Serum GDF15 in β-Thalassemia: A Quantitative Marker of Ineffective Erythropoiesis?

Abstract Introduction: Ineffective erythropoiesis (IE) is a crucial hallmark of β-Thalassemia (β-Thal) and sets the goal for treatment of both Transfusion-Dependent (TDT) and Non Transfusion-Dependent Thalassemia (NTDT) patients. The Growth Differentiation Factor (GDF) group has a relevant role in the molecular regulation of IE. Specifically, GDF11 contributes to the inhibition of RBC maturation and it is targeted by activin traps, such as luspatercept. However, its role is still debated; i.e., in a mouse model of β-Thal, the absence of GDF11 alone is not sufficient to mitigate IE (Guerra A et al., Blood, 2019). GDF15 increases from early until late phases of erythroid differentiation and negatively regulates erythroid cell development in-vitro, modulating maturation and apoptosis. (Ranjbaran R et a.l, Exp Cell Res, 2020). A few clinical studies found elevated serum GDF15 levels in β-Thal (Tanno T et al., Nat Med, 2007; Huang Y et al., Int J Med Sci, 2019), but data are sparse and a clear correlation with the severity of the pathology is still missing. Methods: We run an observational study at our institution. At routine checks, patients were asked to consent to a specific blood sample for GDF15, whereas hemoglobin (Hb), serum erythropoietin (Epo), ferritin (Ftn), iron, and transferrin saturation (TSat) were measured as part of clinical practice. In a small subset of patients, a consent for serial sampling was added. Serum GDF15 was measured by ELISA (DuoSet DY957, R&D Systems). Demographics were collected from clinical records. Statistical analysis was performed using Statistica 10 (Statsoft). Results: GDF15 levels were measured in 458 individuals: 267 TDT, 77 NTDT, 45 β-Thal trait carriers (BTC), and 69 healthy (H) subjects. Median (IQR) levels of GDF15 were significantly different among diagnoses (P<0.0001), and specifically measured 0.22 (0.16-0.34) in H, 0.48 (0.28-0.96) in BTC, 1.35 (0.40-5.46) in NTDT and 5.95 (3.19-10.52) ng/mL in TDT. (FIG 1A). In TDT patients, a mild but highly consistent negative correlation was observed between Hb and GDF15 levels (R=-0.31, P=0.002). GDF15 levels correlated positively with Epo (R=0.60, P<0.0001), TSat (R=0.32, P=0.0003) and serum iron (R=0.27, P=0.003). In addition, they correlated with length of transfusion interval in splenectomized patients (R=0.65, P<0.001). In a small subset of longitudinal data in TDT, the transfusion cycle had a strong and uniform effect on GDF15 levels. FIG 1B shows a single individual (female, 38 years old, splenectomized): mean (±SD) GDF15 levels were 5.27 (± 1.99) and 2.10 (±0.97) ng/mL pre-transfusion and 8 days post-transfusion, respectively (P<0.01). After 21 days post-transfusion, they were 3.74 (±0.76), showing a trend of variation opposite to Hb. In another individual with a prenatal diagnosis of severe TDT (homozygous for IVS I:110), GDF15 levels were high at 5 months and showed a progressive decline after the start of regular transfusion therapy. After reaching the 10 mg/dL threshold for pre-transfusion Hb, GDF15 levels were approximately halved compared to 5 months of age (6.2 vs 2.7 ng/mL) (FIG 1C). In NTDT patients, GDF15 correlated positively with TSat (R=0.40, P<0.0001), serum iron (R=0.39, P<0.0001) and Ftn (R=0.25, P=0.01). Among TDT and NTDT patients, 45 and 19 were paediatrics, respectively. No significant differences were observed at different ages. In BTC, GDF15 correlated negatively with Hb levels (R=-0.43, P=0.002) and positively with Epo (R=0.78, P=0.02), TSat (R=0.77, P<0.0001), serum iron (R=0.62, P<0.0001) and Ftn (R=0.59, P<0.0001). No significant correlations were observed for H subjects. Discussion: GDF15 levels correlated with the severity of β-Thal phenotype, showing a 26-fold (TDT), a 6-fold (NTDT) and a 2-fold (BTC) increase compared to controls. In TDT patients, higher GDF15 levels correlated with lower Hb and higher Epo, which are typically observed as a result of IE in thalassemia. In addition, GDF15 correlated with markers of altered iron metabolism, such as TSat and serum iron. In individual patients, GDF15 showed strong and consistent variation with treatment. GDF15 was also associated with quantitative markers of disease in NTDT and BTC patients. This is to our knowledge the larger sample of patients carrying β-thal mutations in which GDF15 levels were measured and correlated with the severity of the disease. These results show that GDF15 may be a suitable and useful quantitative marker of IE. Figure 1 Figure 1. Disclosures Piga: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Research Funding. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria.

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Removal of Macrophages From the Erythroid Niche Impairs Stress Erythropoiesis but Improves Pathophysiology of Polycythemia Vera and Beta-Thalassemia

Blood ◽

10.1182/blood.v120.21.81.81 ◽

2012 ◽

Vol 120 (21) ◽

pp. 81-81

Author(s):

Pedro Ramos ◽

Carla Casu ◽

Sara Gardenghi ◽

Laura Breda ◽

Bart J Crielaard ◽

...

Keyword(s):

Polycythemia Vera ◽

Research Funding ◽

Serum Iron ◽

Transferrin Saturation ◽

Cell Surface Expression ◽

Beta Thalassemia ◽

Ineffective Erythropoiesis ◽

Erythroid Progenitor ◽

Distribution Width ◽

Stress Erythropoiesis

Abstract Abstract 81 We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC). While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration. In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect. Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P<0.001). BTI mice exhibited normal spleen, amelioration of ineffective erythropoiesis (by accelerating the differentiation of EBs to erythrocytes), improvement of red blood cell (RBC) morphology, red cell distribution width (RDW, P<0.001) and increased hemoglobin levels (∼2g/dL, P<0.01). This effect was due to an increased RBC lifespan following LC administration (P<0.001), which was associated with a decrease in hemichrome formation, but not with a reduction in erythophagocytosis. Our observations indicate that macrophages directly modulate stress- and pathological erythropoiesis. Several adhesion molecules participate in the formation of interactions within the erythroblastic islands, including integrins. Interestingly, βeta1integrin and its associated protein, focal adhesion kinase-1 (Fak1), were reported to be necessary for the compensatory response to anemia, suggesting that this pathway might be involved in the macrophage-EB cross-talk. More EBs co-cultured with macrophages retained cell surface expression of βeta1integrin molecule during the last stage of cell differentiation compared to EBs cultured alone, even though other differentiation markers did not shown any variation. Fak1 phosphorylation in EBs was induced by co-culturing them with splenic macrophages, suggesting that Fak1 signaling is one of the pathways activated in EBs through contact with macrophages. Administration of a FAK1 inhibitor (FAK1i) decreased proliferation of EB co-cultured with macrophages, while delayed recovery from anemia and decreased the spleen size in phlebotomized animals (40% decrease compared to phlebotomized control animals at day 4; P=0.032). Finally, short-term administration of FAK1i to BTI animals rapidly reverted splenomegaly with a concurrent reduction of erythroid expansion in both BM and spleen and led to amelioration of anemia, supported by increased RBCs count. Our data indicate that, while macrophages allow proper erythroid response under conditions of induced anemia or increased erythropoiesis in wt mice, they contribute to the pathological progression of PV and BTI. Activation of Fak1 promotes erythroid proliferation and pathological development, while its inhibition limits ineffective erythropoiesis and splenomegaly in BTI. In conclusion, we identified a new mechanism contributing to the pathophysiology of these disorders, which we believe will have critical scientific and therapeutic implications in the near future. Disclosures: Levine: Agios Pharmaceuticals: Research Funding. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

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RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice

Blood ◽

10.1182/blood.v118.21.1045.1045 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1045-1045 ◽

Cited By ~ 1

Author(s):

Ivanka Toudjarska ◽

Zuhua Cai ◽

Tim Racie ◽

Stuart Milstein ◽

Brian R Bettencourt ◽

...

Keyword(s):

Iron Overload ◽

Serum Iron ◽

Iron Concentration ◽

Transferrin Saturation ◽

Elevated Serum ◽

High Serum ◽

Therapeutic Modality ◽

Deficiency Anemia ◽

Loss Of Function ◽

Hepcidin Expression

Abstract Abstract 1045 The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality. Disclosures: Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.

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Siltuximab Treatment Increases Hemoglobin (Hb) Levels: Preliminary Results From a Prospective Phase 1 Study In Refractory Solid Tumors

Blood ◽

10.1182/blood.v116.21.5150.5150 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5150-5150

Author(s):

R. Kurzrock ◽

E. Angévin ◽

S. Cohen ◽

J. Van Laethem ◽

B. Rijnbeek ◽

...

Keyword(s):

Solid Tumors ◽

Research Funding ◽

Serum Iron ◽

Binding Capacity ◽

Iron Status ◽

Phase 1 ◽

Transferrin Saturation ◽

Maximum Increase ◽

Baseline Serum ◽

Clinically Significant

Abstract Abstract 5150 Introduction: Siltuximab is a chimeric monoclonal antibody with high affinity for the inflammatory cytokine, IL-6, which is currently being studied in hematologic, solid malignancies and multicentric Castleman's disease (MCD). In addition to representing a therapeutic target, IL-6 is reported to play a key role in the etiology and symptoms of anemia of cancer. A possible mechanism is through up-regulation of hepatic production of hepcidin, the central iron-regulatory hormone. Siltuximab treatment has previously been shown to be associated with clinically significant Hb increases in MCD (a disorder caused by deregulated IL-6 production) and renal cell carcinoma. We have prospectively studied the Hb response in the context of a phase I study with siltuximab in patients with advanced solid tumors. Patients and Methods Siltuximab was administered intravenously to patients with any advanced solid tumor at increasing dose levels (2.8 or 5.5 mg/kg every 2 weeks, 11 or 15 mg/kg every 3 weeks). Hepcidin (C-ELISA), Hb, CRP (marker for inflammation) and iron status (serum iron, ferritin, transferrin saturation, total iron binding capacity) were measured at baseline and serially during treatment. IL-6 was not measured since interference of the drug with assay performance prevents accurate measurement of bioactive IL-6. The relationships between these biomarkers and Hb response (defined as a maximum Hb increase of ≥1 g/dL during treatment) were evaluated. Results: Forty-four pts (18 colorectal, 12 ovarian, 5 pancreatic, 9 other) received a median of 3 siltuximab cycles (range 1 – 25). Eight patients were excluded from analysis because they received blood transfusions or ESAs. There were no objective tumor responses (CR or PR). Baseline Hb ranged from 9.4–15.3 g/dL (median 12.2). All 36 evaluable patients had an increase in Hb (median 1.35 g/dL; range 0.1–3.2). Eleven (31%) patients had a maximum increase of ≥2 g/dL. Maximum Hb levels did not exceed the upper limit of normal. Baseline hepcidin (median 118.6 ng/mL; range 9.5–493.3) was positively correlated with baseline CRP (median 13.6 mg/L; range 0.42–152.0) (p<0.05) and ferritin (median 346 pmol/L; range 74.2–8543.1) (p<0.05) but not with baseline Hb or Hb response. For subjects with a Hb response of more than 2 g/dL an association was found with Day 8 hepcidin (p = 0.03) when controlled for baseline serum iron. Early hepcidin percentage change was not correlated with Hb response. Conclusion: Siltuximab treatment was associated with clinically meaningful Hb response in this moderately anemic refractory cancer population. The exact mechanism of action remains uncertain, however correlation with additional markers (e.g., soluble transferrin receptor, inflammatory markers such as IL-6) might also be important to identify patients most likely to respond to treatment and should be evaluated further in randomized trials. Disclosures: Kurzrock: Johnson & Johnson: Research Funding. Angévin:Johnson & Johnson: Research Funding. Cohen:Johnson & Johnson: Research Funding. Van Laethem:Johnson & Johnson: Research Funding. Rijnbeek:Johnson & Johnson: Employment. Vermeulen:Johnson & Johnson: Employment. Tromp:Johnson & Johnson: Employment. Li:Johnson & Johnson: Employment. Reddy:Johnson & Johnson: Employment. Cornfeld:Johnson & Johnson: Employment. Tabernero:Johnson & Johnson: Research Funding.

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HFE Genotyping in Patients with Elevated Serum Iron Indices and Liver Diseases

BioMed Research International ◽

10.1155/2015/164671 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Andreia Silva Evangelista ◽

Maria Cristina Nakhle ◽

Thiago Ferreira de Araújo ◽

Clarice Pires Abrantes-Lemos ◽

Marta Mitiko Deguti ◽

...

Keyword(s):

Liver Disease ◽

Chronic Liver Disease ◽

Serum Iron ◽

Genetic Diseases ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Elevated Serum ◽

Clinical Manifestations ◽

Chronic Liver ◽

Group 2

Iron abnormalities in chronic liver disease may be the result of genetic diseases or secondary factors. The present study aimed to identify subjects with HFE-HH in order to describe the frequency of clinical manifestations, identify risk factors for iron elevation, and compare the iron profile of HFE-HH to other genotypes in liver disease patients. A total of 108 individuals with hepatic disease, transferrin saturation (TS) > 45%, and serum ferritin (SF) > 350 ng/mL were tested for HFE mutations. Two groups were characterized: C282Y/C282Y or C282Y/H63D genotypes (n=16) were the HFE hereditary hemochromatosis (HFE-HH) group; and C282Y and H63D single heterozygotes, the H63D/H63D genotype, and wild-type were considered group 2 (n=92). Nonalcoholic liver disease, alcoholism, and chronic hepatitis C were detected more frequently in group 2, whereas arthropathy, hepatocarcinoma, diabetes, and osteoporosis rates were significantly higher in the HFE-HH group. TS > 82%, SF > 2685 ng/mL, and serum iron > 178 μg/dL were the cutoffs for diagnosis of HFE-HH in patients with liver disease. Thus, in non-Caucasian populations with chronic liver disease, HFE-HH diagnosis is more predictable in those with iron levels higher than those proposed in current guidelines for the general population.

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Association of Body Mass Index with Hemoglobin Concentration and Iron Parameters in Iranian Population

ISRN Hematology ◽

10.1155/2014/525312 ◽

2014 ◽

Vol 2014 ◽

pp. 1-3 ◽

Cited By ~ 10

Author(s):

Akram Ghadiri-Anari ◽

Narjes Nazemian ◽

Hassan-Ali Vahedian-Ardakani

Keyword(s):

Body Mass Index ◽

Body Mass ◽

Serum Iron ◽

Saturation Index ◽

Transferrin Saturation ◽

Elevated Serum ◽

Hemoglobin Concentration ◽

Low Grade ◽

Anemia Of Chronic Disease ◽

Iron Parameters

Background. Studies have reported that obesity has an adverse effect on iron metabolism. Obesity is characterized by chronic, low-grade, systemic inflammation and anemia of chronic disease with elevated serum ferritin and decreased level of serum iron, transferrin saturation, and hemoglobin. Therefore, we examined the association of body mass index with hemoglobin concentration and iron parameters in this study. Methods. This cross-sectional study was conducted in Yazd to assess the relation of body mass index with hemoglobin and iron parameters among 406 adult patients 18–65 years old. Diabetes and conditions that could influence body iron stores were excluded. Results. There is no difference in hemoglobin concentrations, MCV, serum iron, TIBC, transferrin saturation index, and ferritin between normal weight, overweight, and obese persons. Conclusion. Nutritional status of persons and intake of high iron foods by obese persons should be considered. Also, other inflammatory markers should be evaluated in the future studies.

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Mild Iron Overload as Seen in Individuals Homozygous for the Alpha-1 Antitrypsin Pi*Z Variant Does Not Promote Liver Fibrogenesis in HFE Knockout Mice

Cells ◽

10.3390/cells8111415 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1415 ◽

Cited By ~ 2

Author(s):

Nurdan Guldiken ◽

Karim Hamesch ◽

Shari Malan Schuller ◽

Mahmoud Aly ◽

Cecilia Lindhauer ◽

...

Keyword(s):

Liver Fibrosis ◽

Iron Overload ◽

Iron Metabolism ◽

Serum Iron ◽

Transferrin Saturation ◽

Elevated Serum ◽

Hfe Mutations ◽

Alpha 1 Antitrypsin ◽

The Impact ◽

Significant Liver Fibrosis

The presence of the homozygous ‘Pi*Z’ variant of alpha-1 antitrypsin (AAT) (‘Pi*ZZ’ genotype) predisposes to liver fibrosis development, but the role of iron metabolism in this process remains unknown. Therefore, we assessed iron metabolism and variants in the Homeostatic Iron Regulator gene (HFE) as the major cause of hereditary iron overload in a large cohort of Pi*ZZ subjects without liver comorbidities. The human cohort comprised of 409 Pi*ZZ individuals and 254 subjects without evidence of an AAT mutation who were recruited from ten European countries. All underwent a comprehensive work-up and transient elastography to determine liver stiffness measurements (LSM). The corresponding mouse models (Pi*Z overexpressors, HFE knockouts, and double transgenic [DTg] mice) were used to evaluate the impact of mild iron overload on Pi*Z-induced liver injury. Compared to Pi*Z non-carriers, Pi*ZZ individuals had elevated serum iron, transferrin saturation, and ferritin levels, but relevant iron overload was rare. All these parameters were higher in individuals with signs of significant liver fibrosis (LSM ≥ 7.1 kPa) compared to those without signs of significant liver fibrosis. HFE knockout and DTg mice displayed similar extent of iron overload and of fibrosis. Loss of HFE did not alter the extent of AAT accumulation. In Pi*ZZ individuals, presence of HFE mutations was not associated with more severe liver fibrosis. Taken together, Pi*ZZ individuals display minor alterations in serum iron parameters. Neither mild iron overload seen in these individuals nor the presence of HFE mutations (C282Y and H63D) constitute a major contributor to liver fibrosis development.

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Hepcidin Mimetic (PTG-300) Reverses Iron Deficiency While Controlling Hematocrit in Polycythemia Vera Patients

Blood ◽

10.1182/blood-2020-137304 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-41

Author(s):

Yelena Ginzburg ◽

Marina Kremyanskaya ◽

Andrew T. Kuykendall ◽

Abdulraheem Yacoub ◽

Jay Yang ◽

...

Keyword(s):

Iron Deficiency ◽

Serum Ferritin ◽

Research Funding ◽

Serum Iron ◽

Iron Status ◽

Transferrin Saturation ◽

Phase 2 ◽

Publicly Traded ◽

Advisory Committees ◽

Phase 2 Trial

Background. The majority of PV patients are iron deficient at diagnosis [Ginzburg Leukemia 2018]. PV patients are treated with periodic therapeutic phlebotomy (TP) in order to maintain hematocrit levels <45% in an effort to reduce the incidence of thrombotic events [Marchioli NEJM 2013]. Symptomatic iron deficiency represents a challenge in PV as it is commonly present at diagnosis and TP worsens iron deficiency and PV patients can be severely symptomatic from their iron deficiency [Krayenbuehl Blood 2011]. Iron deficiency is defined as insufficient iron stores to meet the needs of cellular homeostasis. We hypothesized that both iron deficiency and expanded erythropoiesis in PV lead to suppression of hepcidin, the body's main negative regulator of iron metabolism, and that hepcidin suppression enhances iron absorption and iron availability for enhanced erythropoiesis in TP-requiring PV patients. We previously demonstrated that PTG-300, a hepcidin-mimetic, caused dose-related anemia in pre-clinical studies. In healthy volunteers PTG-300 decreases serum iron and transferrin saturation (TSAT) by >70% within 12 hours and the effect persists for 3-7 days. In a phase 2 trial in β-thalassemia, PTG-300 also decreased serum iron and TSAT but did not demonstrate off-target effects. The current study aims to compare the iron status in frequent TP-requiring PV patients before, during, and after treatment with PTG-300. Methods. Polycythemia patients who met 2016 WHO criteria for diagnosis were enrolled in the 28-week dose finding part of a Phase 2 trial. All patients required ≥3 phlebotomies with or without concurrent cytoreductive therapy over 6 months prior to enrollment. Patients were given PTG-300 doses of 10, 20, 40, 60 and 80 mg administered subcutaneously weekly in individualized adjustment to maintain hematocrit <45%. Body iron status was quantified by monitoring serum ferritin, serum iron, transferrin saturation (TSAT), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). Results. Thirteen subjects were enrolled to date: 7/13 with low risk, mean age 57.4 years (range 31-74). Six receiving TP alone, 6 on concurrent hydroxyurea, 1 on concurrent interferon; TP in the 24 weeks prior to enrollment = 3-9; median time between TP = 42 days. All subjects-maintained hematocrit <45% after appropriate dose adjustment. Mean baseline values were serum ferritin = 14.2 ng/mL (5, 37); serum iron = 33.0 ug/dL (16.8, 107.8); and TSAT = 7.6% (4, 30). During treatment with PTG-300, serum ferritin levels increased progressively toward normal (Figure 1a) reflecting increase in iron stores. TSAT (Figure 1b) and serum iron values increased modestly but remained below normal ranges, reflecting PTG-300's pharmacodynamic effect of inhibiting iron release from intracellular stores. This was associated with increased MCV (Figure 1c) and MCH (Figure 1d) and decreased hematocrit and erythrocyte counts, together suggesting a normalization of iron distribution. Conclusions. The current results indicate that PTG-300 is an effective agent for the controlling hematocrit and reversing iron deficiency. The effect of PTG-300 on PV-related symptoms and those of iron deficiency, are also being evaluated. Continued patient enrollment will enable more definitive conclusions regarding the efficacy and safety of hepcidin mimetic PTG-300 in PV patients with high TP requirements. Disclosures Kremyanskaya: Incyte Corporation: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding. Kuykendall:Novartis: Research Funding; Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding. Yacoub:Agios: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Novartis: Speakers Bureau; Roche: Other: Support of parent study and funding of editorial support. Yang:AROG: Research Funding; AstraZeneca: Research Funding; Protagonist: Research Funding; Jannsen: Research Funding. Gupta:Protagonist: Current Employment. Valone:Protagonist: Current Employment. Khanna:Protagonist: Current Employment, Current equity holder in publicly-traded company. Hoffman:Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Forbius: Consultancy; Protagonist: Consultancy. Verstovsek:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; ItalPharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; Protagonist Therapeutics: Research Funding; NS Pharma: Research Funding; CTI Biopharma Corp: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; PharmaEssentia: Research Funding; Roche: Research Funding; AstraZeneca: Research Funding.

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Hepcidin Is Elevated in Primary and Secondary Myelofibrosis and Correlates with IL-6 and IL-2Rα but Is High in Patients Treated with Ruxolitinib

Blood ◽

10.1182/blood-2018-99-113661 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1760-1760

Author(s):

Amy Zhou ◽

Maggie J. Allen ◽

Mary Fulbright ◽

Jared S Fowles ◽

Daniel A.C. Fisher ◽

...

Keyword(s):

Sample Size ◽

Inflammatory Cytokines ◽

Research Funding ◽

Serum Iron ◽

Transferrin Saturation ◽

Hepcidin Expression ◽

Stat Signaling ◽

The Relationship ◽

Normal Controls ◽

Rbc Transfusion

Abstract Introduction: Hepcidin, a peptide-hormone produced by hepatocytes, is a key regulator of iron homeostasis in mammals. Hepcidin levels are affected by several factors including inflammation, iron concentration, and erythropoietic signaling, and elevated hepcidin levels can lead to anemia. Inflammatory cytokines such as IL-6 induce hepcidin mRNA transcription via JAK/STAT signaling, whereas high serum iron levels and high erythropoietic drive via erythroferrone (ERFE) suppress hepcidin transcription via BMP/SMAD signaling. Anemia is a common problem in patients with myelofibrosis (MF). In a previous study of primary myelofibrosis (PMF) patients, serum hepcidin levels were found to be elevated compared to normal controls and were associated with increased RBC transfusion requirement and reduced survival (Pardanani et al. Am J Hematol 2013;88(4):312-316). Ruxolitinib is a JAK1/2 inhibitor approved for treatment of patients with MF. Treatment with ruxolitinib reduces spleen volume, constitutional symptoms, and inflammatory cytokines, but does not lead to anemia improvement in MF (Verstovsek et al. NEJM 2012;366:799-807). To further assess the relationship between hepcidin and anemia in MF, we measured hepcidin levels in 99 MF patients including 49 PMF and 50 secondary MF (sMF) patients, as well as 24 patients treated with ruxolitinib. We also evaluated the relationship between hepcidin and hemoglobin levels, inflammatory cytokines, and serum iron markers. Methods: Hepcidin levels (ng/mL) were measured using a commercially available ELISA assay from Intrinsic Lifesciences (Intrinsic Hepcidin IDxTM ELISA) in 99 MF patient plasma samples and 9 normal controls. Plasma cytokine levels (IFN, IL-10, IL-2, IL-2Rα, IL-6, IL-8, MIP-1α, MIP-1β, TNFα, VEGF, TGF-β1, TGF-β2, TGF-β3) were measured via multiplex cytokine analysis (Mesoscale Discovery). CRP, transferrin saturation, and ferritin were measured using standard laboratory methods at our institution. Statistical analyses were performed using GraphPad Prism software. Results: Hepcidin was significantly elevated in MF patients compared to normals (median 81.1 vs 8.5, p <0.001). There was no difference in hepcidin expression between PMF and sMF (median 103.3 vs 80.8, p = 0.380). Hepcidin positively correlated with IL-2Rα and IL-6, but did not correlate with other cytokines evaluated. Hepcidin expression did not differ in samples obtained from patients on treatment with ruxolitinib versus those who were not (median 105.1 vs 80.2, respectively, p = 0.998). A trend between higher hepcidin levels and lower hemoglobin was observed, although it was not statistically significant (p = 0.719). No correlation was found between hepcidin levels and CRP, transferrin saturation, or ferritin. These results are consistent with the previous study confirming that hepcidin is elevated in MF. In addition, we found a correlation between hepcidin and IL-6, which was not observed previously (Pardanani et al. Am J Hematol 2013;88(4):312-316). The lack of significant correlation between hepcidin and hemoglobin may be due to our smaller sample size. Despite ruxolitinib therapy leading to decreases in inflammatory cytokine production, hepcidin levels were markedly elevated in patients treated with ruxolitinib. Conclusion: These findings indicate that abnormal hepcidin production is a hallmark of both PMF and sMF. Hepcidin expression correlated with IL-6 and IL-2Rα, but not with other inflammatory cytokines, nor with CRP. These observations suggest a complex relationship between inflammation and hepcidin in MF. Although a significant correlation between hepcidin and hemoglobin was not found, this may have been related to the relatively small sample size. In addition, RBC transfusion in a subset of patients may have impacted the lack of correlation between iron indices and hepcidin. Hepcidin was high in patients treated with ruxolitinib, suggesting that suppression of JAK-STAT signaling (such as downstream of IL-6) via ruxolitinib may not be sufficient to normalize hepcidin production. Alternatively, inhibition of hepcidin production via ACVR1/ALK2 has been proposed to be the mechanism behind anemia improvement observed with momelotinib therapy (Asshoff et al. Blood 2017;129(13): 1823 - 1830). Therefore, the role of hepcidin as a therapeutic target for treatment of anemia in patients with MF warrants further exploration. Disclosures Zhou: Incyte: Speakers Bureau. Oh:Gilead: Research Funding; Janssen: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; CTI Biopharma: Research Funding.

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Hepcidin Levels and Their Determinants In Different Types of Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.4250.4250 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4250-4250

Author(s):

Valeria Santini ◽

Domenico Girelli ◽

Alessandro Sanna ◽

Nicola Martinelli ◽

Lorena Duca ◽

...

Keyword(s):

Iron Overload ◽

Myelodysplastic Syndromes ◽

Iron Homeostasis ◽

Conflicts Of Interest ◽

Iron Status ◽

Transferrin Saturation ◽

Ineffective Erythropoiesis ◽

Transfusion Therapy ◽

Different Types ◽

Serum Hepcidin

Abstract Abstract 4250 Background and Aims. Iron overload is frequently occurring in patients with myelodysplastic syndromes (MDS), with recent data suggesting an impact on both overall and leukemia-free survival1,2. Though prolonged RBC transfusion therapy appears the main contributor, many patients develop iron overload at an early stage of the disease, before the onset of transfusion dependency. It has been postulated that an altered production of hepcidin, the key hormone regulating iron homeostasis, may play a role at this regard. Until recently, studies have been hampered by problems in the development of reliable hepcidin assays, so that only scanty and conflicting data based on semi-quantitative measurement of urinary hepcidin have been reported3,4. This study mainly focused on analyzing serum hepcidin levels in MDS patients by means of a recently validated and improved Mass-Spectrometry based method5. Patients and Methods. One hundred and thirteen consecutive patients (mean age 72.8 ± 9.2 years; 68.1% males) with different types of MDS according to the WHO classification were included in this study. To be enrolled, patients had to be previously untreated or treated only with transfusions. Besides hepcidin, in all subjects we determined serum ferritin, transferrin saturation (TS), non-transferrin-bound-iron (NTBI), along with some putative determinants of hepcidin, like GDF-156 known to be associated with ineffective erythropoiesis, and C-Reactive Protein (CRP) as a surrogate of systemic IL-6 production. Fifty-four healthy individuals (61.1% males) with rigorous definition of normal iron status were used as controls. Main Results. Biochemical markers of iron overload (ferritin and TS), but also CRP and GDF-15 were significantly higher in MDS patients than in controls, even when considering only non-transfused patients. Patients with RARS and the 5q- syndrome appeared as the most iron overloaded, having the highest levels of ferritin, TS, and NTBI. In the whole MDS population, serum hepcidin levels showed a considerable variability, with overall mean values not significantly different from controls [geometric means (gm) with 95% CIs: 5.31 (3.98-7.08) versus 4.2 (3.53-5.0) nM, P=0.28], while the hepcidin/ferritin ratio was significantly lower than in controls [10.1 (7.53-13.53) versus 52.9 (43.6-64.3), P<0.001]. After stratification according to WHO subtypes, hepcidin levels showed significant differences, with the lowest levels in patients with RARS (gm 1.43 nM) and the highest levels in patients with RAEB 1–2 (gm 11.3 nM) and with CMML (gm 10.04 nM) (P=0.003 by ANOVA). The latter groups had substantial elevation of CRP as compared to other MDS subtypes (P=0.008 by ANOVA), while GDF-15 was consistently but uniformly elevated in all MDS subtypes (P=0.97 by ANOVA). Multivariate linear regression models adjusted also for age, sex, and history of RBC transfusions, showed ferritin (β-coefficient 0.45, P=0.002), CRP (β-coefficient 0.21, P=0.02), and different MDS subtypes as the main independent predictors of hepcidin levels. The different degree of correlation between hepcidin and ferritin among the MDS subtypes were analyzed in a general linear model using the F test for slopes. Hepcidin regulation by iron appeared conserved, though relatively blunted in RA, RARS, and 5q- patients, while it was lost in RAEB 1–2 and CMML. Conclusions. Hepcidin levels are consistently heterogeneous in MDS according to different subtypes, likely as the result of the relative strength of competing stimuli. Relative inhibition by ineffective erythropoiesis (but not mediated by GDF-15) seems to prevail particularly in RARS and 5q- syndrome, and is likely to increase the risk of iron overload in these subgroups. On the other hand, patients with RAEB 1–2 and CMML appears to have hepcidin induction that could be driven by cytokines. If confirmed, these results may be relevant not only for a better understanding of iron pathophysiology in MDS, but also for possible future approach with hepcidin modulators7. References: 1) Sanz G, et al. Blood 2008;112: abs 640. 2) Alessandrino EP, et al. Haematologica 2010;95:476-84. 3) Winder A, et al. Br J Haematol 2008;142:669-71. 4) Murphy PT, et al. Br J Haematol 2009;144:451-2. 5) Campostrini N, et al. J Biomed Biotechnol 2010;2010:329646. 6) Tanno T, et al. Nat Med 2007;13:1096-101. 7) Sasu BJ, et al. Blood 2010;115:3616-24. Disclosures: No relevant conflicts of interest to declare.

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Mitapivat Improves Ineffective Erythropoiesis and Reduces Iron Overload in Patients with Pyruvate Kinase Deficiency

Blood ◽

10.1182/blood-2021-147317 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2005-2005

Author(s):

Eduard J. Van Beers ◽

Hanny Al-Samkari ◽

Rachael F. Grace ◽

Wilma Barcellini ◽

Andreas Glenthoej ◽

...

Keyword(s):

Iron Overload ◽

Board Of Directors ◽

Pyruvate Kinase ◽

Research Funding ◽

Iron Concentration ◽

Transferrin Saturation ◽

Controlled Study ◽

Publicly Traded ◽

Ineffective Erythropoiesis ◽

Advisory Committees

Abstract Background: Pyruvate kinase (PK) deficiency is a rare hereditary disease resulting in chronic hemolytic anemia, which is associated with serious complications, including iron overload, regardless of transfusion status. Ineffective erythropoiesis is linked to iron overload in patients (pts) with hemolytic anemias. Mitapivat is a first-in-class, oral, allosteric activator of the red blood cell PK enzyme (PKR) that has demonstrated improvement in hemoglobin (Hb), hemolysis, and transfusion burden in pts with PK deficiency. This analysis assessed the effect of mitapivat on markers of erythropoiesis and iron overload in pts with PK deficiency enrolled in 2 phase 3 studies, ACTIVATE (NCT03548220) and ACTIVATE-T (NCT03559699), and the long-term extension (LTE) study (NCT03853798). Methods: In ACTIVATE (double-blind, placebo-controlled study), 80 pts (age ≥ 18 years [yrs]) with a confirmed diagnosis of PK deficiency who were not regularly transfused (≤ 4 transfusion episodes in the prior yr; none in the prior 3 months) were randomized to receive mitapivat or placebo. In ACTIVATE-T (open-label, single-arm study), 27 pts (age ≥ 18 yrs) with a confirmed diagnosis of PK deficiency who were regularly transfused (≥ 6 transfusion episodes in the prior yr) were treated with mitapivat. Pts who completed either trial (24 weeks [wks] [ACTIVATE], 40 wks [ACTIVATE-T]) were eligible to continue in the LTE. Erythropoiesis markers included erythropoietin (EPO), erythroferrone, reticulocytes, and soluble transferrin receptor (sTfR). Markers of iron overload included hepcidin, iron, transferrin saturation (TSAT), ferritin, and liver iron concentration (LIC) by magnetic resonance imaging (MRI). In the LTE all pts received mitapivat. Pts from ACTIVATE were categorized into either the mitapivat-to-mitapivat arm (M/M) or the placebo-to-mitapivat arm (P/M). The ACTIVATE-T/LTE analysis includes pts who achieved transfusion-free status in ACTIVATE-T. The ACTIVATE/LTE analysis assessed change in markers from baseline (BL) over time in both study arms. Results: Eighty pts were included in the ACTIVATE/LTE analysis (M/M = 40; P/M = 40). Pts in both arms had abnormal BL erythropoiesis markers consistent with underlying ineffective erythropoiesis, and BL abnormal markers of iron overload. In the M/M arm, mean (SD) EPO, erythroferrone, reticulocytes, and sTfR decreased from BL to Wk 24 of mitapivat treatment by -32.9 IU/L (62.47), -9834.9 ng/L (13081.15), -202.0 10 9/L (246.97), and -56.0 nmol/L (82.57), respectively, while they remained stable or increased in the P/M arm on placebo (Figure). Twenty-four wks after starting mitapivat in the LTE (Wk 48 post BL), pts in the P/M arm had comparable beneficial decreases in mean (SD) EPO, erythroferrone, reticulocytes, and sTfR of -11.6 IU/L (30.74), -9246.1 ng/L (8314.17), -283.7 10 9/L (374.27), and -38.7 nmol/L (48.37), respectively. Improvements in hepcidin, iron, TSAT, and LIC were also observed with mitapivat treatment; ferritin remained stable (Table). Mean (SD) hepcidin increased in the M/M arm at Wk 24 and in the P/M arm 24 wks after starting mitapivat (Wk 48 post BL). At Wk 24, mean (SD) iron and TSAT, and median (Q1, Q3) LIC decreased in the M/M arm, while they increased on placebo. In the P/M arm, iron, TSAT, and LIC decreased 24 wks after starting mitapivat (Wk 48 post BL). Transfusion-free responders from ACTIVATE-T (n = 6) also experienced improvements in markers of erythropoiesis and iron overload in the LTE. Conclusions: In addition to improving Hb, hemolysis, and transfusion burden, data from ACTIVATE, ACTIVATE-T, and the LTE study indicate that activation of PKR with mitapivat improves markers of ineffective erythropoiesis and iron homeostasis in PK deficiency, thereby decreasing iron overload in these pts. Mitapivat has the potential to become the first approved therapy in PK deficiency with beneficial effect on iron overload. Figure 1 Figure 1. Disclosures Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding; Pfizer: Research Funding. Al-Samkari: Amgen: Research Funding; Argenx: Consultancy; Rigel: Consultancy; Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Moderna: Consultancy. Grace: Agios: Research Funding; Dova: Membership on an entity's Board of Directors or advisory committees, Research Funding; Principia: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Barcellini: Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Honoraria; Novartis: Honoraria; Agios: Honoraria, Research Funding. Glenthoej: Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Calgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Research Funding; Novo Nordisk: Honoraria. Judge: Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Xu: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Beynon: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. McGee: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Porter: La Jolla Pharmaceuticals: Honoraria; Protagonism: Honoraria; Agios: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kuo: Celgene: Consultancy; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Bluebird Bio: Consultancy; Apellis: Consultancy.

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