Iron Overload In Myelodysplastic Syndromes Research Articles

Ker-050 Treatment Reduced Iron Overload and Increased Bone Specific Alkaline Phosphatase in Participants with Lower-Risk MDS Supporting Potential to Restore Balance to the Osteohematopoietic Niche

Background: Iron overload (IO) in myelodysplastic syndromes (MDS) contributes to the deterioration of the osteohematopoietic niche (OHN), where hematopoietic and osteogenic precursors interact to regulate hematopoiesis and bone metabolism. Excess iron exacerbates ineffective hematopoiesis, inflammation, and bone loss via direct effects on resident hematopoietic and osteogenic precursors. Iron chelation therapy (ICT) is associated with improved event-free survival in patients with MDS but does not address the underlying cause of IO and introduces patient burden due to tolerability and cost. KER-050 is an investigational, modified activin receptor type IIA ligand trap designed to inhibit select TGF-β superfamily ligands (activins A, B, GDFs 8, 11) and promote differentiation and maturation of erythroid and megakaryocytic precursors. KER-050 has potential to reduce dependence on red blood cell transfusions and increase iron utilization via erythropoiesis, possibly mitigating IO at its source. As previously reported from an ongoing Phase 2 study (NCT04419649), KER-050 treatment in participants with lower-risk (LR) MDS resulted in rates of modified IWG 2006 Hematological Improvement-Erythroid (HI-E) and transfusion independence (TI) of 51.4% and 42.3%, respectively, with a mean decrease in ferritin (322 ng/mL at Week 24) among HI-E and/or TI responders. KER-050 treatment also demonstrated effects on osteogenic precursors, including prevention of bone loss in a mouse model of MDS and dose-dependent increases in bone specific alkaline phosphatase (BSAP), a marker of bone formation, observed in healthy postmenopausal women. Thus, KER-050 has potential to address the multifaceted pathogenesis of MDS, including IO. Here, we present new data from the ongoing Phase 2 study in MDS, exploring the potential of KER-050 to provide benefit beyond hematologic responses and to ameliorate IO in MDS. Methods: This ongoing Phase 2 study is evaluating KER-050 in participants with LR MDS. Data from exploratory assessments of markers of IO, hematopoiesis, and bone turnover are presented as of a cutoff date of April 3, 2023, for all participants who had received the recommended Part 2 dose (RP2D; 3.75-5 mg/kg q4wks). Results: Baseline ferritin levels in RP2D participants (N=59) were generally elevated, while levels of mean corpuscular hemoglobin and reticulocyte hemoglobin were in the normal range (Table 1). After KER-050 treatment, sustained decreases in serum ferritin were observed among participants with baseline levels ≥1000 ng/mL (Figure 1), likely driven in part by reduced transfusion burden in responders. Concomitant increases observed in soluble transferrin receptor (sTfR; Figure 1) suggest that increased erythropoiesis may also have contributed. Mean decreases in ferritin observed in non-transfused (NT) participants (-228 ng/mL at Week 24, n=7) indicate potential for KER-050 to affect iron homeostasis by mechanisms other than transfusion reduction. In 1 NT participant receiving ICT, the baseline ferritin of 1632 ng/mL decreased to <500 ng/mL over 32 weeks, leading to ICT discontinuation and coinciding with an increase in hemoglobin (Hgb) from 9.2 g/dL at baseline to 11.3 g/dL over the same period. In another NT participant not receiving ICT, ferritin decreased from 1094 ng/mL at baseline to 884 ng/mL and 583 ng/mL at Weeks 24 and 68, respectively, while Hgb gradually increased from 9.94 g/dL at baseline to 10.7 and 12.2 g/dL at Weeks 24 and 68. KER-050 treatment was also associated with increases in BSAP, a new finding in this MDS population that is consistent with prior observations in preclinical and healthy volunteer studies. Despite some heterogeneity, mean increases of 8.47% (n=23) and 10.4% (n=13) were observed at Weeks 24 and 48, respectively; 3 participants with erythroid responses had increases in BSAP of ≥30% at these time points. Summary: Exploratory data indicate potential for KER-050 to drive meaningful decreases in ferritin in participants with LR MDS, particularly among those with IO. While transfusion reduction likely contributes, observations in NT participants highlight the potential for enhancement of erythropoiesis to improve IO, irrespective of transfusions. Observed changes in BSAP suggest that KER-050 is also acting on osteogenic precursors, with the potential to provide functional improvements within the OHN. Updated results will be provided at the presentation.

Iron Chelation Improves Ineffective Erythropoiesis and Iron Overload in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Patients are treated with periodic red cell (RBC) transfusions, resulting in iron overload. Iron overload in MDS patients correlates with decreased survival, yet despite the availability of effective, safe, and orally administered iron chelation therapy, the treatment of iron overload in MDS remains controversial. The TELESTO trial, using deferasirox in low risk MDS patients, revealed an increase in event-free survival without impacting overall survival, hemoglobin, or RBC transfusion requirements [Angelucci Ann Int Med 2020]. However, iron chelators have different affinities for iron relative to transferrin; deferiprone has the lowest affinity of available iron chelators [Sohn Blood 2008], consistent with its ability to increase hepcidin expression in mice [Schmidt Am J Hematol 2015; Casu haematol 2016]. We hypothesize that deferiprone (DFP) impacts systemic and cellular iron distribution to enhance erythropoiesis in MDS. To test this hypothesis, we employed a highly penetrant well-established mouse model of MDS, NUP98:HOXD13 fusion transgenic mice. These MDS mice exhibit hypercellular marrow, increased apoptosis in the bone marrow compartment, and impaired survival relative to control; a proportion of mice progress to acute leukemic transformation after 7 months of age [Lin Blood 2005]. Using MDS mice, we further characterize parameters of iron trafficking in erythroblasts, evaluate the effects of DFP on erythropoiesis and iron distribution, and provide correlatory evidence in stem and progenitor cells from MDS patients. MDS mice were treated at 5 months of age with 1.25 mg/mL DFP in the drinking water for 4 weeks and compared with untreated MDS mice. Our results characterize an iron overload phenotype with aberrant erythropoiesis in MDS mice, reversed in DFP-treated MDS mice (Figure 1). Specifically, DFP leads to a redistribution of iron from liver and spleen to increase serum iron concentration and transferrin saturation, increased hemoglobin and RBC concentration in circulation, and normalized hepcidin responsiveness to iron. While MDS mice exhibit relatively decreased WBC count, no changes in WBC count are observed in DFP-treated MDS mice. Importantly, serum erythropoietin (Epo) concentration, splenomegaly, expanded erythropoiesis and erythroblast differentiation in the bone marrow are normalized in DFP-treated MDS mice. As expected when erythropoiesis is reversed, erythroblast expression of Fam132b (erythroferrone) is suppressed but no changes in STAT5 or AKT signaling, pathways downstream of Epo, are evident in DFP-treated MDS mice. These findings suggest that the expected changes in signaling downstream of Epo are unaffected by DFP administration, implicating possible changes in erythroblast Epor expression in DFP-treated MDS mice. We used sorted bone marrow to evaluate Epor expression in progressive stages of terminal erythropoiesis and demonstrate decreased Epor expression in later stage bone marrow erythroblasts from MDS mice, normalized in DFP-treated MDS mice. These findings provide evidence that increased Epor expression is a potential mechanism by which DFP leads to enhanced Epo-responsiveness and enhanced erythroblast differentiation despite decreased serum Epo concentration in MDS mice. In addition, we demonstrate for the first time increased ferritin concentration and normalized Tfr1, Pcbp1 and Ncoa4 expression in late stage erythroblasts from DFP-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts and their normalization with DFP treatment (Figure 2). Importantly, this effect is not observed in DFP-treated WT mice. Our data also provides evidence that reversal of ineffective erythropoiesis in DFP-treated MDS mice occurs independently of effects on erythroblast apoptosis. Finally, we confirm increased expression of genes involved in Epo-mediation as well as iron uptake and trafficking-Epor, Fam132b, Tfr1, Pcbp1 and Ncoa4-in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS and provide a rationale for exploring the effect of DFP in patients with low risk MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinical Significance of Iron Overload and Iron Metabolism in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Myelodysplasticsyndrome (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell diseases leading to an insufficient formation of functional blood cells. Disease-immanent factors as insufficient erythropoiesis and treatment-related factors as recurrent treatment with red blood cell transfusions frequently lead to systemic iron overload in MDS and AML patients. In addition, alterations of function and expression of proteins associated with iron metabolism are increasingly recognized to be pathogenetic factors and potential vulnerabilities of these diseases. Iron is known to be involved in multiple intracellular and extracellular processes. It is essential for cell metabolism as well as for cell proliferation and closely linked to the formation of reactive oxygen species. Therefore, iron can influence the course of clonal myeloid disorders, the leukemic environment and the occurrence as well as the defense of infections. Imbalances of iron homeostasis may induce cell death of normal but also of malignant cells. New potential treatment strategies utilizing the importance of the iron homeostasis include iron chelation, modulation of proteins involved in iron metabolism, induction of leukemic cell death via ferroptosis and exploitation of iron proteins for the delivery of antileukemic drugs. Here, we provide an overview of some of the latest findings about the function, the prognostic impact and potential treatment strategies of iron in patients with MDS and AML.

Dual-layer detector spectral CT versus magnetic resonance imaging for the assessment of iron overload in myelodysplastic syndromes and aplastic anemia.

The purpose of this study is to investigate the performance of dual-layer detector spectral CT for iron deposition compared to magnetic resonance imaging (MRI) T2* imaging. Thirty-one patients with a clinical history of myelodysplastic syndromes and aplastic anemia underwent liver and cardiac T2*-weighted unenhanced MRI on a three-tesla MRI scanner, and underwent unenhanced CT scan laterally on a 128-row spectral detector CT. R2* values of the liver, septal muscle, and paraspinal muscle were calculated. Attenuation differences (ΔH) in the liver and myocardium were calculated between the lower (50keV) and higher (120keV) energy levels. The liver and cardiac T2* values were 9.54 ± 5.63ms and 21.41 ± 2.44ms, respectively. The liver-to-muscle and myocardium-to-muscle T2* value ratios were 0.37 ± 0.23 and 0.79 ± 0.19, respectively. The liver and cardiac ΔH were -1.13 ± 4.24HU and 2.22 ± 4.41HU, respectively. There was a strong linear correlation between the liver R2* and ΔH (r = -0.832, P < 0.001), but weak correlation existed between the cardiac R2* and ΔH (P = 0.041). Dual-layer detector spectral unenhanced CT seemed to be equally valuable to MRI T2* imaging for evaluating liver iron overload.

Treatment of iron overload in myelodysplastic syndromes and other bone marrow failure syndromes

Iron is essential to maintain cellular homeostasis, such as hemoglobin synthesis, mitochondrial respiratory chain formation, DNA replication, DNA demethylation, and histone demethylation. In addition, iron acts as a catalyst to produce reactive oxygen species, including hydroxyl radicals, which induce 8-OHdG production and DNA double strand breaks. Hence, the total body iron level should be strictly regulated. Recently, hepatic hepcidin was found to inhibit iron absorption from the gastrointestinal tract, and hepcidin production is reduced by erythroid factors, such as growth differentiation factor 15 (GDF15) and erythroferrone. Systemic iron kinetics seem to be regulated by cooperation among the liver, gastrointestinal tract, and hematopoietic tissues. However, this cooperation could be disturbed in bone marrow failure syndrome (BMFS). In some anemic disorders, such as β-thalassemia, and some categories of MDS, GDF15 or erythroferrone were overproduced and promoted iron absorption. Frequent blood transfusions rapidly increase iron accumulation in the body and eventually result in irreversible organ damage, such as heart failure. Therefore, the introduction of an early intervention to improve iron overload in BMFS is necessary. In recent years, oral chelators have been introduced for clinical use. Erythropoiesis-stimulating agents and thrombopoietin receptor agonists could also improve refractory anemia or BMFS and improve iron overload.

Assessment By MRI T2*of Iron Accumulation and Removal in Transfusion Dependent MDS Patients

Introduction: Transfusion dependency in Myelodysplastic syndrome (MDS) patients is associated with shortened over-all survival and leukemia-free survival; however, it is not clear whether this detrimental effect is mediated by transfusional iron overload itself or whether the need for RBC transfusion is a surrogate marker of disease severity. Additionally, despite the negative effects of anemia and transfusion dependence on disease outcomes and patient quality of life, the clinical impact of iron overload in MDS remains controversial. Overall, the most common non-leukemic cause of death in MDS is heart failure. Recent T2* cardiac MRI (CMR) studies have shown that cardiac iron accumulation in MDS patients is variable and infrequent. T2* CMR is noninvasive, robust and the best available modality for assessing iron organ quantification. The current study assessed iron overload in transfusion dependent MDS patients using ferritin levels combined with myocardial and liver iron quantification using T2* CMR. The study monitored iron accumulation dynamics while on iron chelation therapy. Methods: We collected retrospectively clinical data in 18 RBC transfusion dependent MDS patients that had a CMR in our institution; some of the patients have serial scans. All scans were obtained using a 1.5 T scanner (Signa HDx ver 15 GE). Scans interpretation was performed using a dedicated workstation (Medis Medical imaging version 7.6, the Netherlands) for left ventricular ejection fraction measurement and T2* investigation as previously described. T2* values were recorded in milliseconds and converted to mg/gr dry tissue of myocardial liver and pancreatic tissue. Results: Ten of 18 patients were chelated. During the study period 4 patients have died from pneumonia, brain tumor, complications of Allo BMT and transformation to AML. Demographic data and prognostic score of MDS are shown in table 1. Before the first CMR, patients were treated with median of 48 PC (14-145) with median ferritin levels 1691.3ng/ml (269.4-3730ng/ml). In Table 2 we show iron accumulation in the first CMR. Iron accumulation in the heart was seen in 2 patients. The results are shown as severity of accumulation and not absolute numbers, as normal and abnormal range is different in the different organs. Seven patients had yearly serial CMR done, 6 of them were treated with iron chelation. We found iron accumulation in the heart in 4 of them. In table 3 we show example of a patient that was treated with chelation. Another patient, that had decrease ejection fraction with only moderate accumulation in the heart in CMR, improved her heart function with chelation. During the study period all patients continue to be transfusion dependent except 1 patient that became transfusion independent. She was transfused with 182 PC with maximal ferritin of 6926ng/ml. She was chelated with deferiprone and then with desferal. She is now transfusion independent for a year. Conclusion: Blood transfusion dependency in MDS patients, leads to high ferritin levels and evidences in CMR of iron accumulation in parenchymal orangs including the heart. During the study 4 patients had evidence of iron accumulation in the heart with 2 of them with severe accumulation that improved with iron chelation. Liver and pancreas accumulation was seen in 12 and 8 patients accordingly. Prospective studies documenting the correlation between chelation therapy and improvement in organ damage and survival are needed. Disclosures No relevant conflicts of interest to declare.

Iron Chelation Improves Erythropoiesis in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis, cytopenias, and transformation to acute leukemia (AML). Low risk MDS patients exhibit a longer median survival, the lowest rate of progression to AML, and account for approximately two-thirds of all MDS patients, requiring recurrent RBC transfusions to alleviate symptomatic anemia. Transfusion-dependence results in iron overload which is associated with reduced overall survival. Methods to diagnose and treat (e.g. iron chelator deferiprone (DFP)) iron overload are available, but because retrospective studies to date only identify a correlative, not a causal, relationship between iron overload and reduced survival, there is little consensus on whether benefits of its diagnosis and treatment outweigh risks in this patient population. We aimed to evaluate and characterize the effect of iron loading and iron chelation on ineffective hematopoiesis in NUP98-HOXD13 transgenic (NHD13) mice, a highly penetrant model with peripheral blood cytopenias and ineffective hematopoiesis with dysplasia, consistent with MDS in humans [Lin Blood 2005]. We hypothesize that iron overload in MDS has deleterious biological effects on hematopoiesis with increased likelihood of worsening disease, reversible with iron chelation therapy. Our preliminary data demonstrates that 1) DFP enhances erythroid differentiation in CD34+ cells from MDS patients; and 2) exogenous iron suppresses erythropoiesis in wild type (WT) mice. The current experiments reveal the effect of exogenous iron and DFP in NHD13 mice. Age and gender matched 5 month old mice, 5-10 mice per group, were injected with 20mg iron dextran and/or treated with DFP (175 mg/mouse) in the drinking water over 4 weeks. Treated mice were analyzed and compared with PBS injected NHD13 mice and WT controls. Our experiments demonstrate that NHD13 mice exhibit anemia and leukopenia, increased serum erythropoietin (Epo) concentration, and higher MCV despite no difference in reticulocyte count relative to WT controls (Table I) and no splenomegaly (Fig 1a). In addition, erythroblast ROS concentration (Fig 1b) and iron load in the liver (Fig 1c) increase without differences in transferrin saturation or hepcidin expression in NHD13 relative to WT mice. These findings demonstrate characteristics consistent with human MDS patients. We then analyzed the effects of additional iron loading, iron chelation, or a combination of both on parameters of iron metabolism and erythropoiesis in NHD13 mice. Iron-treated NHD13 mice exhibit higher WBC count, RBC count, and hemoglobin, both DFP- and iron-treated NHD13 mice exhibit decreased serum Epo, and DFP+iron-treated NHD13 mice exhibit decreased serum Epo while increasing RBC count and hemoglobin (Table I). These findings suggest that exogenous iron increases Epo responsiveness and extramedullary erythroid mass in NHD13 mice in a manner similar to what we observed in iron-treated thalassemic mice [Ginzburg Exp Hem 2009]. Furthermore, spleen size increased (Fig 1a) and erythroblast ROS decreased (Fig 1b) only in iron-treated NHD13 mice. These findings suggest that erythroblast ROS is unrelated to excess iron in NHD13 mice. Liver iron increased in iron-treated and decreased in DFP-treated NHD13 mice (Fig 1c) as expected. In addition, CD71 (TfR1) surface expression on bone marrow erythroblasts is suppressed in all treated NHD13 mice but only significantly decreased in DFP-treated mice (Fig 1d), suggesting expected iron restriction. Lastly, DFP- and DFP+iron-treated (but not iron-treated) NHD13 mice decreased the proportion of bone marrow erythroblasts (Fig 1e) and increased erythroid differentiation in NHD13 mouse bone marrow (Fig 1f). Taken together, our findings demonstrate the robustness of NHD13 mice as a model of MDS to study erythropoiesis, the utility of iron injection in NHD13 mice to mimic robust iron overload in MDS, and the effectiveness of DFP in enhancing erythroid differentiation, reversal of erythroid expansion, and Epo responsiveness in NHD13 mice. Additional experiments (i.e. RBC survival, analysis of bone marrow signaling, erythroferrone expression, and parameters of the iron restriction response) to further explore the dysregulation of iron metabolism in NHD13 mice are ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hepcidin and Erythroferrone in the Anemia of Low-Risk Myelodysplastic Syndromes

BACKGROUND: Anemia is the most common manifestation of low-risk myelodysplastic syndromes (MDS). Iron-overload in MDS can occur before transfusion dependence in the context of ineffective erythropoiesis. Significantly lower hepcidin levels have been described in patients with sideroblastic refractory anemia compared to higher risk MDS, promoting inadequate iron absorption that leads to higher iron overload. Erythroferrone (ERFE) is a hormone that stimulates erythropoiesis and regulates iron homeostasis; in physiological conditions, is stimulated by erythropoietin and increases iron absorption inhibiting hepcidin. There are no studies describing the activity of ERFE in MDS. The objective of this study was to describe the relationship among hepcidin, ERFE and iron overload in 31 patients with low-risk MDS.METHODS: 50 samples were analized, 31 from patients (16 males, 17 females) with low-risk MDS: 10 low IPSS-R and 21 very-low IPSS-R; and 19 from healthy controls. 13 patients showed severe anemia with transfusion dependence, 4 patients received only erythropoiesis stimulating agents (ESA) and 14 patients did not receive any treatment for the anemia. Patient characteristics are summarized in table 1. Hepcidin levels were measured using the DRG Hepcidin 25 (bioactive) HS ELISA Kit (DRG Diagnostics GmbH), and ERFE was measured with the FAM132B (Human) OKEH02395 ELISA kit (Aviva Systems). For the analysis, two groups of patients were considered: 13 with severe and transfusion dependent anemia and 18 with mild/moderate anemia.RESULTS: Patients with severe anemia showed higher serum ferritin levels (median 2143ng/mL vs 204ng/mL, p<0.001) compared to patients without transfusion dependence. Hepcidin levels were significantly higher in patients with transfusion dependent anemia (mean 59.85 vs 16.11ng/mL, p=0.001) compared to patients with transfusion independence, and were also higher in these last patients compared to healthy controls (mean 16.11 vs 10.6ng/mL, p<0.001). Regarding ERFE, patients with transfusion dependent anemia showed significantly higher ERFE levels (mean 281.92 vs. 62.83pg/mL, p=0.016) than patients with mild/moderate anemia. However, there were no significant differences between patients with mild/moderate anemia and healthy controls. There was no correlation between hepcidin and ERFE levels (p=0.46).CONCLUSIONS: To the best of our knowledge, this is the first simultaneous analysis of hepcidine and ERFE in MDS. Patients with severe anemia showed significantly higher ERFE levels compared to those with moderate anemia, suggesting a higher erythropoietic stimulus. Patients with severe anemia showed significantly superior hepcidin levels, hindering iron absorption in situations of massive iron overload. Accordingly, ERFE did not show negative correlation with hepcidin in either cohort, supporting the abnormal iron metabolism in MDS. Larger studies are required to define the relationship between hepcidin and ERFE in low-risk MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Iron Overload in Myelodysplastic Syndromes: Pathophysiology, Consequences, Diagnosis, and Treatment

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic neoplasms varying in severity affecting one or more lines of hematopoiesis. Ineffective erythropoiesis results in dysregulation of iron metabolism. Most MDS patients have anemia, and some require regular red blood cell transfusions. These transfusions, in addition to factors of the disease itself, can result in iron overload (IO). Retrospective analyses suggest that MDS patients with IO have reduced overall survival and poorer outcomes following allogeneic stem cell transplant vs. those without IO. Iron chelation therapy (ICT; deferoxamine, deferasirox, or deferiprone) has been used to alleviate IO in other transfusion-dependent hematologic conditions (e.g., thalassemia), but its role in MDS has not been firmly established. A growing body of evidence suggests that ICT in MDS patients is an effective means for reducing transfusional IO and may significantly improve outcomes such as survival. The orally administered iron chelator deferasirox has been widely studied in MDS, and available studies have shown it to be generally well tolerated and effective in reducing IO in this population. The pathophysiology and clinical consequences of IO in MDS, as well as current methods for diagnosing and treating IO in these patients, are discussed.

Myelodysplastic syndromes and iron metabolism

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by ineffective hematopoiesis in bone marrow and cytopenias in peripheral blood. In patients with MDS, iron overload is frequent due to red blood cell transfusions and ineffective erythropoiesis. Dysplastic erythroblasts in MDS secrete humoral factors such as erythroferrone, which suppress hepatic expression of hepcidin. Hepcidin is the key regulator of systemic iron homeostasis, and suppression of hepcidin expression leads to an increase in iron absorption from the intestines, exacerbating systemic iron overload. Patients with MDS with ring sideroblasts (MDS-RS) are prone to iron overload, with most harboring splicing factor 3B subunit 1 (SF3B1) mutations in hematopoietic cells. SF3B1 mutations may induce ring sideroblasts by downregulating ATP binding cassette subfamily B member 7, which exports iron-sulfur clusters from the mitochondria to the cytoplasm. Iron overload in MDS causes hepatic dysfunction, diabetes, cardiac failure, and atherosclerosis, whereas excess iron may suppress normal hematopoiesis. Though randomized control studies are lacking, results from retrospective and cohort studies indicate that iron chelation therapy is appropriate for lower-risk MSD patients with transfusion-related iron overload, although it is not recommended for higher-risk MSD patients with short life expectancy.

Iron overload in myelodysplastic syndromes (MDS).

Iron overload (IOL) starts to develop in MDS patients before they become transfusion-dependent because ineffective erythropoiesis suppresses hepcidin production in the liver and thus leads to unrestrained intestinal iron uptake. However, the most important cause of iron overload in MDS is chronic transfusion therapy. While transfusion dependency by itself is a negative prognostic factor reflecting poor bone marrow function, the ensuing transfusional iron overload has an additional dose-dependent negative impact on the survival of patients with lower risk MDS. Cardiac dysfunction appears to be important in this context, as a consequence of chronic anemia, age-related cardiac comorbidity, and iron overload. Another potential problem is iron-related endothelial dysfunction. There is some evidence that with increasing age, high circulating iron levels worsen the atherosclerotic phenotype. Transfusional IOL also appears to aggravate bone marrow failure in MDS, through unfavorable effects on mesenchymal stromal cells as well a hematopoietic cells, particularly erythroid precursors. Patient series and clinical trials have shown that the iron chelators deferoxamine and deferasirox can improve hematopoiesis in a minority of transfusion-dependent patients. Analyses of registry data suggest that iron chelation provides a survival benefit for patients with MDS, but data from a prospective randomized clinical trial are still lacking.

Reactive oxygen species levels control NF-κB activation by low dose deferasirox in erythroid progenitors of low risk myelodysplastic syndromes.

Anemia is a frequent cytopenia in myelodysplastic syndromes (MDS) and most patients require red blood cell transfusion resulting in iron overload (IO). Deferasirox (DFX) has become the standard treatment of IO in MDS and it displays positive effects on erythropoiesis. In low risk MDS samples, mechanisms improving erythropoiesis after DFX treatment remain unclear. Herein, we addressed this question by using liquid cultures with iron overload of erythroid precursors treated with low dose of DFX (3μM), which corresponds to DFX 5 mg/kg/day, an unusual dose used for iron chelation. We highlight a decreased apoptosis rate and an increased proportion of cycling cells, both leading to higher proliferation rates. The iron chelation properties of low dose DFX failed to activate the Iron Regulatory Proteins and to support iron depletion, but low dose DFX dampers intracellular reactive oxygen species. Furthermore low concentrations of DFX activate the NF-κB pathway in erythroid precursors triggering anti-apoptotic and anti-inflammatory signals. Establishing stable gene silencing of the Thioredoxin (TRX) 1 genes, a NF-κB modulator, showed that fine-tuning of reactive oxygen species (ROS) levels regulates NF-κB. These results justify a clinical trial proposing low dose DFX in MDS patients refractory to erythropoiesis stimulating agents.

Myelodysplastic Syndromes and Iron Chelation Therapy.

Over recent decades we have been fortunate to witness the advent of new technologies and of an expanded knowledge and application of chelation therapies to the benefit of patients with iron overload. However, extrapolation of learnings from thalassemia to the myelodysplastic syndromes (MDS) has resulted in a fragmented and uncoordinated clinical evidence base. We’re therefore forced to change our understanding of MDS, looking with other eyes to observational studies that inform us about the relationship between iron and tissue damage in these subjects. The available evidence suggests that iron accumulation is prognostically significant in MDS, but levels of accumulation historically associated with organ damage (based on data generated in the thalassemias) are infrequent. Emerging experimental data have provided some insight into this paradox, as our understanding of iron-induced tissue damage has evolved from a process of progressive bulking of organs through high-volumes iron deposition, to one of ‘toxic’ damage inflicted through multiple cellular pathways. Damage from iron may, therefore, occur prior to reaching reference thresholds, and similarly, chelation may be of benefit before overt iron overload is seen. In this review, we revisit the scientific and clinical evidence for iron overload in MDS to better characterize the iron overload phenotype in these patients, which differs from the classical transfusional and non-transfusional iron overload syndrome. We hope this will provide a conceptual framework to better understand the complex associations between anemia, iron and clinical outcomes, to accelerate progress in this area.

Mechanisms of Improvement of Erythropoiesis with Low Dose Deferasirox in Low Risk Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell disorder characterized by dysplasia leading to ineffective hematopoiesis and evolution to acute myeloid leukemia. Anemia is the most frequent cytopenia in MDS and the majority of patients requires red blood cell (RBC) transfusion resulting in the development of iron overload (IO). Deferasirox (DFX) became a standard treatment of IO in MDS and seems to have positive effects on hematopoiesis in some MDS patients leading to reduction of RBC transfusion or even transfusion independence (Gatterman, 2012, Maurillo, 2015). In this study, our aims were to decipher the intrinsic mechanisms explaining the potential positive effects of DFX on erythroid differentiation in low risk MDS.Methods: We report our data about erythroid differentiation, cell cycle, apoptosis and cell signaling pathways concerning CD34+ hematopoietic stem progenitor cells (HSPCs) from low risk MDS samples in a 2-step erythroid differentiation liquid culture with low dose DFX.Results: We observed a higher proliferation rate at the end of the cell culture procedure with DFX 3µM versus control condition (p=0,038). We did not find these effects with DFX > 5µM. This higher proliferation rate is due to the combination of less apoptotic cells and more cycling cells. At day 10 of the culture (D10) (n=7), there were less apoptotic cells for DFX 3μM condition (DFX 3): 13,8% versus 17,5% for control condition (p=0,0325). This lower apoptosis rate was more relevant at D14 (n=9): 19,1% versus 24,7% for control condition (p=0,007). There were more cycling cells with DFX 3 at D10 (n=6): fewer cells were blocked at the beginning of the cell cycle in subG0-G0-G1 phases (68,9% versus 77,6%) (p=0,0001) and more cells (30,5% versus 21,9%) were engaged in cell cycle (S-G2-M) (p=0,0001) for DFX 3. Regarding clonogenic assays (n=10), there was an increased number of CFU-E colonies for DFX 3 versus control condition with on average 72,7 (15-261) and 46,5 (3-174,5) colonies respectively (p=0,04). We did not see any effect of DFX on erythroid differentiation. The percentages of proerythroblasts, basophilic and orthochromatic erythroblasts were similar for DFX 3 and control at D5, D10 and D14 of the cell culture regardless the techniques (flow cytometry or morphological analyses by cytospin).These functional effects were partially related to iron chelation of DFX. As expected, we saw a lower iron intracellular concentration (-12,7%) by mass spectrometry with DFX 3 (p=0,019). Nevertheless this chelation effect was not sufficient to activate iron regulation metabolism (IRP-IRE) in REMSA assay, indeed we saw no difference in IRP activity between DFX 3 and control condition. Moreover, low dose of DFX had a protector effect against mitochondrial reactive oxygen species at D14 (n=9; p=0,03). We aim to complete the Redox profile by analysis of some oxidative stress metabolites like malonaldehyde, carbonyl groups, γH2AX and oxidized form of glutathione.Then, we have investigated which signaling pathways were responsible for these functional effects by flow cytometry (FC) and immunofluorescence (IF) microscopy. By FC, PI3K/AKT, mTOR and MAPK/ERK were not over-activated in DFX 3. However,we found an increased nuclear translocation of NFκB in DFX3 (p=0,0004) by IF. Using RT-qPCR microarrays, we have analyzed the expression of 84 gene targets of NFκB. Five genes were upregulated in DFX 3 with a fold change >2 compared to control: BCL2A1, BIRC3, TNFRSF1B, EGFR, IL1B. This gene profile leads in silico to an anti-apoptotic signal (PCVIz®).To demonstrate a link between the ROS protection effects of DFX and the NFκB activation pathway, we have realized a cellular model of inhibition of thioredoxin (TRX), a ROS regulated protein, by three siRNA anti-TRX1. TRX1 in its reduced form can activate NFκB. By inhibiting TRX1, we hypothesize that NFκB pathway cannot be activated and that could impede cell proliferation. Definitive results will be presented at ASH.Conclusions: Our study described the pro-proliferative effects of low dose of DFX on erythroid progenitors in low risk MDS patients in vitro via a mechanism of mitochondrial ROS protection and NFκB activation. Those data highlight the potential interest of an early introduction of iron chelation therapy with low dose DFX (around 5mg/kg/d corresponding to a plasmatic level of 3µM) in the disease course of transfused low risk MDS patients. DisclosuresMeunier:Novartis: Research Funding. Park:Novartis: Research Funding.

Oxidative Stress Levels Are Correlated with Disease Progression and Iron Overload in MDS Patients with Excess Blasts

Introduction: Oxidative stress is closely related to iron overload in myelodysplastic syndrome (MDS) and induces DNA damage. A recent study has suggested that oxidative stress leads to increased mutation frequency in a murine model of MDS, and that additional mutations lead to the progression of MDS. We have recently reported that oxidative stress marker reflects not only iron overload but also disease progression in a MDS patient (2016, Shimizu et al). So there is a possibility that the increase in oxidative stress markers may have not only caused the iron overload but also resulted in the disease progression of MDS. To clarify this point, we evaluated the association among oxidative stress marker and disease progression in eight patients with MDS. In this study, we first examined the oxidative stress marker, derivatives of reactive oxygen metabolite (dROM), ferritin in serum and Wilms Tumor 1 gene (WT1) in the peripheral blood throughout the course of treatment. Then, we analyzed the correlations of the levels of dROM, ferritin and WT1. Next, we investigated ROS expression in each fraction of WBC by flow cytometry in a healthy donor and some MDS patients.Materials and methods: Levels of serum dROM were measured by colorimetry, using samples of MDS (RA n=3, RAEB-1 n=4, RAEB-2 n=1). Ferritin in serum and WT1 in peripheral blood were also measured. Statistical analyses were performed by Pearsonfs test. The ROS expression was evaluated during the clinical course by flow cytometry.Results: Table 1 shows the relationship between dROM, ferritin and WT1 in MDS patients throughout the treatment. Case 1 to 3 patients showed disease progression with excess blasts in spite of azacitidine treatment. These patients showed a correlation tendency between dROM and ferritin, dROM and WT1. Case 1 showed close relationship between ferritin and WT1. Case 3 shows slight relation between ferritin and WT1. Case 4 to 8 patients showed no disease progression. They were treated with supportive therapy, including blood transfusion or cytokine therapy. These patients showed no relation between dROM and the elevation of ferritin and WT1. In these patients, there was no relation between ferritin and WT1 except Case 6. We also evaluated the score of WPSS, IPSS and dROM at diagnosis. dROM did not correlate with WPSS and IPSS. Figure 1 shows increased ROS expression during disease progression in a MDS patient compared with healthy control. Considering that other MDS patients with excess blasts showed the similar pattern, the present result suggested that the oxidative stress markers were produced by tumor-derived cells.Conclusions: dROM levels were slightly related with the ferritin and WT1 mRNA expression levels in MDS patients with excess blasts. We speculate that the increase in the oxidative stress markers is a cause of not only iron overload but also the disease progression of MDS. Oxidative stress markers may contribute to evaluate the progression of treatment efficacy for MDS patients. [Display omitted] [Display omitted] DisclosuresNakaseko:BMS: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; NOVARTIS: Honoraria.

Study of abnormal iron metabolism parameters and iron overload in patients with myelodysplastic syndromes

目的研究骨髓增生异常综合征(MDS)患者铁代谢参数和临床特征。方法对2015年6月至2016年3月间初诊未经祛铁治疗的94例MDS患者的铁代谢参数和临床特征进行回顾性调查。结果94例初治MDS患者中，71例(75.53%)贫血，56例(59.57%)存在输血依赖，52例(55.32%)存在铁过载。IPSS低危／中危-1较中危-2/高危铁过载多见(P<0.01)。铁过载组和非铁过载组比较，血清铁(SI)升高[36.5(8.5~64.7)µmol/L对25.2(3.7~45.3)µmol/L，P<0.01]，转铁蛋白饱和度(TSAT)降低[43.5%(12.2%~77.2%)对53.4%(14.8%~97.5%)，P<0.01]，不稳定细胞铁(LCI)及细胞内活性氧簇(ROS)差异无统计学意义。靶器官(心、肝、胰岛等)功能临床资料比较中，铁过载组ALT及B型钠尿肽前体分别为25(3~158)U/L及190(6~4 281)ng/L，均明显高于非铁过载组的16(5~80)U/L及84(12~2 275)ng/L(P=0.03及P=0.05)。难治性贫血伴环状铁粒幼红细胞(RARS)患者与非RARS患者铁代谢参数差异均无统计学意义(P>0.05)，输血依赖程度和输血量显著影响SI、TSAT及SF(P值均<0.01)，但LCI及ROS组间差异无统计学意义。无输血依赖、非铁过载的38例患者中，骨髓红系病态造血、幼红细胞比例增高组LCI水平显著增高。结论初治MDS患者贫血及输血依赖现象较为常见，输血频率和输血量显著影响铁代谢参数。而无输血依赖的非铁过载患者骨髓红系病态造血、幼红细胞比例增高影响LCI水平，LCI可以作为衡量铁过载和铁分布重要的早期指标。

Labile plasma iron, more practical and more sensitive to iron overload in myelodysplastic syndromes

Objectives: In order to gain an insight into labile plasma iron (LPI) in iron metabolism microenvironment in MDS.Methods: We performed ELISA, quantitative real-time polymerase chain reaction, flow cytometry, MRI T2* assays to test LPI, iron biochemical parameters, and liver iron concentration (LIC) among 22 MDS patients.Results: LPI has a statistical difference (P < 0.001 by analysis of variance (ANOVA)), which decreased gradually, among three groups, while no difference was found in adjusted serum ferritin (ASF) (P = 0.086 by ANOVA). After DFO treatment, serum hepcidin expression increased from 301.26 ± 59.78 to 340.33 ± 49.78 µg/l (P = 0.032), while hepcidin/ASF was upregulated gradually from 0.16 ± 0.08 to 0.22 ± 0.03 (P = 0.045). APAF-1 expression (P = 0.047) and erythroid apoptosis rate (P = 0.009) decreased significantly, respectively. No statistical difference was found in EPO (P = 0.247) and GDF15 expression (P = 0.172). LIC dropped from 9.83 ± 4.84 to 6.28 ± 4.01 mg/g dry weight (P < 0.001). No significant difference was found in cardiac T2* (P = 0.594). LPI has a closer connection to LIC than ASF (r = 0.739, P < 0.001 vs. r = 0.321, P = 0.034).Discussion: LPI seems to be a real-time indicator which reflects body iron loading status instantaneously. Despite the limited knowledge available on LPI speciation in different types and degrees of IO, LPI measurements can be and are in fact used for identifying systemic IO and for initiating/adjusting chelation regimens.

CONIFER - Non-Interventional Study to Evaluate Therapy Monitoring During Deferasirox Treatment of Iron Toxicity in Myelodysplastic Syndrome Patients with Transfusional Iron Overload

Background: The non-interventional study CONIFER was designed to assess the safety and clinical practicability of deferasirox for the treatment of transfusional iron overload in myelodysplastic syndrome (MDS) patients. Methods: Patients included in the study were diagnosed with MDS and received at least 1 treatment with deferasirox. The observation period covered the time from the initial visit until the last follow-up. Results: The data of 99 patients with MDS scored mainly as International Prognostic Scoring System (IPSS) low and intermediate 1 were evaluated. The mean age of the participants was 75 years and 58% of the patients were male. Iron overload was assessed by serum ferritin level (mean baseline serum ferritin 2,080 ± 1,244 µg/l). Patients were treated for a mean duration of 16 months (mean daily dose at baseline 11.8 ± 7.0 mg/kg). Stratification of serum ferritin levels by deferasirox dose showed a reduction at the higher but no reduction at the lower dose (< 15 mg/kg vs. ≥ 15 mg/kg and < 20 mg/kg vs. ≥ 20 mg/kg). The majority of patients (81%) were affected by at least 1 adverse event, with decreased renal creatinine clearance being the most frequent. Conclusion: Higher doses (≥ 15 mg/kg) of deferasirox effectively and safely reduced serum ferritin levels in MDS patients with transfusional iron overload.

Impact of medication adherence on the effectiveness of deferasirox for the treatment of transfusional iron overload in myelodysplastic syndrome.

Regular blood transfusions in the management of myelodysplastic syndrome (MDS) often lead to iron overload. The main objective of this study was to evaluate the impact of medication adherence on the effectiveness of deferasirox for the treatment of transfusional iron overload in patients with MDS. Secondary objectives were to describe treatment effectiveness and safety in daily clinical practice. A longitudinal, retrospective, observational study was carried out in a university hospital. The inclusion criteria were age over 18 years, MDS diagnosis and treatment with deferasirox for transfusion-dependent iron overload during the period of study (from January 2011 to April 2015). Treatment effectiveness was estimated by serum ferritin (SF), and adherence was measured by medication possession ratio (MPR). Clinically relevant analytical alterations during the treatment and reasons for treatment discontinuation were also assessed. Thirty-five patients were included in the study. Median SF at baseline was 1636 μg/L, and it decreased to 1399 μg/L during follow-up. The median adherence rate was 92%, although only 54·8% of the patients maintained deferasirox adherence ≥90% during the whole duration of treatment. Adherence rate was inversely correlated to SF (r = -0·288, P = 0·004). The median (p25, p75) duration of treatment was 11 (3·0, 37·8) months. The most common reasons for treatment discontinuation were renal toxicity (35%) and patient's death (25%). Deferasirox's effectiveness, measured by the decrease in SF, was significantly better in adherent patients. The most frequent reason for treatment discontinuation was renal toxicity. Developing strategies to improve deferasirox treatment adherence and monitoring renal function in those patients should be key points in pharmaceutical care.

Transient Elastography for the Detection of Hepatic Iron Overload in Patients with Myelodysplastic Syndrome

Background: In myelodysplastic syndrome (MDS), anemia often leads to red blood cell transfusion dependency and iron overload (IOL). Serum ferritin (SF) is used as a surrogate marker for IOL. IOL can lead to liver failure. Transient elastography (TE) also known as fibroscan is an easy and noninvasive procedure for liver stiffness measurements (LSM). Methods: Sixty patients with either MDS, chronic myelomonocytic leukemia with dysplastic features, acute myeloid leukemia progressed from MDS or myelodysplastic/ myeloproliferative neoplasm, unclassifiable were included. All patients underwent a fibroscan, had their SF measured and disease duration calculated. Patients were grouped according to transfusion dependency or independency status. Results: Transfusion dependent patients had significantly higher LSM than those who were transfusion independent (p=0.003). There was no positive correlation between SF and LSM (p=0.103) or time since diagnosis and LSM (p=0.886). Patients with elevated SF did not have significantly higher LSM compared to those with normal SF (p=0.583). Conclusion: These data indicate that transfusion dependency has an impact on liver stiffness in MDS. Longitudinel studies are needed to conclude whether TE is of value in monitoring IOL in MDS.