Hematopoietic Research Articles

N-3 Polyunsaturated Fatty Acid (N-3 PUFA) Derived Lipid Mediators Increase Hematopoiesis during Zebrafish Embryogenesis

Polyunsaturated fatty acids (PUFAs), which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. The richest source of PUFAs is fish oil. N-3 PUFAs have been used in the prevention and treatment of coronary artery disease, diabetes, hypertension, arthritis, cancer, and other inflammatory and autoimmune disorders in humans. Lipid mediators derived from n-3 PUFAs are implicated in mediating the resolution of inflammation. However, their effect on hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation and on hematopoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-derived lipid mediators. We exposed wild-type zebrafish embryos to different n-3 PUFA-derived lipid mediators. After fixation, we performed whole mount in situ hybridization (WISH) using antisense RNA probes such as c-myb (hematopoietic stem cell marker), gata1 (erythroid progenitor cell marker), hbbe1 (erythrocyte marker), l-plastin (macrophage and neutrophil cell marker ), mpx (neutrophil cell marker), lyz (neutrophil cell marker), and rag1 (immature lymphocyte marker); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and fluorescence-activated cell sorting (FACS) to understand the effect of lipid mediator exposure on the proliferation of HSPCs, erythroid progenitor cells, erythrocytes, granulocytic cells, neutrophils, macrophages, and T lymphocytes in different development stages during vertebrate hematopoiesis. In addition, we also used o-dianisidine staining to study red blood cell (RBC) formation and transgenic zebrafish to observe different blood lineages including HSPCs, neutrophils, macrophages, erythroid progenitors, and erythrocytes with fluorescent microscopy. We identified two novel lipid mediators which promote HSPC proliferation, enhance erythroid progenitor cell and erythrocyte production, and increase neutrophil cell number. These two molecules could be applied as novel therapeutics for blood disorders and blood cancers such as anemia and leukemia in humans. These two compounds can be intensely investigated to become candidate supplements to enhance HSPC amplification before HSPC transplantation for leukemia patients.

Autoimmune Cytopenia Associated with Indolent B-Cell Clones: A Large Multicentric Cohort Study

Introduction Autoimmune cytopenias (AIC) are a group of rare diseases sharing immune-induced accelerated destruction and/or impaired production of blood cells. AIC can be associated with overt B-cell lymphoid neoplasia. In that case, the management is based on the treatment of the hematological malignancy. However, AIC can be associated with indolent B-cell clones (IBC). In that case, the treatment of the AIC is not consensual. We aimed to describe the treatments used in France for IBC-associated AIC, and to compare their effectiveness, safety, and impact on IBC progression. Methods We conducted a multicenter, retrospective observational study including adults treated between 2000 and 2022 for a definite AIC (immune thrombocytopenia, ITP; autoimmune hemolytic anemia, AIHA; or Evans syndrome, ES) associated with an IBC, namely: chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma, monoclonal B lymphocytosis, and mantle cell lymphoma. Patients who required a specific treatment of the underlying IBC for one or several of the following reason(s) were excluded: extensive marrow infiltration leading to impaired megacaryopoiesis and/or erythropoiesis, massive organ infiltration and/or compressive lymphadenopathies, lymphocytosis doubling within 6 months (with a lymphocytes count >30 x 10 9/L), doubling of lymphadenopathy size within 2 months, hyperviscosity, and any other IBC-related condition requiring treatment (amyloidosis, neuropathy...). Patients were classified according first-line treatment for AIC: group 1 for those treated without chemotherapy (corticosteroids, intravenous immunoglobulin, rituximab...); group 2 for those treated with chemotherapy (cyclophosphamide, ibrutinib...). Only the first-line treatment was assessed. Response to treatment was defined according to standard international criteria. An infection was defined as serious if graded >2, according to the Common Terminology Criteria for Adverse Events. The progression was defined as the need to urge lymphoma-specific therapy. Subgroup analyses were performed by type of AIC (ITP, warm/mixed AIHA, cold agglutinin disease, and ES). Overall survival was estimated using the Kaplan-Meier method and comparison was performed using Log-rank test. Results In total, 187 patients were included throughout 30 French university and general hospitals using the French reference center network for immune cytopenias in adults. The median age at diagnosis of AIC was 75 years (range 43 to 97 years) with a male/female ratio of 1.47 (Table 1). We observed 105 (56.1%) patients with AIHA, 80 (42.8%) with CLL and 44 (23.5%) with MZL. In most cases (58.8%), the time elapsed between IBC and AIC diagnosis was £6 months. We included 132 (72.2%) patients in group 1. Among them, 75 (55.6%) patients were treated with only corticosteroids +/- intravenous immunoglobulin, 52 (38.5%) with rituximab, 2 with splenectomy, and 1 with dapsone. We included 52 (27.8%) patients in group 2. Among them, 46 (88.5%) patients were treated as first-line with a rituximab-cyclophosphamide-dexamethasone combined regimen, 2 with rituximab-bendamustine, 2 with chloraminophen, 1 with fludarabine-cyclophosphamide-dexamethasone, and 1 with ibrutinib. Both overall response and relapse rates were similar between groups: 86.6% versus 86.5% and 45.5% versus 41.5%, respectively. We did not find any difference in subgroup analyses by type of AIC. Serious infections were observed in 17.6% patients: 17.8% in group 1, 17.3% in group 2. With a median of follow-up of 39.8 months (95% IC 30.0 to 51.3), the overall survival did not differ between group 1 (84.0% at 4 years, 95% IC 73.7 to 90.5) and group 2 (81.8% at 4 years, 95% IC 66.1 to 90.7), nor the progression rate of IBC was similar in both groups (9.6% and 7.7%, respectively). Conclusion We report the largest cohort of AIC associated with IBC. So far, very few patients with lymphomas such as MZL have been described in this situation. We observed that treatments were heterogeneous. Whatever the strategy used (treating AIC only or also using chemotherapy for the IBC), the effectiveness and safety profiles of first-line treatments were not different. Additional analyses are ongoing to better clarify the impact of rituximab alone versus rituximab and chemotherapy, and IBC' characteristics on the overall progression-free survival.

Real-World Impact of Luspatercept on Patients with Myelodysplastic Syndromes Requiring Red Blood Cell Transfusions and with Prior Exposure to Erythropoietin-Stimulating Agents: A United States Healthcare Claims Database Study

Background: Myelodysplastic syndromes (MDS) are among the most common hematologic cancers in older US adults. MDS are characterized by abnormal bone marrow function leading to impaired production of blood cells, persistent cytopenia, and variable risk of progression to acute myeloid leukemia (AML). Since anemia is a common complication of MDS, many patients (pts) become dependent on red blood cell transfusions (RBCTs) and erythropoiesis-stimulating agents (ESAs) are the standard of care for patients with lower-risk MDS. In a randomized, double-blind, placebo-controlled phase 3 MEDALIST clinical trial, luspatercept, a first-in-class erythroid maturation agent, was shown to reduce the severity of anemia among transfusion-dependent (TD) pts with lower-risk MDS with ring sideroblasts (RS) status and who were refractory to prior ESA treatment (Fenaux P, et al. N Engl J Med 2020; 382:140-151). However, real-world evidence regarding the effect of luspatercept on transfusion burden among pts with MDS requiring RBCTs and with prior ESA exposure is currently limited. Aim: To describe clinical outcomes and MDS-related treatment patterns before and after the initiation of luspatercept among real-world pts with lower-risk MDS in the USA who required RBCTs before luspatercept initiation and were previously exposed to ESAs. Methods:In this retrospective claims-based cohort study, data from Symphony Health, a nationally representative database, (Jan 1, 2010 to June 30, 2022) were used to identify adults with ≥1 claim with a diagnosis of MDS (initial diagnosis on or after Jan 1, 2015) before the first claim for luspatercept (index date). Pts were considered TD at luspatercept initiation if they had ≥1 claim for RBCT in the 8 weeks before index date, and ESA-exposed if they had ≥1 ESA claim at any point before or at luspatercept initiation. Pts with chemotherapy treatments, with a diagnosis for refractory anemia with excess blasts, or with AML during the study period were excluded as they were presumed to have higher-risk MDS. Pts were also excluded if they had prior use of hypomethylating agents (HMAs) or a diagnosis for β-thalassemia, myelofibrosis, aplastic anemia, or breast cancer in the 6 months prior to index date (baseline period). Pt demographics and clinical characteristics were assessed during the baseline period. Pts' RBCT status (transfusion independent [TI] or TD) was measured in the 6 months after index date. A pt was considered 8-week TI if they had 0 RBCT in ≥1 rolling 8-week period in the 6 months after index date. 12-week, 16-week, and 24-week TI pts were defined similarly, using increasingly longer periods without RBCT. Pts' use of MDS-related treatment classes (ie, chemotherapy, ESAs, granulocyte-colony stimulating factor [G-CSF], HMA, and immunomodulatory imide drug [IMiD] agent) was measured before and after index date until end of data availability or clinical activity (follow-up). Results:The population comprisedof240 pts with MDS who were TD and had ESA exposure before luspatercept initiation. The mean (standard deviation [SD]) age was 75.4 (5.4) years and 56.7% were male. About one fifth of pts received ≥2 treatment classes before luspatercept initiation (2: 17.5%; 3+: 1.7%). Most pts (64.2%) achieved 8-week TI in the 6 months after luspatercept initiation. Similarly, 53.8%, 43.3%, and 29.6% of pts achieved 12-, 16-, and 24-week TI in the 6 months after luspatercept initiation, respectively (Figure 1). The percentage of pts achieving 8-, 12-, 16-, and 24-week TI was numerically higher among pts with positive RS status than those with negative/unknown RS status (Figure 1). Most pts (64.2%) did not require any additional MDS-related treatments during the study follow-up period (mean (SD) 15.6 (7.3) months). The most common treatment classes used after luspatercept initiation were ESA (22.5%) and HMA (16.3%; Figure 2). Conclusions:The findings of this real-world study strongly corroborate clinical trial results, showing that luspatercept is highly effective in reducing transfusion dependence among ESA-exposed pts with lower-risk MDS who were TD before luspatercept initiation. In this study, over half of pts achieved 8-week and 12-week TI status during the follow-up period, and more than a quarter did not require any RBCT over a 24-week period. Further, most pts treated with luspatercept did not require any additional MDS-related treatments during the follow-up period.

Ruxolitinib Versus Best Available Therapy for Polycythemia Vera: A Systematic Review and Meta-Analysis

Background: Polycythemia vera (PV) is characterized by dysregulated Janus kinase (JAK) activation, leading to excessive production of blood cells, increased risk of thrombotic and hemorrhagic events, and an impairment on the quality of life of patients. Ruxolitinib, a JAK1/JAK2 inhibitor, has shown promising results in recent trials, but its comparative efficacy to best available therapy (BAT) remains unclear. We aimed to perform a systematic review and meta-analysis comparing the efficacy and safety of Ruxolitinib versus BAT for patients with PV. Methods: We systematically searched PubMed, Embase, and Cochrane Central for clinical studies published up to July 1st comparing Ruxolitinib to BAT. BAT was defined as hydroxyurea, interferon or pegylated interferon, pipobroman and anagrelide. Efficacy outcomes assessed were complete response to therapy and reduction in the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) score; safety outcomes were thromboembolism, anemia, and herpes. We pooled outcomes as risk ratios (RR) with 95% confidence intervals (CI). Statistical analysis was performed with Review Manager version 5.4.1. Heterogeneity was assessed with I² statistics. The protocol was registered in PROSPERO (ID: CRD42023427977). Results: A total of five studies met our inclusion criteria, of which four were randomized trials. Our analysis encompassed a population of 684 patients, including 336(49%) allocated to the Ruxolitinib group, and 348(51%) allocated to the BAT group. Ruxolitinib had a significantly greater response to treatment compared to BAT (RR 1.97; 95% CI 1.03 -3.77; p=0.04; I²=60%; Fig 1A) and significantly reduced MPN-SAF score by at least 50% (RR 2.50; 95% CI 1.29-4.86; p=0.007; I²=76%; Fig 1B). Additionally, use of Ruxolitinib showed a significantly higher incidence of herpes infection (RR 3.57; 95% CI 1.27 -10.04; p=0.02; I²=0%; Fig 1C) and anemia (RR 1.97; 95% CI 1.15-3.37; p=0.01; I²=50%; Fig 1D). There were no significant differences between the two treatment groups regarding thromboembolic events (p=0.07; Fig 1E). Conclusion: Our meta-analysis reveals that Ruxolitinib exhibits superior efficacy in achieving a complete response for PV patients compared to BAT and in reducing MPN-SAF score by ≥50%. While no significant difference was observed in thromboembolic events, Ruxolitinib demonstrated significant risk of anemia and herpes infection.

The Effect of SCD-1 Inhibition on Human Hematopoietic Stem Cell Mitochondrial Metabolism, Cell Proliferation, and Differentiation Potential

Hematopoietic stem cells (HSC) give rise to all blood lineages and sustain the production of blood cells throughout life. Due to their inherently high regenerative potential, HSC are used in a variety of clinical settings, including bone marrow transplantation (BMT) directly to cure a variety of hematologic and oncologic disorders often with gene therapy. During regeneration, HSC are activated into cycle. For this, HSC undergo drastic mitochondrial and metabolic remodeling to meet the bioenergetic and biosynthetic needs of activated HSC. A growing body of evidence indicates that HSC sustain injury during activation. This remodeling is important for optimal HSC function but is permanently changed after HSC activating stress. It is thus important to identify the metabolic needs of activated HSC to improve HSC functions for therapeutic purposes. In both murine and human HSC, numerous metabolic enzymes get upregulated during activation, including those involved in de novo lipid synthesis. This metabolism is poorly understood in human HSC. Our overarching question is to understand the metabolic needs of HSC within the human system. Stearoyl-co-A-desaturase 1 (SCD-1), an enzyme responsible for conversion of stearic to oleic acid within the de novo lipid synthesis pathway, is suspected to play a role in metabolic reprogramming after stress. Using CD34+ mobilized human peripheral blood, we analyzed the effect of SCD-1 inhibition on HSC and progenitor proliferation, mitochondrial metabolism, and lineage differentiation potential. Cells were cultured either in vehicle control conditions or with SCD-1 inhibitor (SCDi). Subsequently, cells were counted and analyzed by flow cytometry on Days 4-5 and 10-15. More primitive or stem cell markers were utilized on Days 0 and 5 whereas differentiation markers were utilized on Day 10-15. Both tetramethylrhodamine-ethyl ester dye (TMRE) and MitoSOX reagents were used to assess mitochondrial membrane potential and mitochondrial ROS, respectively. Our results demonstrate that the size of the HSC population generated by SCDi-treated CD34+ cells within 5 days of culture was similar to vehicle treated cells and was composed of similar proportion of CD34+CD38+, CD34+CD38-, and CD38+CD34- cells, indicating that SCD-1 inhibition does not alter HSC expansion under these culture conditions. However, TMRE levels were lower in all HSC populations whereas mitoSOX levels were unchanged by SCD-1 inhibition compared to vehicle. In differentiation conditions, SCDi cultures were composed of a larger proportion of myeloid progenitors and a smaller proportion of erythroid cells after 10-15 days, compared to vehicle-treated cultures. These findings suggest that de novo lipid synthesis is necessary for HSC differentiation but dispensable for proliferation/expansion in vitro. We then used a xenotransplant model to assess the effect of SCDi on HSC regenerative potential in vivo. CD34+ mobilized human peripheral blood cells were cultured for 3 days under conditions that maintain HSC functions in vitro and transplanted into immunodeficient, sublethally irradiated NSG mice. Peripheral blood of xenotransplanted mice was analyzed monthly for human cell chimerism and mature blood lineage potential from 1 to 6 months post-transplant. Bone marrow analysis was performed at the 6 month time point as well, assessing for both HSC markers and lineage markers. Mice that received SCDi-treated cells exhibited human cell chimerism at a level similar to mice that were transplanted with vehicle treated cells, at about 3 to 30%. However, SCDi-treated cells gave rise to a lymphoid-biased graft, in particular T cells, both in the peripheral blood and in the bone marrow of xenotransplanted mice, compared to a more balanced myeloid-lymphoid graft from vehicle treated cells. In conclusion, these findings suggest that de novo lipid synthesis is critically important for HSC lineage fate and balanced differentiation in vitro and in vivo. This is especially clinically relevant as this work may implicate a possible therapeutic target as improving de novo lipid synthesis may aid in patients who suffer from persistent cytopenias after BMT and graft failure.

The Iron Chaperone Poly C Binding Protein 1 Regulates Iron Efflux through Intestinal Ferroportin in Mice

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin (FPN). Here we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through FPN in the intestine of mice. PCBP1 binds iron-glutathione complexes in the cytosol of cells and delivers the iron to ferritin, the iron storage protein, to iron centers in enzymes, and to iron-sulfur cluster chaperones. PCBP1 also limits the chemical reactivity of iron, thereby limiting iron-catalyzed oxidative damage in cells. To explore the role of PCBP1 in dietary iron absorption, we developed mice with an inducible, enterocyte-specific deletion of PCBP1. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through FPN. Kinetic studies of iron transport using a stable isotope ( 57Fe) coupled with a mini-hepcidin that blocks FPN activity demonstrated that PCBP1 deletion had no effect of intestinal iron uptake but caused high rates of iron efflux through FPN. Surprisingly, excess iron efflux occurred despite lower levels of FPN protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low iron diet and liver oxidative stress with iron loading on a high iron diet. To investigate the roles of PCBP1 more precisely in enterocytes, we developed 3-D organoid cultures from intestinal epithelium which functionally expressed the major iron trafficking proteins: divalent metal transporter 1, PCBP1, ferritin, transferrin receptor 1 and FPN. Ex vivo culture of wild-type and PCBP1-depleted enteroids demonstrated normal patterns of FPN expression and hepcidin-mediated turnover. However, fluorescence measurements of kinetically labile iron pools in enteroids competent or blocked for FPN iron efflux indicated that PCBP1 dramatically altered efflux activity. PCBP1 functioned as a cytosolic buffer for iron, binding and retaining iron and thereby keeping “free” iron at very low levels. This low concentration of free iron limited its availability for FPN-mediated efflux and allowed hepcidin-mediated changes in FPN levels to regulate the amount of iron exported from the enterocyte. Thus, by binding enterocyte iron, PCBP1 reduces the concentration of unchaperoned, “free” iron to a low level. This low level of cytosolic free iron is necessary for hepcidin-mediated regulation of FPN activity.

Early Response Transcription Factors in Erythroid Regeneration

Acute anemia activates distinct mechanisms from physiological erythropoiesis to increase red blood cell production. While erythroid regeneration in this context requires some critical cytokines (e.g., BMP-4), little is known about transcription factors (TFs) connecting growth factor stimulation to anemia-specific gene expression and cellular responses. We previously discovered that GATA1 and TAL1 control a cohort of composite cis-elements which are anemia-activated in erythroid progenitors and precursors during regeneration. Intriguingly, cis-regulatory activity and gene activation at these loci are delayed by several days following initial anemia onset by the red blood cell lysing chemical phenylhydrazine. This delay suggested that these elements were utilized for erythroid precursor expansion but not activation. We hypothesize that distinct TF mechanisms are early responders to erythroid regeneration. To identify early anemia response TFs, we compared transcript levels in mouse spleen Kit + erythroid progenitors at days 0 and 1 post-anemia (anemia upregulated genes; p<0.05, fold change>1.5). Among upregulated genes, only 10 were TFs, and 3 of them ( Jun, Junb, Fos) are components of Activator protein 1 (AP-1) transcription factor. All 3 transcripts were subsequently downregulated at 3 days after anemia. In physiological hematopoiesis, AP-1 is highly expressed in most cells but low within the erythroid lineage. These findings suggested transient anemia-activated AP-1 may initiate erythroid regeneration. While AP-1 is a big family, only JUN and FOS family exhibited transient anemia-activation. Genes containing AP-1 binding motifs were cell intrinsically upregulated post-anemia in Jun and Fos-expressing cells. To determine Jun TF activity in anemia, we performed phospho-flow cytometry in Lin- spleen cells from mice after inducing anemia. We discovered that at Day 1 vs Control, the median fluorescence intensity (MFI) of Kit +CD71 +Ter119 + cells stained with total Jun or phospho-Jun (p-Jun) antibodies increased 2.0-fold (p=0.03) and 2.9-fold (p=0.04), respectively. Concurring with transcript levels, both total and p-Jun protein increased between 6- and 24-hours post-anemia and decreased at 72 hours. These changes post-anemia were absent in non-erythroid (CD71 -Ter119 -) and Kit - cells. These data demonstrated a transient increase in JUN TF activity early during the anemia response in erythroid progenitor/precursors. AP-1 activity is commonly elevated in response to a variety of stressors, including hypoxia and inflammation. Given the limited sets of transcripts upregulated at early anemia time points, direct AP-1 targets may be required for establishing an anemia-dependent transcriptome. To identify AP-1 target genes in erythroid regeneration, we selected genetic loci nearby (distance <100kb) AP-1-occupied cis-elements in human erythroleukemia (K562) or mouse erythroid progenitor cells (GEO: GSE31477, 208960, 170378, 172818). 50 potential AP-1 target genes were upregulated in stress erythroid precursors on day 3 vs day 1 post-anemia. Seven anemia-regulated transcription factors (SOX6, E2F2, TAL1, TFDP2, FOXO3, GFI1B, MAFG) reside at loci containing candidate AP-1 cis-elements. The T-cell Acute Lymphoblastic Leukemia-1 (TAL1) TF cooperates with GATA1 at E-box-GATA composite cis-elements to promote transcription of a number of erythroid-specific genes, several of which are needed for effective erythroid regeneration. Knockdown approaches will be used to test whether Jun expression and activity promotes the anemia-dependent transcriptome, stress erythroid precursor differentiation, and cell survival during erythroid regeneration. Given that Jun can be activated by BMP-4 signaling via the Jun kinase (JNK) pathway, and the requirement for BMP-4 in erythroid regeneration, we are investigating whether BMP-4 stimulation (or other cytokines) phosphorylates Jun to establish the regenerative transcriptome at early time points post-anemia. These studies will improve our understanding of transcriptional mechanisms driving erythroid regenerative responses, which can be useful for identifying new therapeutic targets for acute/chronic anemias.

Soluble PD-L1 Predicts Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a spectrum of myeloid malignancies typified by regular genetic abnormalities, clonal hematopoiesis, dysfunctional myelopoiesis, inefficient blood cell production, peripheral-blood cytopenia, and an elevated risk for progressing to acute myeloid leukemia (AML). Mounting evidence suggests that the dysregulation of immune checkpoints (ICs) plays critical roles in immune evasion of MDS, which thus led to a series of trials using HMAs in combination with immune checkpoints inhibitors (ICIs). Of these, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway has been studied most extensively. Recently, forms of cell-free soluble PD-1/PD-L1 (sPD-1/sPD-L1) detection in plasm of patients with solid tumors has open a new paradigm for PD-1/PD-L1 investigations. However, within the context of MDS, the potential dysregulation and prognostic impact of sPD-1/sPD-L1 remains uncertain. This study was aimed to assess roles of sPD-1/sPD-L1 in newly diagnosed MDS patients. Method: Between July 2020 and March 2023, 130 MDS/sAML patients matched the inclusion criteria and 55 healthy individuals with no familial or personal history of autoimmune or cancer disorders were included. The criteria for patient inclusion included: (1) a diagnosis of MDS confirmed pathologically according to 2016/2022 World Health Organization (WHO) classification, or sAML patients with a verified prior MDS history; (2) absence of other conditions like autoimmune diseases or different types of tumors; (3) availability of complete clinical information and survival data. MDS patients were categorized by IPSS-R as: lower-risk (very low risk/ low risk; higher-risk (intermediate risk/high risk/very high risk. Follow-up was conducted until June 16th, 2023. The median follow-up was 14.32 months (range 3.2-34.63 months). Profile of sICs (sPD-L1, sPD-1, sCTLA-4, sGITR, sTIM-3, sOX40, sLAG-3, s4-1BB, sICOS) were tested by ELISA, cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were detected using Luminex analysis, and the expression levels of PD-1 on T cells (CD3 +, CD4 +, CD8 +, DPT, DNT）were measured using flow cytometry (FCM). Result: In the plasma of 98 newly diagnosed MDS patients, sPD-L1 levels (median 66.80 pg/ml, range 25.00-219.5 pg/ml) were significantly elevated compared to 55 healthy blood donors (median 49.22 pg/ml, range 12.5-218.1 pg/ml). sPD-L1 level was higher in higher-risk IPSS-R groups ( P=0.0380). In addition, higher sPD-L1 levels also showed a correlation with lower hemoglobin concentration, implying a potential link between higher sPD-L1 levels and a more advanced MDS disease state. The AUC for sPD-1 was found to be 0.6333 [95% CI 0.5331-0.7334, P=0.0100] and for sPD-L1, it was 0.6785 [95% CI 0.5836-0.7733, P=0.0002]. The optimal cut-off values were established at 187.5 pg/mL for sPD-1 and 55.08 pg/mL for sPD-L1. Based on these cut-off values, patients with increased sPD-L1 experienced shorter OS ( P=0.0305). By using multivariate Cox model, high expression of sPD-L1 ( P=0.019, HR = 4.172, 95% CI: 1.265-13.755) and high IPSS-R scores ( P=0.030, HR = 2.898, 95% CI: 1.111-7.565) were found to be independent risk factors for newly diagnosed MDS patients. In addition, compared to healthy controls, the PD-1 + T cell subpopulations in MDS patients were significantly reduced, sPD-1 levels were positively correlated with proportion of CD4 + PD-1 + T cells, absolute count of CD4 + PD-1 + T cells and PD-1 + DPT cells, suggesting that CD4 + T cells and DPT cells may be potential sources of sPD-1 in plasma. Subsequently, we analysed the potential roles of cytokines and HMAs in sPD-1/sPD-L1 regulation. It was found that sPD-1 levels had a positive correlation with sPD-L1, IL-2Rα, IP-10, MIG, TNFα, GROα, and TRAIL. sPD-L1 was positively correlated with IL-2Rα, IP-10, MCP-1, MIG, MIP1α, and SCF (Figure 1 A). Notably. We found a reduced sPD-L1 levels in MDS patients who achieved remission after HMA treatment compared to treatment-naïve patients. ( P=0.0302) (Figure 1 B). Conclusions: In conclusion, we found sPD-L1 is an independent risk factors for overall survival in newly diagnosed MDS patients. The elevation of plasma sPD-L1 levels is associated with disease progression in MDS, and may be a potential target for MDS immunotherapy.

Severe Chronic Idiopathic Neutropenia Managed with Chronic Filgrastim: A Case Report

Myelodysplastic syndrome (MDS) represents a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis and abnormal blood cell production. Chronic neutropenia is one such MDS and is classified into mild (Absolute Neutrophil Count (ANC): 1,000 to 1,500/µL), moderate (ANC: 500 to 1,000/µL), or severe (ANC: below 500/µL), with the latter posing the highest risk of infection-related complications. In an attempt to alleviate neutropenia, patients with MDS are often treated with colony stimulating factors (CSFs), such as filgrastim to promote growth and maturation of neutrophils. The target ANC is 1,500 to 2,000/µL for adequate protection against most infections. However, CSF treatments have the potential to stimulate the transformation of MDS into acute myeloid leukemia (AML) so CSF is often used with caution. Presented below is a case of severe chronic idiopathic neutropenia responsive to chronic filgrastim that has yet to show evidence of transformation. A 79 year-old female with a past medical history of distant tobacco use, hypertension, acute cerebrovascular accident, and Hashimoto's thyroiditis presents to the clinic for routine follow-up of her now stable chronic idiopathic neutropenia being managed with weekly filgrastim injections for three weeks with one week off in between cycles. She had originally presented to medical attention in 2004 complaining of chronic mouth sores and ulcers with leukopenia and a severely decreased ANC of <500/µL. Continued monitoring with more than 3 complete blood counts (CBC) with a differential consistently noted a decreased ANC in the setting of persisting oral mucosal erosion. Leukocytes were not noted to be dysplastic, quantitative IgG, IgA, IgM levels were noted to be within normal limits, anti-nuclear antibodies/ anti-neutrophil antibodies were noted to be negative argued against an etiology of large granular lymphocytic leukemia, immunodeficiency, and/or autoimmune disorders, respectively. A trial of glucocorticoids did not significantly increase her ANC which led to her ultimately receiving a bone marrow biopsy (BMB) in 2008. A mildly hypercellular marrow with trilineage hematopoiesis was found, with maturation of all lines, negative flow cytometry, and myelodysplasia. Comforted by the lack of MDS pathologically, the patient was started on filgrastim with significant improvement. Over the course of years the frequency and duration of filgrastim was optimized to her current regimen of weekly filgrastim injections for 3 weeks followed by one week off which allowed her to maintain an adequate ANC and avoid oral ulcers to this day. Despite the feared complication of kickstarting the transformation of MDS into AML with CSF therapy, patients such as the one mentioned here can benefit greatly from judicious use of CSF when blood work can be monitored for complications. The use of G-CSF in severe chronic neutropenia to raise levels from below 500/µL to the ideal target ANC is 1,500 to 2,000 cells per microliter (µL) for adequate protection is in line with our current understanding that even long-term use of G-CSF does not appear to increase the risk of AML (Dale et al., 2022). G-CSF should continue to be considered in the management of severe chronic neutropenia as the risk for transformation may be insignificant compared to the clinical benefit of its use when properly monitored.

Transferrin and Erythropoietin Increased Levels Correlate with Thrombosis at High Altitude

Living at high altitude involves great interest because of the challenging multiple adaptive responses to a hypoxic environment. Native Bolivian Andeans have lived at an altitude of 4000 m in The Andes mountains for about 14,000 years exposed to a low atmospheric pressure (453 mmHg) and low oxygen concentration (12,7%). It was described Andeans adaptation to high altitude is mainly related to cardiovascular issues (PMID: 29100088), with a low significantly genetic selection in HIF pathway components responsible of erythropoiesis regulation (PMID: 36980912), so that they have adapted to exist with increased hemoglobin concentrations. Thus, Andeans can be prevalent to undergone hematological and thromboembolic disorders. Thrombotic events have been reported to be increased at high altitude, and this recently associated to increased transferrin (Tf) (PMID: 36040436), likewise to increased erythropoietin (Epo) (Amaru, RevMed 2022). Increased Tf were described to be induced by both iron deficiency and hypoxia via HIF (PMID: 9242677). In addition, iron deficiency was postulated to inhibit the function of prolyl hydroxylase 2 (PHD2) in the hydroxylation of HIF-2, necessary for recognition and degradation by VHL (PMID:22304911). Under normal conditions, Tf is bound to fibrinogen at a molar ratio of 4:1, leading to plasma Tf sequestration by fibrinogen and leaving little Tf free in the circulation. While at hypoxia conditions, abnormally upregulated Tf potentiates thrombin, factor XIIa and inhibits antithrombin, inducing hypercoagulability (PMID:36040436). Similarly, increased HIF1a at high altitude increases Tf promoting the transferrin gene expression, which contains HIF-1α binding sites in its enhancer region (PMID:36844187). HIF is a DNA-binding transcription factor for activating expression of Epo gene (PMID:32561149). Epo, in turn, is increased due to the hyperegulation of HIF as expected at low atmospheric pressure environments. Epo activates the hypoxia-inducible factor that leads to Epo synthesis to stimulate red blood cell production, which also increases the demand for iron to synthesize hemoglobin (PMID:21078592). This, added to a decreased iron and increased HIF 1a, gives rise to a hyperregulation of Tf. Epo has been described to have prothrombotic properties and be related to thrombotic events (PMID:10779449; Amaru, RevMed 2022). In this sense, to further observe a correlation of transferrin and erythropoietin increased levels with the risk of thrombotic events at high altitude, we studied patients with erythrocytosis, anemia and polycythemia vera. We analyzed clinical, lab tests and epidemiological data of 920 patients with Chronic Mountain Sickness erythrocytosis (CMS-e) (n=560), anemia (n=372), and Polycythemia Vera (PV) (n=20), all Bolivian Andeans born and residing at 4000 m. Considering at this altitude normal hemoglobin concentrations vary from 14-17 g/dl in women and 15-18 g/dl in men, erythrocytosis condition comprises Hb levels >18 g/dl in women and >19 g/dl in men (Amaru et al, Rev Hematol Mex, 2016); similarly, it embraces Hb <12 g/dl for women and <13 g/dl for men in anemias (PMID: 23317073). Positive JAK F617V tests were corroborated in PV patients. Iron deficiency correlation considered serum ferritin <30 ug/L and MCV <80 fL (Clark et al, Nutrition, 2008). Eco-doppler study records confirmed the occurrence of thrombotic events. Data analysis was performed through Excel 16.29.1 and SPSS program, Chi-squared, Fishers and Pearson tests were done. We observed significative increased Tf levels in erythrocytosis, anemia, and PV patients mainly on those presenting iron levels deficiency. Epo was highly increased in erythrocytosis patients with iron deficiency. These results correlated with the incidence of thrombotic events in erythrocytosis (CMS-e) and anemia patients with iron deficiency. Age was also relevant in both groups. (Table 1). The increase of Epo levels correlated with thrombosis in erythrocytosis and PV patients with iron deficiency (Table 1). Transferrin and Erythropoietin increased levels at high altitude correlated with thrombosis in Chronic Mountain Sickness erythrocytosis and iron deficiency anemia patients. However, Increased Tf did not correlate with thrombosis in PV patients at high altitude. Our data confirm that increased Tf and Epo have prothrombogenic properties in CMS-e, iron deficiency anemia patients at high altitude.

Abnormalities in red blood cell production and pathogenesis of anemia in the progression of rock bream iridovirus (RBIV)

Rock bream iridovirus (RBIV), belonging to Megalocytivirus, causes severe mortality in rock bream. Almost all deaths associated with RBIV are accompanied by splenic enlargement and anemia. Although red blood cells (RBCs) are involved in the immune response against viral infections, their involvement in rock bream has not yet been studied in terms of the immune response against RBIV. In this study, the viral replication patterns, blood characteristics and anemia-related factors were evaluated in rock bream post RBIV infection. The virus-infected RBCs of rock bream demonstrated similarities in the expression levels of hemoglobins (HGB) (α and β), cytokine-dependent hematopoietic cell linker (CLNK) and hematopoietic transcription factor GATA (GATA), with significantly decreasing levels from 4 days post infection (dpi) to 17 (dpi), when the viral replication was at its peak. This suggests that the expression of blood-related genes is inadequate for HGB synthesis and RBC production, thereby causing anemia leading to death. Moreover, the levels of complete blood cell count (CBC) indicators, such as RBCs, HGB and hematocrit (HCT), significantly decreased from 10 to 17 dpi. This phenomenon suggests that blood-related gene expression and/or RBC-, HGB- and HCT-related levels are critical factors in RBIV-induced anemia and disease progression. These results highlight the significance of blood-mediated immune responses against RBIV infection in rock bream. Understanding blood-related gene levels to identify blood-related immune response interactions in rock bream will be useful for development of future strategies in controlling RBIV diseases in rock bream.

Case Report: Severe ischemic priapism as the clinical presentation in patient with chronic myeloid leukemia (CML) treated by proximal shunt

Background: Priapism refers to an erection of the penis that persists for four hours or more and is not associated with sexual arousal or stimulation. This condition is thought to be caused by abnormal production of blood cells in the bone marrow, which leads to the release of certain chemicals into the bloodstream. The current article presents a severe ischemic priapism case, and assesses and contrasts the documented instances of patients with chronic myelogenous leukemia (CML) who experienced priapism as their primary clinical symptom over the last two decades. Case presentation: The 21-year-old male patient presented with persistent and painful erections for three days. Further tests revealed he had CML and ischemic priapism. He was treated with distal shunting and proximal shunting with cytoreductive therapy to resolve the symptoms. Conclusions: Priapism is a medical emergency that can cause permanent damage to the erectile tissue if not treated promptly. Prolonged and painful erections can cause ischemia, leading to tissue damage and decreased function. If priapism is caused by an underlying medical condition; delaying treatment can allow the underlying condition to progress and become more difficult to treat.

Determination of bone marrow cell morphology in rat.

The skeletal system of the body is responsible for important functions in the human body. In addition to causing movement, this system also plays a role in the production of blood cells and fat storage. Bone marrow is a spongy or viscous tissue that fills the inside of the body's bones. The basic structure of bone marrow is of two types. Red bone marrow and yellow bone marrow. Red bone marrow contains blood stem cells that can become red blood cells, white blood cells, or platelets. Yellow bone marrow is made mostly of fat and contains stem cells that can turn into cartilage, fat, or bone cells. Human bone marrow mesenchymal stem cells (HBMSCs) are widely used cell sources for clinical bone regeneration. Achieving a therapeutic effect depends on the osteogenic differentiation potential of the stem cells. The purpose of judging the morphology of bone marrow cells is to diagnose leukemia or bone marrow disorders, determine the cause of severe anemia or thrombocytopenia and low platelet count, identify abnormal chromosomes to prevent hereditary diseases, and plan their treatment. In this study, we examined the morphological characteristics of bone marrow cells, mesenchyme cells, and osteoblasts in a laboratory environment. The results of the morphological investigations showed changes such as the change of the position of the nucleus and the rounding of the cytoplasm in the differentiated cells compared to the mesenchyme cells. Therefore, to identify and diagnose as many of these cells as possible, molecular genetic techniques such as network algorithms and fluorescence staining can be used for hematological and cytomorphological investigations.

Complement involvement in sickle cell disease

Sickle Cell Disease (SCD) is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, leading to the formation of sickle-shaped red blood cells, causing vaso-occlusion. Inflammation is a key component of the pathophysiology of SCD, contributing to the vascular complications and tissue damage. This review is centered on exploring the role of the inflammatory complement system in the pathophysiology of SCD. Our goal is to offer a comprehensive summary of the existing evidence regarding complement activation in patients with SCD, encompassing both steady-state conditions and episodes of vaso-occlusive events. Additionally, we will discuss the proposed mechanisms by which the complement system may contribute to tissue injury in this pathology. Finally, we will provide an overview of the available evidence concerning the effectiveness of therapeutic interventions aimed at blocking the complement system in the context of SCD and discuss the perspective of complement inhibition.

Modulations by L-Carvone and Thymoquinone to Exert Protection on Bone Marrow Cells Against Benzene-induced Toxicities Through Anti-inflammatory Pathways in SD Rats

Background Adverse effects of benzene (BZ) exposure toxicities were documented by many researchers worldwide. BZ exposure causes many hematological abnormalities that might be treated with naturally occurring phytocompounds. Aim In this experimental study, we evaluated the anti-inflammatory effect of L-carvone and thymoquinone together (LCTQ) against BZ-induced inflammatory toxicities in Sprague-Dawley (SD) rats. Introduction BZ exposure could cause an excess formation of immature blood cells to enter the peripheral bloodstream. Phytocompounds namely L-carvone (LC) and thymoquinone (TQ) have confirmed anti-inflammatory effects against various diseases. Materials and Methods Rats were divided into four different groups such as control, LCTQ group, BZ pathologic group, and treatment group (LCTQ+BZ). After 10 weeks of the experimental period, body weight changes, hematological parameters, pro-inflammatory cytokines, oxidative stress, RBC antioxidants, bone marrow cellular abnormalities, and bone marrow DNA fragmentation were evaluated. Results BZ toxicity showed abnormal loss of body weight, altered hematological parameters, increased pro-inflammatory cytokines, abnormal cellular oxidative status, and DNA damage. LCTQ treatment showed significant ( p < 0.05) increase in body weight, normalized hematological parameters such as red blood cells, hemoglobin, white blood cells (lymphocytes and eosinophils), platelets (PLT), and hematocrit with RBC parameters, reduction of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), controlled oxidative stress, normalized enzymatic antioxidants in the RBC cells, normalized nucleated cells, megakaryocyte cells, and controlled DNA fragmentation were observed. Conclusion The current study showed an anti-inflammatory effect of LCTQ through the control of inflammation against benzene-induced toxicities in rats.

Abstract 14829: Inflammatory Cells and Cytokines Are Increased in Mice With Pulmonary Arterial Hypertension Associated With Hematopoietic Dnmt3a Depletion

Background: Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodeling of the precapillary pulmonary arteries. Increasing evidence suggests that inflammation plays an essential role in PAH development. Understanding the involvement of immunity in this context can provide valuable insights into the pathogenesis of PAH. DNA Methyltransferase 3A ( DNMT3A ) mutations can lead to altered DNA methylation patterns in immune cells, potentially affecting the expression of immune-regulatory genes. Here, we investigate the role of inflammation in PAH driven by hematopoietic Dnmt3a depletion in mice. Methods: We used male and female conditional, Vav-Cre-driven hematopoietic Dnmt3a knockout mice ( Dnmt3a –/– ) and compared them to controls (n=3/group). Lung tissue and plasma were collected. Single-cell suspensions of digested lungs were stained to detect total leukocytes (CD45+), T cells (CD3+), B cells (CD19+), macrophages (Ly6G-, F4/80+), and neutrophils (Ly6G+, F4/80-) via flow cytometry. Plasma samples were screened for 32 distinct cytokines using a cytokine array. Results: Dnmt3a –/– mice spontaneously developed PAH at 4.5 months. PAH was associated with a 19% increase in total CD45+ leukocytes in the lungs compared to controls. T cells, and B cells were increased in Dnmt3a –/– mice compared to controls (7.9% and 3.1%, respectively). In contrast, there was no difference in lung neutrophil count. Two subsets of macrophages were observed based on granularity. Compared to controls, Dnmt3a –/– mice had a 29% increase in agranular macrophages and a 19.7% increase in granular macrophages. IL-13, known for its role in alternative monocyte/macrophage activation in PAH patients associated with systemic sclerosis (SSc-PAH), was significantly increased in the plasma of Dnmt3a –/– mice compared to controls (1.8 times higher, p=0.02). IL-12p40, a chemoattractant for macrophages, showed a trend towards being increased in Dnmt3a –/– mice compared to controls (5.7 times higher, p=0.14). Conclusion: Deletion of DNMT3A in the hemopoietic system triggers an inflammatory form of PAH in mice, associated with increased macrophages, B- and T-cells in the lung. IL-13 may be a potential diagnostic marker in PAH.

Tropomyosin 1 Deficiency Facilitates Epithelial-to-Mesenchymal Transitions to Promote Hemogenic Endothelial Specification and Hematopoietic Progenitor Cell Formation

Tropomyosins coat actin filaments and impact many actin-related biology, including signaling and cell morphogenesis. Prior work suggested that TPM1 regulated blood cell formation in vitro, but it was unclear how or when TPM1 affected hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC)model systems, single cell transcriptomics, and a novel murine model, we aimed to define how and when TPM1 regulated developmental hematopoiesis. We used CRISPR/Cas9 to create TPM1 knockout (KO) iPSC lines in a primitive hematopoiesis system, in which hemogenic endothelial cells spawn hematopoietic progenitor cells (HPC). Western blot showed that TPM1 was highly expressed in iPSCs and endothelial cells but virtually absent in HPCs. TPM1KO enhanced endothelial cell and hematopoietic progenitor cell (HPC) formation without augmenting cell cycle kinetics. RNA sequencing analysis showed that TPM1KO altered epithelial-to-mesenchymal transition (EMT)-related gene expression and functionally enhanced EMT during in vitro hematopoiesis, without generally disrupting normal cell development. Single-cell transcriptomics showed that TPM1 was highly expressed in human and mouse hemogenic endothelium, and abrogated in HPCs, suggesting that TPM1 might also regulate developmental hematopoiesis in vivo similar to our in vitro findings. Indeed, TPM1 expression coincided with EMT-related gene expression during in vivo hematopoiesis (p<1x10-16). To investigate the role of TPM1 in vivo, we used a novel murine TPM1 GeneTrap-Reporter mouse model. By western blot, TPM1 protein was decreased ~50% in peripheral blood. TPM1 GT/GTmice displayed embryonic lethality by E9.5, due to cardiac dysmorphology previously seen in TPM1 knockout mice. TPM1 reporter expression (LacZ) was observed in E9.5 endothelium, temporally consistent with Runx1+/CD31+ hemogenic endothelial cell emergence. Whole-mount imaging of TPM1 GT/+ embryos showed an enhanced percentage (%) of Runx1 + HE at E9.5 and Runx1 +cKit + HPCs at E10.5. Consistent with increased embryonic HPC production, TPM1 GT/+mice showed increased percentages of Lin-Sca1+cKit+ HPCs in bone marrow at 6-8 weeks of age. Complete blood counts in 6-8 weeks-old mice were normal. Conclusions These findings illuminate novel effects of TPM1, a cytoskeletal regulatory molecule, on developmental hematopoiesis. TPM1KO disinhibits EMT to promote hemogenic endothelial specification and HPC production during developmental hematopoiesis, without compromising mature blood cell function. Temporal TPM1 inhibition may offer innovative approaches to enhance blood cell formation in translationally relevant cell culture systems. Our findings may explain genetic associations linking polymorphisms in the TPM1 gene locus with altered human blood traits. More broadly, the radical changes required to form HPCs from endothelial cells during hematopoiesis represent an exciting area of study for actin and tropomyosin biology.

Red Cells Distribution Width and Prognosis in Myelofibrosis Patients Treated with Ruxolitinib

Introduction Myelofibrosis (MF) is a BCR::ABL1-negative myeloproliferative neoplasm characterized primarily by genetic abnormalities and an aberrant production of pro-inflammatory cytokines (including IL-2, IL-6, TNF-α, and TGF-β among others) released from pathological hematopoietic stem cells leading to the deposition of reactive fibrosis in the bone marrow (BM), at the same time the development of extramedullary hematopoiesis, mainly in the spleen and liver, and finally the production of red blood cells (RBC) of often bizarre shape, i.e. dacryocytes. Consequently, increased variability in RBC volume, so-called anisocytosis, is a hallmark of MF. A small number of studies have already demonstrated that quantitative measurement of anisocytosis in terms of red cells distribution width (RDW) may have a negative prognostic role in newly diagnosed MF patients. Aim In this study we further evaluate the prognostic impact of RDW in a series of patients with primary (PMF) and secondary myelofibrosis (SMF) at diagnosis and during treatment with ruxolitinib (RUX). Methods Between October 2014 and May 2023, the RDW of 49 consecutive patients with PMF or SMF (32 males and 17 females; median age at RUX initiation, 70.5 years) were retrospectively assessed at MF diagnosis, at RUX start and after 3, 6, 12, 24 and 36 months from therapy initiation. All patients were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. Red cells distribution width was routinely reported by automated cell counters as part of RBC count, with an upper limit of normal of 14.5% used as a cut-off in our laboratory. At the time of RDW evaluation, complete blood cells count and spleen size [expressed as the distance from the left costal margin (BCM) in cm] were available for all patients. Results Among 49 patients, 24 had PMF (10 prefibrotic and 14 overt fibrotic) and 25 SMF [15 had post-polycythemia vera (PPV)- and 10 post-essential thrombocythemia (PET)-MF]. The JAK2V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%), while 2 patients (4.1%) were “triple-negative”. Elevated RDW values were present in all but 3 patients at MF diagnosis with a median RDW of 18.7% (range, 13.3% - 25.7%). RDW was higher in patients with splenomegaly ( p=0.04) and with JAK2V617F mutation ( p=0.09). Furthermore, looking specifically at JAK2V617F-mutated cases, the greater the RDW values, the higher the JAK2V617F allele burden. Interestingly, a higher RDW value at diagnosis also correlated with a shorter time from MF diagnosis to RUX start (-3.5 months per one RDW unit, 95%CI: -7.5 to +0.4; p=0.08). We observed a modest (<1%) increase in RDW during the first 6 months of RUX treatment. In a multivariable random-intercept model that considers all time points and contains the covariates time, RUX dose, and hemoglobin (Hb), we also observed a clear decrease in RDW with increasing Hb (slope: -0.35% per g/dL of Hb, 95%CI: -0.56 to -0.14; p=0.001). Median overall survival (OS) from MF diagnosis was 6.5 years with a median duration of RUX treatment of 33.3 months. The median RDW at diagnosis of 18.7% was used as a cut-off to identify two subgroups of patients [Group 1 (N=22): RDW 18.7% to 25.7%; Group 2 (N=23): RDW 13.3% to 18.6%] showing a difference in OS [Group 1 vs. 2: Hazard Ratio (HR) 2.74, 95%CI: 1.13 to 6.65; p=0.03] ( Figure 1); the median OS was 8.5 (14 deaths) and 11.8 (9 deaths) years for the Group 1 and 2, respectively. Using continuous RDW at diagnosis, the crude HR was 1.21 per one RDW unit (95%CI: 1.06 to 1.37; p=0.004). In a Cox model adjusted for gender, age, and Hb, the HR was 1.13 (95%CI: 0.99 to 1.29; p=0.06). Notably, RDW at diagnosis was associated with a 35% increase in infectious disease mortality in a crude analysis (HR 1.35, 95%CI: 1.07 to 1.71, 8 deaths; p=0.01) and a 27% increase (HR 1.27, 95%CI: 0.99 to 1.63; p=0.07) in the adjusted analysis. Conclusions Red cells distribution width may represent a good integrative measure of several disease processes and also has prognostic significance at the time of MF diagnosis and during RUX treatment. Even though our preliminary observations are limited by the small number of patients and need to be further confirmed by larger studies, we can speculate that RDW may help in the rapid detection of MF patients with a poor prognosis, which can only partially be improved from currently available targeted therapies with JAK inhibitors.

GDF11/SMAD Regulated Splicing of GATA1 Is Associated with Response to Luspatercept in Lower-Risk Myelodysplastic Syndromes (LR-MDS)

Introduction: Luspatercept is an erythroid maturation agent that promotes late-stage erythropoiesis in patients with LR-MDS. While it is known that luspatercept, a modified activin receptor ligand trap, binds to GDF11 and Activin B, the mechanism by which these ligands trigger a decrease in red blood cell (RBC) formation is unknown. Luspatercept was approved by the FDA following the phase 3 MEDALIST study, but biomarkers of response are currently unknown. Results:In a large cohort of primary MDS CD34+ samples, GDF11 expression was significantly higher in MDS samples than in age-matched healthy controls (183 MDS samples vs 17 controls, P < 0.05). GDF11 was detected in primary MDS sera and was substantially elevated in the refractory anemia (RA) and RA with ring sideroblasts (RARS) MDS subgroups. Activin receptor 2B (ACVR2B), the receptor of GDF11, was also overexpressed in MDS samples, and its expression was confirmed by immunohistochemistry (IHC) in the bone marrows of primary MDS patients. GDF11/ACVR2B activates SMAD2, and we found that the overexpression of SMAD2 in CD34+ cells was associated with more severe anemia in MDS patients. RNA-seq and SMAD2 ChIP-seq analyses were then performed to determine the downstream effects of GDF11 in erythroid progenitors. Human CD34+ primary cells were utilized to generate BFU-E stage erythroid progenitors. We discovered that GDF11 promotes an abnormal erythroid gene expression program characterized by increased expression of pro-apoptotic and early erythroid differentiation-related genes. SMAD2 binding was detected in regulatory regions of early erythroid (including GATA1), proinflammatory, and apoptotic transcripts using ChIP-seq analysis with p-SMAD2. In a zebrafish model, administration of GDF11 caused a decrease in erythroid cells, which was rescued by treatment with luspatercept (Wobus et al. Leukemia 2021 35(10):2936-47). Luspatercept inhibited the phosphorylation and activation of SMAD2 in response to GDF11 stimulation in primary hematopoietic cells and leukemia cell lines. Importantly, luspatercept could reverse the growth-inhibiting effects of MDS-derived sera on primary hematopoietic progenitors. Finally, to identify biomarkers associated with clinical responses, we analyzed phase 3 MEDALIST study samples collected before and after treatment with lupatercept. RNA-seq analysis was performed on bone marrow samples from responders (R; n = 14) and non-responders (NR; n = 9) (Figure). Analysis of pre-treatment/screening bone marrow transcriptomes revealed no significant differences between R and NR in global gene expression. Analysis of splice isoforms between the two cohorts revealed strikingly greater differences. Most differential splicing events were observed in NR (702 in R vs 5232 in NR). The most prevalent splicing modifications were exon skipping events, and most functional pathways affected by differential exon usage were RNA splicing related. GATA1, the master erythroid transcription factor, was spliced differently in R at baseline, which was an intriguing observation. Response to luspatercept was found to be associated with exon 2 skipping, resulting in a shortened isoform of GATA1. R had a greater proportion of the short GATA1 isoform than NR (12% in R versus 6% in NR; P = 0.04). We analyzed ChIP-seq data from primary erythroid cells to determine the effect of GDF11/SMAD2 activation on GATA1 splicing. GDF11 stimulation resulted in SMAD2 binding to the second intron of GATA1. CRISPR-mediated deletion of the SMAD2 binding region in primary erythroid progenitors increased expression of the GATA1 long isoform. Next, we performed a CRISPR-mediated deletion of exon 2 of GATA1, resulting in a predominance of GATA1 short isoform expression and a decrease in erythroid output. In CRISPR-modified primary erythroid cells with a predominant short GATA1 isoform (exon 2-deleted cells), luspatercept was able to reverse the inhibition induced by GDF11. Conclusions: These results demonstrate that MDS cases that respond to luspatercept are associated with a relatively high level of expression of functionally deficient GATA1 short isoform. We further demonstrate that GDF11 stimulates the production of the GATA1 short isoform via SMAD2 binding to intron 2 of the GATA1 gene. The regulation of a GATA1 splicing isoform by GDF11/SMAD2 is associated with clinical responses to luspatercept, according to these novel findings.

Hematological Parameters: Comparative Evaluation of Performance between Dxh 500 and XN-10 Equipments

Introduction The development of hematopoietic stem cell transplantation (HSCT) was an extremely important milestone for treating certain malignant and non-malignant hematological diseases. The HSCT process contemplates collecting hematopoietic progenitor cells (HPC) from the donor, conditioning chemotherapy, infusion of HPC, and engraftment. Mobilized peripheral blood is the most commonly used HPC source for HSCT due to its ease of obtaining through an apheresis procedure and high progenitor cell count yields compared to bone marrow source, which requires a bone marrow aspiration procedure, usually performed in a surgical center under general anesthesia, and requires the collection of higher volumes to reach the amount of HPC desired. The total nucleated cell count (TNC) corresponds to the sum of all nucleated cells in the sample and is used as a reference for the target dose for both sources of HPC. The TNC provides essential information on the quality of the sample obtained, since an infusion of insufficient number of cells affects the ability to reconstitute the recipient's hematopoietic system. An insufficient TNC can result in poor engraftment, leading to a slow recovery of blood cell production and increasing the risk of complications such as infections and bleeding. Determination of the number of nucleated cells in bone marrow and peripheral blood samples can be performed automatically by hematological analyzers such as the Beckman Coulter DxH 500 and Sysmex XN-10. This study aims to compare the performance of those equipments in determining hematological parameters in bone marrow and apheresis material. Materials and methods We compared the results of 40 HPC samples (20 bone marrow samples and 20 peripheral blood samples collected by apheresis) analyzed in the Sysmex XN-10 and Beckman Coulter DxH 500 equipments. All these specimens were analyzed without dilution and diluted 1:10. The evaluated parameters were: TNC, hematocrit, platelets, lymphocytes, monocytes, and granulocytes. Results The analysis of the results showed a positive Pearson correlation coefficient (above 0.9) between XN-10 and DxH 500 equipment for the evaluation of TNC in diluted samples obtained by apheresis (figure 1) and by bone marrow (figure 2). In pure samples obtained by apheresis the DxH 500 equipment showed non-linearity for TNC but revealed a positive correlation with the XN-10 when evaluating pure bone marrow samples (figure 2). On the other hand, XN-10 equipment showed linearity when correlating TCN between pure and diluted samples obtained by apheresis (figure 1) and bone marrow aspiration (figure 2). Hematocrit analysis in samples obtained by apheresis showed 90% and 100% of agreement for diluted samples between the equipment and for diluted versus pure samples, respectively, when using the cutoff value of 12%. Hematocrit validation using the Beckman Coulter equipment was impaired due to the sample's viscosity and limited linearity. Also, there was a satisfactory hematocrit correlation for pure bone marrow samples and comparing diluted versus pure bone marrow samples with both equipment. The results also showed a good correlation when analyzing the platelet count in pure and diluted samples (bone marrow and apheresis) with XN-10 or DxH 500, as well as comparing the diluted samples versus the pure samples. The use of differential count of nucleated cells (lymphocytes, monocytes and granulocytes) realized by the two evaluated devices was not approved for bone marrow or apheresis samples. Conclusion The determination of TNC, platelet and hematocrit counts using the Sysmex XN-10 equipment were satisfactory for both pure and diluted samples obtained by apheresis or bone marrow. For pure samples obtained by apheresis analyzed by DxH 500, we could observe absence of linearity for TNC and hematocrit. This situation could be explained by the high number of cells in this sample and the consequent high viscosity.