Non Invasive Evaluation of Bone Marrow Activity in Patients with Sickle Cell Disease: Correlation with Disease Features, Genotype, Markers of Erythropoiesis, Iron Metabolism and Hydroxyurea Treatment

Background: Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by pathological polymerization of hemoglobin, increased red cell rigidity and poor microvascular blood flow with consequent tissue ischemia and infarction. Thus, hemolytic anemia, vaso-occlusion and vasculopathy are the hallmarks of its clinical presentation. The transferrin receptor (TfR) mediates the transport of iron into cells and the circulating TfR can be measured as soluble transferrin receptor (sTfR). sTfR levels are frequently used to establish the diagnosis of iron deficiency anemia, especially in the context of inflammation, but they also reflect bone marrow erythropoietic activity (BMA) and mass. Erythropoietic activity has been found to be the most important determinant of sTfR levels. In this context, we aimed to study and evaluate bone marrow activity in patients with compound heterozygous HbS and beta-thalassemia (HbS/βthal) based in sTfR measurements and explore possible correlations with of key features of the disease such as: the hemolytic component, vaso-occlusive crises (VOC), acute chest syndrome, venous thrombosis, arterial thrombosis including stroke, avascular necrosis, pulmonary hypertension, hydroxyurea therapy, inflammation and renal injury. along with other biomarkers of erythropoiesis and iron metabolism such as Placental Growth Factor (PlGF), Growth Differentiation Factor-15 (GDF-15), Ferritin and Hepcidin-25. Patients and Methods: Ninety adult Caucasian patients with HbS/βthal [49 patients under hydroxyurea (HU+) treatment and 41 patients without hydroxyurea (HU-) treatment], were included in this study, while 22 apparently healthy individuals of similar age and gender served as controls. None of the patients has received any transfusions at least 6-monthes before enrollment in the study. Along with hematologic and blood chemistry parameters determination, levels of circulating sTfR, PlGF, GDF-15 and Hepcidin-25 were measured in patients with HbS/βthal and controls using RUO and IVD immunoenzymatic techniques. BMA activity was calculated from the established formula: patient-sTFR/meanControl-sTFR. Results: We found that: sTfR levels were markedly elevated in all patients with HbS/βthal compared to controls (4.8±2.2 vs. 1.0±0.2 mg/L, p<0.001), resulting in a 1.6-11.9 fold increase of BMA. No correlation was found between BMA and disease features as well as regarding hydroxyurea treatment BMA (p>0.434). BMA correlated significantly with the markers of the erythropoietic and hemolytic component such as: Hemoglobin (r=-0.434, p<0.001); Reticulocyte Production Index (r=0.645, p<0.001); LDH (r=0.570, p<0.001); Billirubin (r=0.540, p<0.001), PlGF (r=0.597, p<0.001) and Hb A levels (r=-0.493, p<0.001), while no correlation was found between BMA and Hb F levels. Furthermore, BMA values correlated significantly only with GDF-15 (r=0.466, p<0.001), while interestingly no correlation was found between BMA and Ferritin and Hepcidin-25 levels alone (r=0.101, p>0.351 and r=-0.043, p>0.710, respectively), but a negative correlation was found between BMA and Hepcidin-25/Ferritin ratio, (r=-0.330, p=0.005). Conclusions: Our findings demonstrate that all patients with HbS/βthal studied have a significantly increased degree of erythroid BMA as assessed by measurements of sTfR levels. Erythroid BMA correlated significantly with Hepcidin/Ferritin ratio, which is an index of the degree of Hepcidin expression relative to iron overload. The correlation of erythroid BMA with Hb A levels, indicate the important role of βthal genotype in HbS/βthal disease. Furthermore, BMA is not related to hydroxyurea therapy and/or iron metabolism parameters in these patients. This implicates a likely complex action of hydroxyurea, which causes intermittent cytotoxic suppression of erythroid progenitors and cell stress signaling. The latter affects erythropoiesis, leading to recruitment of erythroid progenitors with increased HbF levels, although the number of erythroid progenitors -the main source of sTfR- remains stable. Disclosures Voskaridou: Genesis: Consultancy, Research Funding; Protagonist: Research Funding; Celgene Corporation: Consultancy, Research Funding; Acceleron: Consultancy, Research Funding; Addmedica: Membership on an entity's Board of Directors or advisory committees.

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Clathrin Assembly Protein CALM Plays a Key Role In Erythroid Differentiation Via Regulating Transferrin Receptor Endocytosis

Blood ◽

10.1182/blood.v116.21.4256.4256 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4256-4256

Author(s):

Yuichi Ishikawa ◽

Manami Maeda ◽

Min Li ◽

Sung-Uk Lee ◽

Julie Teruya Feldstein ◽

...

Keyword(s):

Transferrin Receptor ◽

Mammalian Cells ◽

Research Funding ◽

Molecular Mechanisms ◽

Erythroid Differentiation ◽

Global Changes ◽

Erythroid Progenitors ◽

Erythroid Development ◽

Terminal Erythroid Differentiation

Abstract Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

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Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

Blood ◽

10.1182/blood-2021-146747 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 938-938

Author(s):

Barbora Kralova ◽

Ondrej Jahoda ◽

Jihyun Song ◽

Katarina Hlusickova Kapralova ◽

Lucie Sochorcova ◽

...

Keyword(s):

Bone Marrow ◽

Iron Deficiency ◽

Mouse Model ◽

Systemic Inflammation ◽

Erythropoietin Receptor ◽

Iron Accumulation ◽

Erythropoietic Activity ◽

Erythroid Progenitors ◽

Gain Of Function ◽

Progressive Decline

Abstract We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis. We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage. Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation. Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis. Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR. Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. Disclosures No relevant conflicts of interest to declare.

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Effect of Hydroxyurea Therapy on the Incidence of Infections in Ugandan Children with Sickle Cell Anaemia

Blood ◽

10.1182/blood-2021-153559 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 765-765

Author(s):

Ruth Namazzi ◽

Andrea L. Conroy ◽

Caitlin Bond ◽

Micheal J. Goings ◽

Dibyadyuti Datta ◽

...

Keyword(s):

Sickle Cell ◽

Research Funding ◽

Infection Prevention ◽

Sickle Cell Anaemia ◽

Risk Of Infection ◽

African Children ◽

Hydroxyurea Treatment ◽

Before And After ◽

The Mean ◽

Hydroxyurea Therapy

Abstract Hydroxyurea is efficacious against sickle cell anaemia (SCA)-related complications in African children. Prior studies demonstrated conflicting results on the effect of hydroxyurea on risk of infection, the most common cause of morbidity and mortality in African children with SCA. We evaluated the incidence of infections before and after starting hydroxyurea in 117 children aged 1-5 years with SCA enrolled in the Zinc for Infection Prevention in Sickle cell anaemia (ZIPS) clinical trial that received zinc or placebo treatment for one year (Clinicaltrials.gov, NCT03528434). Children were enrolled between March 2018 and November 2019 and initiated on hydroxyurea (20 mg/kg/day) if they met Uganda SCA guideline criteria for hydroxyurea treatment at any time during the study. We compared the incidence of infections before and after hydroxyurea therapy, adjusting for zinc treatment. Overall, the mean duration on hydroxyurea was 223.8 (85.2) person days. The mean(SD) incidence of any severe/invasive infections (infections meeting strict clinical and laboratory or radiological diagnostic criteria) was 6.2(9.0) vs. 1.9(2.3) infections per child per year before and after hydroxyurea (incidence rate ratio [IRR]: 0.40, 95%CI: 0.29-0.54, p<0.001), including a decrease in the incidence of bacteremia [IRR: 0.05] malaria, [IRR 0.28], cellulitis [IRR: 0.24], gastroenteritis [IRR: 0.41], pharyngitis [IRR: 0.59] and sinusitis [IRR: 0.38] (all p<0.05). Similarly, the incidence of clinically defined infections (infections meeting clinical but not laboratory/radiological criteria) decreased after hydroxyurea, (IRR: 0.49, 0.41-0.59), influenced primarily by large reductions in the incidence of lower and upper respiratory tract infections (p<0.001 for both). As expected, hydroxyurea treatment was also associated with a decrease in complications of SCA, including vaso-occlusive crises, hospitalizations and transfusions (all p<0.001). There was no interaction between zinc and hydroxyurea therapy on risk of infection and SCA-related complications. In Ugandan children with SCA, hydroxyurea therapy not only decreases the incidence of SCA-related complications, but also substantially reduces the incidence of infections. Research to understand the underlying mechanisms of protection from hydroxyurea against infection and exploration of its potential use for infection prevention is warranted. Disclosures Ware: Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Hemex Health: Research Funding; Nova Laboratories: Research Funding; Novartis: Other: DSMB Chair; Editas: Other: DSMB Chair.

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Decreased Iron Utilization Leads to Dysregulation of Iron Homeostasis in Aplastic Anemia

Blood ◽

10.1182/blood-2018-99-118010 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3872-3872

Author(s):

Aruna Barade ◽

Sreenithi Santhakumar ◽

Biju George ◽

Eunice Sindhuvi Edison

Keyword(s):

Bone Marrow ◽

Serum Ferritin ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Bone Marrow Failure ◽

Iron Regulation ◽

Soluble Transferrin Receptor ◽

Erythropoietic Activity ◽

Serum Hepcidin ◽

Iron Utilization

Abstract Aplastic Anemia (AA) arises due to specific failure of bone marrow precursor cells or stem cells to produce adequate amount of mature hematopoietic cells. Erythropoiesis utilizes majority of iron in humans. Erythropoietic activity is fine-tuned with iron regulation since they are both intrinsically regulated. Hepcidin, the main iron regulator is the target of erythropoietic regulators like GDF15, TWSG1 and erythroferrone. Even though the dysregulation of iron balance is well understood in beta thalassemia where ineffective erythropoiesis is the main cause, regulation of iron is poorly elucidated in bone marrow failure where erythropoiesis is minimal. In this study, we analysed several iron regulatory molecules in a cohort of patients with AA and tried to understand iron regulation in bone marrow failure. The current study investigated 77 patients with newly or recently diagnosed AA and had received less than ten transfusions from the year 2016 to 2018. Their haematological parameters were documented. Serum hepcidin was assessed using ELISA kit (DRG Diagnostics) and serum ferritin and soluble transferrin receptor (sTfR) was measured by chemiluminescent immunoassay. DNA was extracted from peripheral blood and telomere length was measured using quantitative real-time PCR (qPCR) using relative quantitation method. The association between different iron regulators were analysed using SPSS Software. The median age of AA patients was 38 (5-72) years and the number of males and females were 49 (64%) and 28 (36%) respectively. Patients were grouped based on severity into non severe AA (NSAA) (n=17, 22%), Severe AA (SAA) (n=51, 66%) and Very Severe AA (VSAA) (n=9, 12%). Out of 77 AA patients, 50 had not received any red cell transfusion in the last one month prior to the sample collection; twenty seven had received transfusions ranging from 1 to 9. The mean haemoglobin was 7.6±1.87g/dL, with a median WBC count of 2900 (100-8400)/cu.mm and a median platelet count of 9000 (2000-66000)/ul. Serum ferritin levels ranged from 12.8 to 5728ng/ml (Median- 1208ng/ml). The median soluble transferrin receptor (sTfR) levels were 1.29mg/L (0.52-6.34). Fifty four patients (54/77, 70%) had sTfR values less than the lower limit of normal (Normal range 1.8-4.6 mg/L). Median serum hepcidin was 40.66ng/ml (1.15-81.2). Serum ferritin levels positively correlated with serum hepcidin (r=0.61, p=0.000) whereas it correlated negatively with sTfR (r=-0.408, p=0.000). No significant association was observed between the number of transfusions and ferritin levels (r=0.087, p=0.453). There was no significant difference in the ferritin levels when patients were grouped according to severity (Median Ferritin, Range - 1208, 12.8-2984ng/ml in NSAA; 1099, 61.8-5728ng/ml in SAA; 1288, 268-3824; p= 0.83). Significantly higher hepcidin levels were observed in VSAA as compared to NSAA (Figure.1, p= 0.014). There was no significant association observed between response and biochemical parameters. A quartile analysis based on ferritin values was carried out to study the effect of iron overload on haematological parameters and iron regulators (Table.1). With increasing ferritin values, hepcidin levels significantly increased (p=0.001) whereas sTfR levels were reduced (p=0.002). In AA, decrease in erythropoietic activity as evidenced by reduced sTfR levels leads to minimal iron utilization and accumulation of iron in the tissues as ferritin. This stimulates the production of hepcidin and further exacerbates iron loading and leads to dysregulation of iron homeostasis. Increased iron in the marrow can lead to oxidative stress and may further be detrimental to the limited intrinsic haematopoiesis. Hence better understanding of iron regulation in AA may advance to effective therapeutic options. Disclosures No relevant conflicts of interest to declare.

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Predictors of Hydroxyurea Use in Children with Sickle Cell Disease

Blood ◽

10.1182/blood-2018-99-111377 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4934-4934

Author(s):

Chisom Ifeoma Okwor ◽

Robert J Klaassen ◽

Mary-Ann Harrison ◽

Ken Tang ◽

Isabelle Laforest ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Small Sample Size ◽

Small Sample ◽

Care Utilization ◽

Lower Income ◽

Cell Disease ◽

Hematological Indices ◽

Hydroxyurea Treatment

Abstract Background: Since hydroxyurea emerged as an effective therapy for sickle cell disease (SCD), there have been numerous studies that have demonstrated its safety and efficacy in children and adults with SCD. In their 2014 guidelines, the NHLBI recommended that hydroxyurea treatment should be offered to all infants and children with sickle cell anemia (HbSS and HbS/beta0 thalassemia) starting at 9 months of age. However, hydroxyurea is underused among children and adolescents with SCD and to date, there have been no studies that have identified the specific determinants that may predict hydroxyurea adherence in these patients. Objectives: 1. To identify predictors of hydroxyurea adherence in children with SCD. 2. To measure the rate of hydroxyurea use among CHEO patients with SCD who were born between January 1, 2003 and December 31, 2015; and 3. To compare the rates of SCD-related complications between patients who were not prescribed hydroxyurea, patients who were adherent to hydroxyurea and patients who were not adherent to hydroxyurea Methods: We extracted medical chart data to identify patients with SCD who were born between January 1, 2003 and December 31, 2015. Patients were classified as either "Not prescribed hydroxyurea" or "Prescribed hydroxyurea" based on clinical documentation and the presence of at least one hydroxyurea outpatient prescription. For those patients who were prescribed hydroxyurea, hematological indices were collected and analyzed over time to estimate adherence to hydroxyurea. To measure the adherence of children prescribed hydroxyurea, we examined the trends in the patient's hematological indices after their first prescription of hydroxyurea. Adherence was defined as increased hematological indices (from baseline) by greater than or equal to any 2 of the following: Mean corpuscular volume (MCV) by 10 fL; Hemoglobin levels (g/L) by 10 g/L and/or %HbF (fetal hemoglobin) by 10%. We measured the frequency of disease-related complications among CHEO patients with SCD according to their use of hydroxyurea and used multivariate analyses to evaluate immigration status, newborn screening status, SCD subtype, SCD complications, income, age and sex as predictors for hydroxyurea adherence. Results: Children with HbSS were more likely to have been prescribed hydroxyurea compared to children with HbSC (87.8% vs. 9.5%). Canadian citizenship, newborn hemoglobinopathy screening and lower familial income were associated with better hydroxyurea adherence (Table 1). Although the association was not statistically significant, patients were more likely to be prescribed hydroxyurea if they were from a lower income background (61.9% for lowest and second lowest quartiles vs. 38.1% for third and highest quintiles). Patients were also more likely to adhere to hydroxyurea if they did not have private medical insurance for hydroxyurea coverage (Table 1). Finally, hydroxyurea adherence was associated with reduced rates of health care utilization and SCD-related complications (Table 2). Conclusions: In line with previous studies of hydroxyurea for the treatment of SCD, patients who were adherent to hydroxyurea had fewer complications compared to those patients who were either non-adherent to or not prescribed hydroxyurea. Similarly, patients had fewer complications after being prescribed hydroxyurea compared to before they started hydroxyurea with a reduction in the rate of ED visits, acute chest syndromes, complications, transfusions and hospitalizations. Patients from non-immigrant families, patients who were identified through newborn hemoglobinopathy screening and patients from lower income families were more likely to be adherent to hydroxyurea. Although the results of this study were limited by its small sample size, further studies will clarify these determinants of hydroxyurea adherence among SCD patients and enable clinicians to improve hydroxyurea adherence for SCD patients. Disclosures Klaassen: Shire: Consultancy; Novartis: Research Funding; Hoffman-La Roche: Consultancy; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; Octapharma AG: Consultancy, Honoraria; Agios Pharmaceuticals Inc.: Consultancy; Cangene: Research Funding.

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Optimizing Hydroxyurea Therapy with Reduced Laboratory Monitoring for Children with Sickle Cell Anemia in Sub-Saharan Africa: The Reach Experience

Blood ◽

10.1182/blood-2020-134180 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Banu Aygun ◽

George A. Tomlinson ◽

Patrick T. McGann ◽

Leon Tshilolo ◽

Thomas N. Williams ◽

...

Keyword(s):

Sickle Cell Anemia ◽

Research Funding ◽

Reticulocyte Count ◽

Sub Saharan Africa ◽

Laboratory Monitoring ◽

Hydroxyurea Treatment ◽

Thalassemia Trait ◽

Treatment Responses ◽

Sub Saharan ◽

Hydroxyurea Therapy

Introduction: Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) is an open-label study of hydroxyurea for children with sickle cell anemia (SCA) in sub-Saharan Africa (Angola, DR Congo, Kenya, and Uganda). Initial results documented the feasibility, safety, and benefits of hydroxyurea for SCA in sub-Saharan Africa but guidance for optimizing hydroxyurea therapy is needed. We describe 5 years of hydroxyurea dosing and monitoring in the largest prospective cohort of children with SCA receiving hydroxyurea to date. Methods: Children 1-10 years of age with SCA were enrolled. The hydroxyurea dose was fixed at 15-20 mg/kg/day for the first 6 months with monthly complete blood counts (CBCs) to ensure safety. From month 6-24, the dose was escalated (5 mg/kg every 8 weeks) to maximum tolerated dose (MTD), defined as mild myelosuppression with absolute neutrophil count (ANC) <4.0 x 109/L on 2 consecutive CBCs without hematological toxicities. CBCs were performed monthly until MTD or a stable dose was achieved, then subsequently every 3 months. Dose limiting toxicities (DLT) requiring a temporary treatment hold were defined as ANC <1.0 x 109/L, Hb <4.0 g/dL, reticulocyte count <80 x 109/L unless Hb >7.0 g/dL, or platelets <80 x 109/L. Known genetic modifiers of SCA, including G6PD deficiency and α-thalassemia trait, were determined for all participants. Results: A total of 606 children initiated hydroxyurea and currently 555 (92%) remain on treatment, with average treatment duration of 48 ± 12 months and a total of 2,441 patient-years of hydroxyurea treatment. Over 85% achieved MTD with an average hydroxyurea dose of 22.5 ± 5.0 mg/kg/day, ranging from 19.0 mg/kg/day in Angola to 25.4 mg/kg/day in Uganda. With dose increases over time, the most recent average dose is 23.9 ± 5.4 mg/kg/day (site range 22.9-24.6 mg/kg/day). Lab benefits have been sustained; Hb increased from 7.3 g/dL at baseline to 8.4 g/dL at MTD and remains 8.3 g/dL at Month 60. Similarly, the average HbF increased from 11% baseline to 25% at MTD and remains 23% at Month 60. The average ANC decreased from 6.8 x 109/L at baseline to 3.2 x 109/L at MTD and remains 3.5 x 109/L at Month 60. Lab toxicities are infrequent, transient, and mostly incidental. Of 19,730 CBCs obtained during the treatment phase, 421 (2.1%) in 225 participants included a DLT. The most common DLT was thrombocytopenia (33%), with only 4 platelet counts <20 x 109/L and no bleeding. Anemia was the second most common DLT (26%), most commonly associated with a high reticulocyte count and malarial infection, unrelated to hydroxyurea. Severe neutropenia (ANC <0.5 x 109/L) was rare (5 events) with no neutropenic infections. Over 2/3 of DLT events were identified incidentally during a scheduled visit when the study participant was asymptomatic, including all 5 severe neutropenic episodes. Weight-for-age and height-for-age Z-scores were not associated with higher rates of DLT during hydroxyurea treatment. Children with two-gene deletional α-thalassemia trait tolerated significantly lower hydroxyurea doses than the normal genotype (MTD dose 20.0 vs. 24.0 mg/kg/day, p <0.001) and had significantly different treatment responses at Month 60 including lower HbF (19.5 vs 24.3%, p <0.0001) and MCV (72 vs 99 fL, p<0.001) but higher hemoglobin (8.5 vs 8.1 g/dL, p=0.016). DLT frequency was unaffected by α-thalassemia status. Males with G6PD A- deficiency did not demonstrate significant differences in dosing, response, or toxicity. Conclusions: Hydroxyurea is safe, well-tolerated, and effective for children with SCA living in sub-Saharan Africa. Treatment responses are robust and sustained in REACH across all 4 clinical sites and unaffected by baseline Z-score. Hydroxyurea optimization requires periodic dose escalation for weight gain and titration to mild myelosuppression. Deletional α-thalassemia trait significantly influences the hydroxyurea dose and treatment responses, but the lab benefits with optimized dosing are still robust regardless of the α-globin genotype. Lab toxicities from hydroxyurea are uncommon and typically asymptomatic, suggesting that routine CBC monitoring is needed only at 3-month intervals once a stable dose is achieved, more to optimize the dose than to identify incidental toxicities. This approach to optimizing hydroxyurea therapy will allow more widespread utilization in low-resource settings with limited laboratory monitoring. Disclosures Aygun: National Heart, Lung, and Blood Institute: Research Funding; National Institute of Nursing Research: Research Funding; Patient-Centered Outsomes Research Institute: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Thrombin-Mediated Activation of PAR-1 Contributes to Microvascular Stasis in Mouse Models of Sickle Cell Disease Via Increased Endothelial Expression of P-Selectin and VWF

Blood ◽

10.1182/blood-2018-99-116238 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 266-266

Author(s):

Erica Sparkenbaugh ◽

Chunsheng Chen ◽

Julia Nguyen ◽

Shaobin Wang ◽

Gregory M. Vercellotti ◽

...

Keyword(s):

Bone Marrow ◽

Sickle Cell Disease ◽

Mouse Model ◽

Mouse Models ◽

Sickle Cell ◽

Research Funding ◽

Bleeding Complications ◽

Cell Physiology ◽

Dependent Manner ◽

Cell Disease

Abstract Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene, resulting in an altered red cell physiology that causes vascular complications such as hemolytic anemia, chronic inflammation, activation of coagulation, and vaso-occlusion. We have shown that infusion of hemin results in tissue factor (TF)-dependent activation of coagulation in mice. Furthermore, TF-dependent activation of coagulation contributes to inflammation in a mouse model of SCD. Interestingly, thrombin-dependent inflammation in sickle cell mice was not attenuated by deficiency of the main thrombin receptor protease activated receptor-1 (PAR-1). However, others have shown that the activation of endothelial cell PAR-1 with agonist peptide enhances interactions of these cells with sickle RBCs in a P-selectin-dependent manner. Importantly, P-selectin inhibition reduces microvascular stasis in mouse models of SCD and prevents vaso-occlusive crisis in sickle cell patients. We propose that thrombin-mediated PAR-1 activation promotes microvascular stasis in mouse models of SCD via increased expression of P-selectin and VWF on the endothelium, triggered by exocytosis of Weibel-Palade bodies. To investigate if the TF/thrombin/PAR-1 pathway contributes to microvascular stasis, dorsal skinfold chambers were implanted in NY1DD sickle mice 3 days before the experiment. Microvascular stasis was determined in 20-25 preselected micro-vessels in response to intravenous infusion of stroma-free hemoglobin (SFH, 1.6 µmol/kg) and was expressed as % non-flowing vessels (mean ± SEM). We previously demonstrated that infusion of SFH results in microvascular stasis that is inhibited by hemopexin in NY1DD mice, indicating that hemoglobin releases heme into the circulation. In NY1DD mice treated with control IgG antibody, SFH infusion caused stasis in 28.3 ± 1.7% and 36.7 ± 1.7% of preselected vessels at 1 and 4 hrs after infusion, respectively. Treatment with an inhibitory anti-TF antibody 1H1 (25 mg/kg; IP) 30 minutes before SFH infusion significantly reduced stasis to only 3.3 ± 1.5% and 2.8 ± 1.6% of vessels at 1 and 4 hrs, respectively (p<0.01). To investigate if TF contributes to SFH-induced microvascular stasis via generation of downstream coagulation proteases, NY1DD mice were fed with control chow or chow containing either Factor Xa (FXa) inhibitor rivaroxaban (0.4 mg/g chow) or thrombin inhibitor dabigatran (10 mg/g chow) ad libitum for 4 days prior to stasis experiments. We previously showed that these doses efficiently anticoagulated sickle mice without bleeding complications. FXa inhibition significantly reduced stasis at 1 hr (4.9 ± 0.1% versus 22.4 ± 3.8%, p<0.001) and 4 hrs (1.7 ± 1.6% versus 12.8 ± 1.9%, p<0.001) after SFH injection. Similarly, dabigatran also significantly attenuated SFH-induced stasis (3.1 ± 1.5% non-flowing vessels at 1 hour and 1.6 ± 1.6% non-flowing vessels at 4 hours). Next, to investigate if thrombin contributes to SFH-induced stasis through activation of PAR-1, we used bone marrow transplantation to generate sickle mice lacking PAR-1 expression in all non-hematopoietic cells. PAR-1+/+ and PAR-1-/- mice were lethally irradiated and transplanted with bone marrow from Townes sickle (SS) mice. Efficient reconstitution of bone marrow was confirmed by hemoglobin electrophoresis. We found that PAR-1-/- SS mice were significantly protected from SFH-induced stasis compared to PAR-1+/+ SS mice at both 1 hr (13.2 ± 3.4% versus 37.6 ± 3.9% stasis; P<0.001) and 4 hrs (6.6 ± 1.7% versus 18.0 ± 1.5% stasis; p<0.05) after hemoglobin challenge. We have previously shown that a monoclonal antibody to murine P-selectin or a polyclonal antibody to VWF markedly inhibit stasis in sickle mice. After the 4 hr stasis measurement, lungs were harvested from PAR-1+/+ SS and PAR-1-/- SS the mice and stained for P-selectin, VWF, and CD31 (a marker for endothelial cells). We found that intensity of P-selectin and VWF staining was markedly reduced in the lungs of PAR-1-/- SS mice compared to the staining observed in lungs of PAR-1+/+ SS mice. Our data indicates that SFH-induced microvascular stasis is significantly diminished by inhibition of TF, FXa, or thrombin activity or knockout of endothelial PAR-1, which leads to reduced expression of P-selectin and VWF on endothelium in a mouse model of SCD. We speculate that inhibiting the TF/thrombin/PAR-1 axis may reduce vaso-occlusive crises in SCD patients. Disclosures Vercellotti: CSL Behring: Research Funding. Belcher:CSL Behring: Research Funding.

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Initial Change Of Iron Metabolic Flux Prior To Differentiation Of Erythroid Progenitors After Epoetin Beta Pegol (C.E.R.A.) Treatment

Blood ◽

10.1182/blood.v122.21.3444.3444 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3444-3444

Author(s):

Yusuke Sasaki ◽

Mariko Noguchi-Sasaki ◽

Yukari Matsuo ◽

Junko Fukumura ◽

Fumi Sato-Inomata ◽

...

Keyword(s):

Bone Marrow ◽

Iron Metabolism ◽

Serum Iron ◽

Metabolic Flux ◽

Iron Status ◽

Erythroid Progenitors ◽

Epoetin Beta Pegol ◽

Epoetin Beta ◽

Serum Hepcidin ◽

Initial Change

Abstract Introduction Erythropoiesis and iron metabolism are inextricably linked. Developing immature erythroblasts have an extremely high iron requirement, especially during hemoglobin synthesis. Hepcidin, which inhibits iron efflux by binding to iron exporter ferroportin, is suppressed to promote the supply of required iron. Suppression of hepcidin after erythropoiesis-stimulating agent (ESA) treatment is mainly in an indirect manner, especially via erythropoietic activity, but the nature of suppressive mechanism of hepcidin is still unknown. Epoetin beta pegol (C.E.R.A.) is a novel long-acting ESA, which potentially has intensive and continuous effects on reduction of hepcidin. In the present study, we investigated change of iron metabolic flux associated with enhanced erythropoiesis by C.E.R.A. to analyze the mechanism underlying suppression of hepcidin. Methods Initial change of iron metabolism was analyzed in C57BL/6N mice intravenously treated with 10 μg/kg of C.E.R.A. or vehicle. Hematological indices such as reticulocyte counts and iron indices including serum hepcidin and iron levels were measured. Reticulocyte hemoglobin equivalent (Ret-He) which reflects the iron status of reticulocyte was also determined. Ter119 and transferrin receptor (CD71) expression on bone marrow cells was evaluated by flow cytometry for analysis of the maturation status of bone marrow erythroblasts. Results C.E.R.A. suppressed serum hepcidin levels after 9 hours, while serum iron levels were significantly decreased at 9 hours followed by recovery to the control levels at 24 hours. Ter119(+)CD71(high) immature erythroblasts were decreased and CD71 expression levels on the same cells were increased at 9 hours after C.E.R.A. treatment. C.E.R.A. elevated reticulocyte counts and Ret-He levels at 48 hours. Discussion and Conclusion Transient decrease in serum iron levels and continuous suppression of serum hepcidin levels were observed in early phase after C.E.R.A. treatment, prior to increase in erythroblasts through proliferation and differentiation of erythroid progenitors. Furthermore, considering decrease in immature erythroblasts followed by increase in hemoglobin-rich reticulocytes, it is possible that initial change of iron metabolic flux occurred by the accelerated iron incorporation into immature erythroblasts through CD71 recycling after C.E.R.A. treatment. These results suggest that sensing initial change of iron metabolic flux leads to suppression of hepcidin after C.E.R.A. treatment, but further analysis is needed for the mechanism of increase in iron absorption into immature erythroblasts immediately after C.E.R.A. treatment independent of differentiation of erythroid progenitors. Disclosures: No relevant conflicts of interest to declare.

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Upregulation of Renal Iron Metabolism in Sickle Cell Disease Mice

Blood ◽

10.1182/blood.v128.22.1276.1276 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1276-1276

Author(s):

Guelaguetza Vazquez-Meves ◽

Namita Kumari ◽

Nowah Afangbedji ◽

Alfia Khaibullina ◽

Zena Quezado ◽

...

Keyword(s):

Sickle Cell Disease ◽

Epithelial Cells ◽

Iron Metabolism ◽

Transferrin Receptor ◽

Globin Gene ◽

Proximal Tubules ◽

Iron Accumulation ◽

Cell Disease ◽

Intracellular Iron ◽

Iron Storage

Abstract BACKGROUND: Hemolysis and frequent blood transfusions lead to the iron overload and organ iron accumulation in patients with red blood cells disorders. The pattern of iron accumulation within different organs is disease specific. Abnormalities of renal iron metabolism and cortical iron deposition is characteristic for sickle cell disease (SCD) but not for β-thalassemia. Renal iron deposition does not correlate with iron overload and blood transfusion. Iron is reabsorbed from primary urine in the renal proximal epithelial cells and released into the renal intersitium by ferroportin. Iron-regulating hormone, hepcidin controls ferroportin expression. Binding of hepcidin to the ferroportin induces ferroportin degradation and intracellular iron accumulation. Low concentrations of circulating hepcidin are common in SCD patients and do not explain paradoxical renal iron accumulation. SCD mice accumulate iron in the epithelial cells of proximal tubules and may be a suitable model to study iron metabolism in SCD. OBJECTIVES: To characterize proteins of the renal iron metabolism in SCD mouse model. METHODS: The SCD (Townes) mice do not express mouse α- or β-globin alleles, but carry two copies of a human α1-globin gene and two copies of a human Aγ-globin and βS-globin genes. These animals synthesize approximately 94% human sickle (HbS) and 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals carry two copies of the human α1-globin gene and two copies of the human hemoglobin gamma (Aγ) gene and the human wildtype hemoglobin beta (βA) gene. Kidneys were collected from 5 months old SCD and control mice. Renal cortex was used for RNA and protein isolation. Levels of renal hepcidin, ferroportin, transferrin receptor (TFR1), divalent cation receptor (DMT1), ferritin and hepheastin were determined by q-RT-PCR, WB and ELISA. Paraffin-embedded sections were used for immunostaining. Perl's Prussian blue staining was used for detection of renal iron accumulation. RESULTS:We detected significant accumulation of iron in the epithelial cells of proximal tubules in SCD mice. Expression of renal hepcidin was increased in SCD mice compared to controls. Surprisingly mRNA levels of all other proteins involved in renal iron metabolism (ferroportin, TFR1, DMT1, ferritin and hephaestin) were decreased in SCD mice kidney. In contrast, we found increased protein levels of transferrin receptor (iron importer), ferritin (iron storage protein) and slightly increased level of ferroportin (iron exporter). We also observed significant renal macrophages infiltration in SCD mice. CONCLUSIONS: Increased levels of renal hepcidin expression in SCD mice may be associated with renal inflammation. Higher levels of locally expressed hepcidin may lead to the partial degradation of the iron exporter (ferroportin). Increased levels of iron importers (TFR1 and DMT1) and no significant change in ferroportin expression can cumulatively saturate iron storage in ferritin and lead to the accumulation of intracellular iron. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Disclosures Quezado: IONIS Pharmaceuticals: Research Funding.

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Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010535 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3906-3917 ◽

Cited By ~ 2

Author(s):

Aaron M. Wortham ◽

Devorah C. Goldman ◽

Juxing Chen ◽

William H. Fleming ◽

An-Sheng Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Iron Overload ◽

Transferrin Receptor ◽

Function Analysis ◽

Hereditary Hemochromatosis ◽

The Body ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Deficient Mice ◽

Transferrin Receptor 2

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit–erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit–erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

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