid-derived suppressor cells (MDSCs) represent phenotypically heterogeneous populations that suppress tumor-specific T-cell responses. MDSCs are produced from myeloid precursors in emergent states and are increased in several hematologic malignancies. We evaluated the differences in the levels and prognostic significance of MDSCs according to the clinical status of chronic myeloid leukemia (CML). The percentages and numbers of granulocytic (g)MDSCs and monocytic (m)MDSCs in peripheral blood (PB) and bone marrow (BM) aspirates were determined by five-color flow cytometry (HLA-DR/CD11b/CD15/CD33/CD14). The median BM-gMDSC% and PB-gMDSC% of the CML group were lower than those of the complete hematologic response (CHR) and control groups (P<0.001). In the CHR group, patients with major molecular response (MMR) showed higher median BM-gMDSC% than those without MMR (P=0.039). Conversely, the PB-mMDSC number of the CML group was higher than those of the CHR and control groups (P<0.001). Patients with high PB-gMDSC number exhibited superior survival to those with low PB-gMDSC number (P=0.021), and patients with high PB-mMDSC% showed inferior survival to those with low PB-mMDSC%, but there was no statistical significance (P=0.182). Increased gMDSCs at CHR may reflect non-leukemic granulopoiesis, and a high number of PB-gMDSCs suggests better prognosis in CML. However, mMDSCs may be associated with malignant conditions and poor prognosis.

Evaluation of the Expression Levels of Notch Signaling Pathway-Related Genes; JAG1, CXCR4, and MIB1 in Acute Myeloid Leukemia Patients

Background: Acute myeloid leukemia (AML) is a complex disease characterized by clonal expansion of undifferentiated myeloid precursors, resulting in impaired hematopoiesis and bone marrow failure. Different genetic and environmental factors are believed to be involved in the pathogenesis of AML. Notch signaling with a tumor suppressing plays a role in myeloproliferative disorder and is a negative regulator of myeloid progenitor commitment. Crosstalk between Notch signaling and CXCR4 axis is a matter of debate in AML. Method: In the current case-control study, we evaluated the expression level of CXCR4, JAG1, and MIB1, which are all involved or related to Notch signaling in adult AML patients. Blood samples were obtained from 25 AML and 17 healthy individuals and the expression level of the selected genes was evaluated via the real-time PCR. Results: Our results revealed the increased expression of JAG1, but decreased expression of CXCR4 in AML patients in Iranian population of AML patients. Moreover, some gender-associated effects on the expression of JAG1 and CXCR4 were detected, which may be related to sex hormones. The expression level of MIB1 did not change significantly. The correlation analysis showed no correlations between the age of the patients and the expression levels of the genes. Conclusion: Herein, for the first time, we suggested some new evidence regarding the complex role of Notch signaling-related genes (CXCR4 and JAG1) in the pathogenesis of AML in Iranian patients.

Diagnostic dilemma and challenges of granulocytic sarcoma: a review

The maleficent tumour granulocytic sarcoma is an unusual cancer indite of progressive myeloid precursor cells. It is a Chloroma basically present at an extramedullary location spread through immature granulocytic cells. It can affect any part of the body but the primary site is soft tissues, outside bone marrow, lymph nodes. The root cause may be many factors i.e. genetic, environmental, carcinogens etc. This proliferative type of cancer is sometimes misdiagnosis in contradictory to other acute/chronic tumours. For a histopathologist, it is a challenge for an accurate diagnosis. The tissue biopsy is the known diagnostic technique used since days in cancer diagnosis. To address this review, our main focus is to investigate, analyse the diagnostic limitations or research gaps in granulocytic sarcoma recognition at the clinical level and what can be the possibilities that have developed recently in tumour detection at an early stage. Next, we will try to develop a hypothesis from pinpoint methods used in it as molecular diagnosis, immune-histochemical biomarkers, flow cytometry (FACS) for confirmation of sarcoma so that specificity and sensitivity can be well examined. The prognosis/diagnosis in myeloid tumour detection depends on key factor innovation and medical devices and that should be filled in research and so a gold standard diagnostic kit should be developed.