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Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

In β-thalassemia, imbalanced globin synthesis causes reduced red blood cell survival and ineffective erythropoiesis. Suppressed hepcidin levels increase ferroportin-mediated iron transport in enterocytes, causing increased iron absorption and potentially iron overload. Low hepcidin also stimulates ferroportin-mediated iron release from macrophages, increasing transferrin saturation (TSAT), potentially forming non-transferrin-bound iron, which can be toxic. Modulating the hepcidin-ferroportin axis is an attractive strategy to improve ineffective erythropoiesis and limit the potential tissue damage resulting from iron overload. There are no oral β-thalassemia treatments that consistently ameliorate anemia and prevent iron overload.The preclinical and clinical development of vamifeport (VIT-2763), a novel ferroportin inhibitor, was reviewed. PubMed, EMBASE and ClinicalTrials.gov were searched using the search term 'VIT-2763'.Vamifeport is the first oral ferroportin inhibitor in clinical development. In healthy volunteers, vamifeport had comparable safety to placebo, was well tolerated and rapidly decreased iron levels and reduced TSAT, consistent with observations in preclinical models. Data from ongoing/planned Phase II studies are critical to define its potential in β-thalassemia and other conditions associated with iron overabsorption and/or ineffective erythropoiesis. If vamifeport potentially increases hemoglobin and reduces iron-related parameters, it could be a suitable treatment for non-transfusion-dependent and transfusion-dependent β-thalassemia.

Intravenous iron-carbohydrate complex preparations (IVIP) are noninterchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of three IVIP we identify a two-pathway model of transient non-transferrin-bound iron (NTBI) generation following single dose administration. Twenty-eight hypoferremic non-anemic patients randomized to 200 mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside- 1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by high-performance liquid chromatography in combination with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, NTBI and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150 hours (h), except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13 μM at 8 h), rapidly increased with Fe-sucrose (0.8 μM at 2 h, 1.25 μM at 4 h), and delayed for Fe-carboxymaltose (0.57 μM at 24 h). NTBI area-under-curve (AUC) were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIP. We propose two mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIP generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Elevation of endothelial microparticles (EMPs) play an important role in the progression of inflammation-related vascular diseases such as cardiovascular diseases (CVDs). Thai perilla (Perilla frutescens) nutlets are rich in phenolic compounds and flavonoids that exert potent antioxidant and anti-inflammatory effects. We found that the ethyl acetate (EA) and ethanol (Eth) extracts of Thai perilla nutlets contain phenolic compounds such as luteolin, apigenin, chryseoriol and their glycosides, which exhibit antioxidant activity. The goal of the present study was to investigate the effects of the extracts on endothelial activation and EMPs generation in tumour necrosis factor-α (TNF-α)-induced EA.hy926 cells. We found that TNF-α (10 ng/ml) activated EA.hy926 cells and subsequently generated EMPs. Pre-treatment with the extracts significantly attenuated endothelial activation by decreasing the expression of the intracellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Only the Eth extract showed protective effects against overproduction of interleukin-6 (IL-6) in the activated cells. Furthermore, the extracts significantly reduced TNF-α-enhanced EMPs generation in a dose-dependent manner. In conclusion, Thai perilla nutlet extracts, especially the Eth extract, may have potential to protect endothelium against vascular inflammation through the inhibition of endothelial activation and the generation of endothelial microparticles (EMPs).

International audience; Our intention is to demystify the MR quantification of hepatic iron (i.e., the liver iron concentration) and give you a step-by-step approach by answering the most pertinent questions. The following article should be more of a manual or guide for every radiologist than a classic review article, which just summarizes the literature. Furthermore, we provide important background information for professional communication with clinicians. The information regarding the physical background is reduced to a minimum. After reading this article, you should be able to perform adequate MR measurements of the LIC with 1.5-T or 3.0-T scanners. KEY POINTS • MRI is widely accepted as the primary approach to non-invasively determine liver iron concentration (LIC). • This article is a guide for every radiologist to perform adequate MR measurements of the LIC. • When using R2* relaxometry, some points have to be considered to obtain correct measurements-all explained in this article.

Introduction: Luspatercept inhibits select ligands of the TGF-β superfamily implicated in thalassemic erythropoiesis and promotes late-stage erythroid maturation (Suragani RN, et al. Nat Med 2014;20:408-414). This leads to greater red blood cell (RBC) output from thalassemic marrow and reduces transfusion dependence in TDT (Cappellini MD, et al. N Engl J Med 2020;382:1219-1231). The underlying mechanisms for this clinical outcome are not well understood in a syndrome involving significant iron overload and cyclical stimulation of erythropoiesis between transfusions. Here we report novel physiological and clinical insights from a BELIEVE trial (NCT02604433) biomarker analysis, which demonstrate that hepcidin and erythropoietic changes in TDT lead to iron redistribution from macrophages to hepatocytes on luspatercept. Methods: 336 TDT patients ≥ 18 years of age who took part in the BELIEVE study, a multicenter, randomized, double-blind, placebo-controlled phase 3 trial (Cappellini et al. 2020), were randomized 2:1 to receive luspatercept or placebo subcutaneously every 21 days for 48 weeks. This ethically approved study was conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent. Transfusion iron loading rate (ILR) was calculated assuming 1 unit = 200 mg Fe. Liver iron content (LIC) was measured by R2 MRI and R2* MRI, and total body iron stores by Angelucci formula. Serum was assayed for ferritin (SF) nephelometrically (Covance lab), and hepcidin, erythroferrone (ERFE), growth differentiation factor (GDF)15, and transferrin receptor (sTfR1) by ELISA at Intertek (San Diego, CA). Median and interquartile ranges are shown; P value < 0.05 was deemed significant. Results: Within 48 weeks on luspatercept, transfusion iron loading fell by 1.6 g (8 RBC units, ILR difference −0.08 mg Fe/kg/d ± 0.07, range −0.4 to 0.2). Regardless of thalassemic genotype, SF fell by 269.3 ± 963.7 and earliest at 12 weeks by 103.6 ± 690.3 µg/L (both P < 0.0001), with no change on placebo, indicating reduced macrophage iron. However, despite unchanged chelation exposure, no reduction in LIC (5.7 to 6.7 mg/g dw) or calculated body iron stores (3.6 to 4.2 g, not significant) occurred on luspatercept, even though saved iron loading from transfusion was 44% (1.6 g/3.6 g) of baseline body iron stores. On luspatercept, but not placebo, hepcidin fell by 53%, while erythropoietin (EPO), ERFE, GDF15, sTfR1, and reticulocytes rose by 93%, 51%, 59%, 66%, and 112%, respectively (all P < 0.0001). 71% (120/169) patients with and 47% without (17/36) ILR reduction had negative SF trends (P < 0.0001). In patients with SF reduction on luspatercept, bilirubin and LDH rose 50% and 67% (P < 0.0001) indicating increased RBC hemolysis from residual (effective and ineffective) erythropoiesis. ILR change correlated with changes in EPO, hepcidin, sTfR1, and bilirubin, but not with changes in ERFE, GDF15, reticulocytes, or LDH (Figure A). Hepcidin reduction was related to LIC increase post splenectomy (Figure B), suggesting a role for the spleen in preventing hemolytic iron redistribution to the liver. Decrease in SF thus associates with erythroid iron incorporation (sTfR1), hence RBC production that reduces transfusion needs (falling ILR) but enhances hemolytic rerouting of iron to the liver. In a mixed-effect linear regression analysis, transfusion, LIC, baseline SF, time, and treatment predicted SF changes in a benchmark model. We found high baseline LIC lessens the SF outcome, and hepcidin, EPO, and bilirubin jointly explained 66% of the treatment effect of luspatercept on SF. Conclusions: Luspatercept-increased ERFE, likely as a result of increased production or higher frequency of late-stage erythroblasts, partially reduces hepcidin production. Lower hepcidin mobilizes iron stores to facilitate bulk hemoglobinization, erythropoietic response, and transfusion burden reduction. Luspatercept leads to SF reduction that marks hepcidin-dependent iron egress from macrophage compartment to plasma. This relocated iron, variably utilized by thalassemic erythron, refluxes back to plasma for hepatocyte and extrahepatic iron uptake, or as heme iron shunts into the liver through hemolytic pathways from intramarrow ineffective erythropoiesis or peripheral breakdown of newly made thalassemic RBC. Macrophage to hepatocyte iron redistribution in TDT appears to be a mechanism of luspatercept. Figure 1 Figure 1. Disclosures Garbowski: Imara: Consultancy; Vifor: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ugidos: Bristol Myers Squibb: Current Employment. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Suragani: Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Shetty: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Vodala: BMS: Current Employment, Other: stock options. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Schwickart: BMS (Celgene): Current equity holder in publicly-traded company, Ended employment in the past 24 months, Other, Patents & Royalties; Exelixis: Current Employment, Current equity holder in publicly-traded company. Porter: 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; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla Pharmaceuticals: Honoraria; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: The rhesus (Rh) and Kell blood group systems are the most common of over 50 different antigens capable of causing maternal red blood cell (RBC) alloimmunization and severe fetal hemolytic disease. Anti-K and anti-D are responsible for a significant proportion of fetal anemia requiring intrauterine transfusion (IUT). Whilst IUT of packed RBCs improves neonatal survival and morbidity, clinical prognostic indicators are lacking. Our primary objective was to identify predictors of adverse outcome. Methods: We conducted a retrospective single-center study at Mount Sinai Hospital (MSH), Toronto, Canada. All pregnant patients alloimmunized with anti-K and anti-D as a single antibody, between 1991 and 2018 were included. Data were obtained from patient medical records, ultrasound reports and information from the transfusion medicine laboratory. Data included maternal demographics, antibody titers, pregnancy history, number of IUTs, hemoglobin (HB) concentration at the beginning and end of all IUTs. Neonatal outcomes included survival, mode of delivery, gestational age at delivery, birth weight, HB at birth and need for neonatal transfusion, phototherapy or intravenous immunoglobulins (IVIG). Our primary outcome was the composite outcome of stillbirth or neonatal death (SB/NND). We also constructed a secondary outcome consisting of top-up neonatal transfusion, exchange transfusion, phototherapy, or use of IVIG. Medians and interquartile ranges (IQR) or mean±SD were used as summary statistics and compared by Mann-Whitney or t-test; p Results: 116 women with 128 pregnancies and 425 IUTs with anti-K or anti-D as a single antibody were identified. Median maternal age was 31 years (27.0-35.0) for anti-K and 32 years (23.6-40.6) for anti-D. The gestational age at 1st IUT differed significantly between anti-K and anti-D (24.3 vs 28.7 weeks respectively, p=0.004). Women with anti-K antibodies required more IUTs than women with anti-D (3.84 vs 3.12 IUTs, p=0.036) and HB at 1st IUT was significantly lower in the anti-K group (5.10 vs 7.05 g/dL, p=0.001) (Table 1). Following initiation of IUT, the time from 1st IUT to delivery was 69.6 days in the anti-K group and 54.6 in the anti-D group (p=0.06). The daily decrease of HB between 1st and 2nd IUT (as a marker of disease severity), development of fetal hydrops and severe preterm birth did not differ significantly between the two groups. Mean gestation age at delivery was 35.0 weeks in the anti-K and 36.0 weeks in the anti-D group (p=0.28), with 87.1% and 93.9% survival (p=0.37), respectively. The proportion of neonates requiring phototherapy, IVIG and exchange/top-up transfusion was comparable across the two antibody groups (Table 1). Regression analysis showed that delivery occurred sooner if HB dropped more rapidly between the first two IUTs (p=0.01). Each additional transfusion gained on average 22.5 days in utero (Table 2). In multivariable analysis, gestational age at 1st IUT was the only predictor of a SB/NND outcome (adjusted OR 0.79 [95%CI 0.67-0.93]; p=0.006). With 1st IUT at 23 weeks, the risk of SB/NND was 8%, but only 2.5% at 28 weeks and Conclusion: The earlier in gestation that IUTs are implemented, the higher the odds of a SB/NND; however the later the gestation at delivery, the greater the odds of the neonate requiring blood products post-partum. The greater the HB drop between the 1st and 2nd IUT, the shorter the 'time between the first IUT and delivery', which increases the odds of a SB/NND outcome. Disclosures Garbowski: Vifor Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Imara: Consultancy. Shehata:Ferring: Honoraria.

β-thalassemia, a hereditary blood disorder caused by defective synthesis of hemoglobin β globin chains, leads to ineffective erythropoiesis and chronic anemia that may require blood transfusions. Sotatercept (ACE-011) acts as a ligand trap to inhibit negative regulators of late-stage erythropoiesis in the transforming growth factor β superfamily, correcting ineffective erythropoiesis. In this phase II, open-label, dose-finding study, 16 patients with transfusion-dependent β -thalassemia and 30 patients with non-transfusion-dependent β-thalassemia were enrolled at seven centers in four countries between November 2012 and November 2014. Patients were treated with sotatercept at doses of 0.1, 0.3, 0.5, 0.75, or 1.0 mg/kg to determine a safe and effective dose. Doses were administered by subcutaneous injection every 3 weeks. Patients were treated for ≤22 months. Response was assessed as a ≥20% reduction in transfusion burden sustained for 24 weeks in transfusion-dependent β-thalassemia patients, and an increase in hemoglobin level of ≥1.0 g/dL sustained for 12 weeks in non-transfusion-dependent β-thalassemia patients. Sotatercept was well tolerated. After a median treatment duration of 14.4 months (range 0.6-35.9), no severe life-threatening adverse events were observed. Thirteen percent of patients reported serious but manageable adverse events. The active dose of sotatercept was ≥0.3 mg/kg for patients with non-transfusion-dependent β-thalassemia and ≥0.5 mg/kg for those with transfusion-dependent β-thalassemia. Of 30 non-transfusion-dependent β-thalassemia patients treated with ≥0.1 mg/kg sotatercept, 18 (60%) achieved a mean hemoglobin increase ≥1.0 g/dL, and 11 (37%) an increase ≥1.5 g/dL, sustained for ≥12 weeks. Four (100%) transfusion-dependent β-thalassemia patients treated with 1.0 mg/kg sotatercept achieved a transfusion-burden reduction of ≥20%. Sotatercept was effective and well tolerated in patients with β-thalassemia. Most patients with non-transfusion-dependent β-thalassemia treated with higher doses achieved sustained increases in hemoglobin level. Transfusion-dependent β-thalassemia patients treated with higher doses of sotatercept achieved notable reductions in transfusion requirements. This trial was registered at ClinicalTrials.gov with the number NCT01571635.

Aims Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE−/− FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). Methods and results Compared with normo-ferremic ApoE−/− mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE−/− FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE−/− FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. Conclusion Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

The relationship between blood transfusion intensity, chelatable iron pools, and extrahepatic iron distribution is described in thalassemia. Risk factors for cardiosiderosis are discussed with particular reference to the balance of transfusional iron loading rate and transferrin-iron utilization rate as marked by plasma levels of soluble transferrin receptors. Low transfusion regimens increase residual erythropoiesis allowing for apotransferrin-dependent clearance of non-transferrin-bound iron species otherwise destined for myocardium. The impact of transfusion rates on chelation dosing required for iron balance is also shown.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log β(2)=35). Clinically achievable concentrations (1 μM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators.

Introduction Elevated serum ferritin (hyperferritinemia, HF) can result from increased iron load, increased inflammation or from liver damage. In the most common forms of hereditary hemochromatosis (HH), elevated ferritin is usually seen with elevated transferrin saturations, [1,2]. In patients with HF but without raised liver iron concentration (LIC, mg/g dry wt [dw]), increased liver fat (hepatic steatosis, HS), either in isolation or as part of a metabolic syndrome, needs to be considered. HS is important to identify as it may progress to chronic liver damage and is potentially reversible with lifestyle interventions. Hepatic fat can now be quantified with magnetic resonance imaging (Hepafat-Scan®)[3]. Here, we evaluate the usefulness of this method in the diagnosis and subsequent management of patients referred with HF. Methods A total of 132 patients (median age 48 years, range 21-86 ; female: male ratio 1:2.5) referred for investigation of HF and who underwent estimation of liver iron concentration (LIC) by R2-MRI (FerriScan®) over a four-year period (January 2015 - December 2018) are included in this analysis. Data on patient demographics, presenting serum ferritin (SF) and transferrin saturation were obtained. Genetic testing for C282Y and H63D was also performed. Genetic testing for rarer forms of haemochromatosis was confined to patients with unexplained raised LIC. Patients with iron overload secondary to chronic iron ingestion or blood transfusion were excluded from this analysis. Results Patients were subdivided by genetic diagnosis: C282Y homozygote (26, 19.7%), C282Y/H63D (18, 13.6%), H63D homozygote (10, 7.6%), C282Y heterozygote (8, 6.1%), H63D heterozygote (18, 13.6%), autosomal dominant ferroportin disease (4, 3%) and negative HFE gene testing (48, 36.4%). There was no statistical difference between mean presenting SF (p= 0.307) between the different groups. C282Y homozygotes, and those with autosomal ferroportin disease had a significantly higher LIC than others (p After genetic testing and LIC quantification, 32 patients underwent a HepaFat-Scan®. Median age was 63 years, range 23 - 74 years; female: male ratio 1:3.5. 54.5% had a volumetric liver fat fraction (VLFF) > 5%. 7.4% of patients, who did not undergo Hepafat-Scan®, had HS based on sonographic findings. Patients with definitive evidence of HS as defined by VLFF >5% had mean LIC of 1.36mg/g dw, and a higher mean presenting SF of 926 µg/L than those with HS In patients with LIC ≤3mg/g dw (n= 91, mean 1.6), mean presenting SF was 728 mg/ml. 28 of these patients went onto have a Hepafat-Scan®, 16 of which (57.1%) had confirmed steatosis with VLFF >5%. In patients with LIC >3mg/g dw (n= 41, mean LIC 6.9), mean presenting SF was significantly higher at 1056 mg/ml (p=0.021). 4 patients with LIC >3mg/g dw also had a Hepafat-Scan®, with 1 patient having VLFF >5%. Patients with confirmed steatosis were referred for specialist hepatology consultation. Conclusion In patients referred for investigation of HF, demonstrable steatosis (VLFF >5%) by Hepafat-Scan® is remarkably high (13.3% of all referred patients and 54% of patients who received Hepafat-Scan®). The Hepafat-Scan® data show that HS is generally confined to patients with low LIC (≤3mg/g dw) (Figure 1) reflecting the selective decision pathway adopted of excluding hepatic siderosis before proceeding to Hepafat-Scan®. SF is significantly higher in non-iron overloaded patients with HS (VLFF >5%) than those without HS (Figure 1). Estimation of HS should be considered in the diagnostic pathway of patients referred for investigation of HF, particularly when LIC values are not found to be meaningfully increased. References 1. Cullis JO et al. Investigation and management of a raised serum ferritin. British Journal of Haematology 2018; 181:331-340 2. St Pierre TG et al. Noninvasive measurement and imaging of liver iron concentration using proton magnetic resonance. Blood. 2005; 105(2):855-61 3. St Pierre TG et al. Stereological Analysis of Liver Biopsy Histology Sections as a Reference Standard for Validating Non-Invasive Liver Fat Fraction Measurement by MRI. PLoS ONE August 2016; 11(8):e0160789 Disclosures Eleftheriou: Novartis: Honoraria. Garbowski:Imara: Consultancy; Vifor Pharma: Consultancy. St. Pierre:Resonance Health Ltd: Consultancy, Other: Share ownership. Porter:Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria; Bluebird bio: Consultancy, Honoraria. Drasar:Novartis: Honoraria.

Background: Kell and Rhesus (Rh) maternal red blood cell (RBC) alloimmunization are the most common causes of severe fetal haemolytic disease. Widespread use of anti-D immune globulin has dramatically reduced the incidence of Rh(D) alloimmunization, leaving K alloimmunization responsible for a significant proportion of cases of fetal anemia requiring intrauterine transfusion ( ). K antigens are expressed on fetal erythrocytes at 10-11 weeks and k antigens at 6-7 weeks gestation (Tovey et al, 1986). In alloimmunized women, erythroid specific antibodies traverse the placenta, causing immune destruction of fetal erythroid cells leading to progressive haemolytic anaemia. The mechanism of fetal anaemia in K alloimmunization differs somewhat from that in Rh-D, in that anti-K antibodies cause suppression of fetal erythropoiesis (Gariod et al, 2004; Weiner et al, 1996). Whilst IUT of RBCs has improved fetal and neonatal survival, important information such as the critical anti-K titres to guide appropriate timing and frequency of IUT, is somewhat conflicting. Currently anti-K alloimmunized pregnancies do not have a standardized protocol for titer monitoring throughout pregnancy and, once alloimmunized, patients are usually referred to a regional fetal center for close ultrasound (US) surveillance. Our primary objective is to determine from a retrospective analysis of our population whether there is a critical anti-K titer that should trigger intensive US monitoring or intervention and to investigate the rate of progression of fetal anemia following IUTs. Methods: This is a retrospective single-center study at Mount Sinai Hospital (MSH), Toronto, Canada, of all pregnant patients with anti-K as the primary alloimmunizing antibody, between 1991 and 2018. MSH is the largest fetal medicine center in Canada and the largest referral center for IUTs. Ethical approval was granted by the Research Ethics Board (REB # 12-0113-C). Data were obtained from a database of patient medical records, US reports and the transfusion medicine laboratory, including maternal demographics, pregnancy history, presence of other alloantibodies and hemoglobin concentration before and after all IUTs. Neonatal outcomes included survival, mode of delivery, gestational age (GA) at delivery, birth weight and need for neonatal exchange transfusion, phototherapy or IVIG. Data were analyzed using GraphPad Prism 6 and linear correlations are expressed as a p-value. Results: Thirty-eight women underwent 163 IUTs in 44 pregnancies where K was the predominant antibody. Two patients in whom anti-K was a secondary antibody were excluded. In 5 of these pregnancies, 2 had a total of three alloantibodies and 5 had 2 alloantibodies each. The median maternal age was 31 (29 - 35) years. Four women had a history of intrauterine fetal death (IUFD) and 9 of neonatal haemolytic disease. The median GA at 1st IUT was 24.2 weeks (14.9-34.7), and there was a median of 4 IUTs per patient. There were seven cases of hydrops fetalis. The number of IUTs a patient received throughout pregnancy was correlated directly with the anti-K titer (Figure 1). Every 4-fold dilution resulted in a further increase in the IUT number by 2.2 above the mean of 2.5 at a titer of 1:32 (p=0.0137). Figure 2 illustrates the correlation between the GA at 1st IUT and antibody titer. IUTs were required at earlier GAs if the titers were higher. Following a 1:32 titer, every 2-fold titer increase reduced the mean gestational age at 1st IUT by 2 weeks (p Conclusion: Our data support a critical anti-K titer of 1:32 and support a role for anti-K titer monitoring as a predictor of disease severity, counselling women appropriately and establishing a balance between paternal K antigen typing, US middle cerebral artery peak systolic velocity monitoring of the fetus and IUTs. Disclosures Garbowski: Vifor Pharma: Consultancy; Imara: Consultancy.

Background The prodrug-like intravenous iron-carbohydrate complex preparations (IVIPs) should not be considered interchangeable but with due regard to their different pharmacodynamics (PD) which is determined by the semi-crystalline iron core and carbohydrate shell structures (Bhandari et al, 2018). The pharmacokinetics (PK) of IVIPs influences PD profiles and thus efficacy and safety. Here we examine the complex PK/PD relationships with 3 IVIPs, for the first time identifying a 2-compartment model of transient NTBI generation following a single dose administration. Patients and Methods 28 hypoferremic patients randomized (n=8/arm) to 200mg (iron) of ferric carboxymaltose (FCM), iron sucrose (IS) and iron isomaltoside 1000 (IIM) or placebo (n=4), were followed up for 2 wks on an exploratory PK/PD study. Serum samples (0-312h) were analyzed for total serum iron (Fe), plasma IVIP-iron (IVIP-Fe), transferrin (Tf)-bound iron (TBI) by inductively coupled plasma mass spectrometry, Tf saturation (TSAT) by Tf ELISA and a tripyridyl triazine assay, serum ferritin (SF) by turbidimetry, and non-TBI (NTBI) and hepcidin (hepc) as published (Garbowski et al, Transl Med 2016), (Bansal et al, Rapid Commun Mass Spectrom 2010). A p value Results Pts were hypoferremic but non-anemic at baseline: SF 12.4±7.4µg/L, TSAT 18.6±8.5%, hemoglobin 128±8g/L. IVIP-dependent rises in Fe, IVIP-Fe, TSAT, SF, hepc, and NTBI returned to baseline in 2 wks or sooner, except for SF and TSAT. Large differences in the Fe AUCs on study, explainable by large differences in the IVIP-Fe AUCs, were consistent with the known relative differences of plasma half-lives (t1/2) of the IVIPs compared. IVIP-Fe at 4h was highest for IIM and smallest for IS, as was their AUC. With IIM, TSAT reached 80% at 16h, with IS 100% by 4h, and with FCM 98% at 24h; but AUC differences were small. NTBI was low with IIM (0.13µM at 8h), rapidly increased and high with IS (0.8µM at 2h, 1.25µM at 4h), and delayed for FCM (0.58µM at 24h). NTBI appeared after near- (for IIM) or full saturation of Tf but disappeared when TSAT fell Discussion We propose 2 mechanisms for the observed kinetic NTBI profiles. For IS these have 2 distinct components: a rapid onset and an early peak (2-4h), then decay and a secondary peak/shoulder (16h), disappearing at 36h (Fig). The 1st peak reflects the rapid release of labile iron in plasma directly from circulating IVIPs, occurring 4 t1/2, when >90% of IS entered macrophages (SF half-maximal), the 16h peak likely represents iron re-released from macrophages (indirect NTBI). For IIM with long t1/2, the small NTBI peak at 8h thus represents direct NTBI, while the indirect release at 24h occurs at a rate where Tf does not fully saturate, with NTBI being consequently virtually absent (Fig). FCM first follows the IIM profile with minimal generation of direct NTBI, but displays a starkly different behavior reflecting indirect iron release shown as a strong NTBI peak at 24h, when ~4 plasma t1/2 have passed (Fig). This difference between IIM and FCM is accounted for by the difference in t1/2 of FCM and IIM i.e. by the amount of polymer having been taken up by macrophages (so only 6% of FCM and 30% of IIM still circulates). We conclude that, following IVIP administration, NTBI can arise by 2 mechanisms (direct early release and indirect later release). Different IVIPs generate different NTBI and serum ferritin signatures that are broadly PK-dependent, and may have a bearing both on the iron bio-availability and safety. Longer-term studies with common doses (1g for FCM and IIM) are warranted to link the initial NTBI exposure to subsequent safety and response parameters. Disclosures Garbowski: Vifor Pharma: Consultancy; Imara: Consultancy. Porter:Celgene: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria; Bluebird bio: Consultancy, Honoraria. Burckhardt:Vifor Pharma (previously): Employment. Hider:Vifor Pharma: Research Funding; Renapharma AB: Research Funding.

The aims of this review are to highlight the mechanisms and consequences of iron distribution that are most relevant to transfused sickle cell disease (SCD) patients and to address the particular challenges in the monitoring and treatment of iron overload. In contrast to many inherited anemias, in SCD, iron overload does not occur without blood transfusion. The rate of iron loading in SCD depends on the blood transfusion regime: with simple hypertransfusion regimes, rates approximate to thalassemia major, but iron loading can be minimal with automated erythrocyte apheresis. The consequences of transfusional iron overload largely reflect the distribution of storage iron. In SCD, a lower proportion of transfused iron distributes extrahepatically and occurs later than in thalassemia major, so complications of iron overload to the heart and endocrine system are less common. We discuss the mechanisms by which these differences may be mediated. Treatment with iron chelation and monitoring of transfusional iron overload in SCD aim principally at controlling liver iron, thereby reducing the risk of cirrhosis and hepatocellular carcinoma. Monitoring of liver iron concentration pretreatment and in response to chelation can be estimated using serum ferritin, but noninvasive measurement of liver iron concentration using validated and widely available MRI techniques reduces the risk of under- or overtreatment. The optimal use of chelation regimes to achieve these goals is described.

Background Chronic iron overload is an important complication of long-term blood transfusions for severe beta-thalassemias, sickle cell disease and other blood disorders. Iron chelation therapy (ICT) is required to bind and excrete excess iron, which would otherwise accumulate and lead to organ damage or failure. Deferasirox is a once-daily, orally administered ICT approved for the treatment of chronic iron overload due to frequent blood transfusions in patients with beta-thalassemia major and other anaemias. A film-coated tablet (FCT) formulation was launched in the UK in 2016 and replaced the dispersible tablet (DT) formulation. In the context of a randomised clinical trial, the FCT formulation showed greater adherence and patient satisfaction, better palatability and fewer tolerability concerns than the DT. Furthermore, treatment compliance by pill count was higher with FCT (92.9%) than with DT (85.3%) (Taher et al, 2017). Little information exists however about compliance, efficacy and tolerability outside of a clinical trial setting. Objectives We wished to assess in a 'real world' situation, the effects of switching the deferasirox formulation from DT to FCT on patient adherence to ICT, iron overload and renal function. Methods Patients receiving ICT with deferasirox who were switched from the DT to FCT formulations were followed over a 12-month period and results audited using hospital dispensing and biochemistry records. The date of the first FCT prescription was defined as baseline. The initial daily dose used for switching from DT to FCT was as per manufacturer's recommendations: 70% of the DT daily dose. The impact on iron overload was assessed by comparing serum ferritin levels at 3, 6, 9 and 12 months post-switch with baseline values. The impact on renal function was assessed by comparing serum creatinine levels at 3, 6, 9 and 12 months post-switch with baseline values as well as the number of serum creatinine increases of 30% or greater above baseline. The changes in serum ferritin and creatinine were subsequently analysed by paired t-test. The Proportion of Days Covered (PDC) was calculated as a measure of patient adherence to ICT in the 12 months before and after switching formulations. Results 74 patients switched from deferasirox DT to FCT with the following diagnoses: beta-thalassemia (n = 45), sickle-cell disease (9), thalassemia-intermedia (6), HbE-thalassemia (5), other transfusion-dependent disorders (9). The median age was 36 (range: 1-78yo), mean baseline serum ferritin was 2767µg/L (range: 412-8742), mean baseline creatinine was 64.5 umol/L (range: 17-140) and the median prescribed daily dose of DT was 1250mg (range: 62.5 - 3500). The mean PDC in the 12 months prior to switching formulations was 0.80 (range: 0.31-1.00). This increased to 0.91 (range: 0.21-1.00) in the 12 months following the switch to FCT. The median prescribed daily dose of FCT was 900mg (range: 90 - 2520) The mean changes in ferritin and creatinine at 3, 6, 9 and 12 months post-switch are shown in the table. 6 out of 74 patients (8%) had a creatinine increase of >30% from baseline whilst receiving the FCT, occurring after an average of 120 days (range: 30-260). All 6 patients were managed by dose adjustment of FCT and creatinine returned to the normal range in 5 out of 6 cases. Conclusions The switch from deferasirox DT to FCT resulted in improved patient adherence to chelation, a reduction in mean serum ferritin and a modest rise in mean serum creatinine. Some patients showed a reversible rise in creatinine from baseline. The median daily dose of FCT prescribed was 72% of the DT formulation, approximately equivalent according to the known bioavailability of the different preparations and suggesting that improvements in serum ferritin were due to the more consistent daily administration of the FCT rather than an increased daily deferasirox dose. We suggest that when the fall in ferritin is abrupt and/or to levels Reference Taher, A. T., et al. (2017). "New film-coated tablet formulation of deferasirox is well tolerated in patients with thalassemia or lower-risk MDS: Results of the randomized, phase II ECLIPSE study." American journal of hematology 92(5): 420-428. Table Table. Disclosures Garbowski: Vifor: Consultancy. Porter:Novartis: Consultancy; Cerus: Honoraria; Agios: Honoraria.

Background The hemoglobin (Hb) response to the activin receptor type IIA ligand trap ACE-011 (Sotatercept) in non-transfusion-dependent thalassemia (NTDT), and the transfusion requirement response in TDT are described, but the mechanisms of action, clinical predictors and markers of response, are unclear. In principle, ACE-011 may act on early and/or late erythroblasts to decrease ineffective erythropoiesis (IE), both in healthy subjects and in thalassemia. As erythropoiesis intimately links to iron metabolism, changes in markers of iron metabolism relative to those of erythropoiesis may inform the mechanisms and developmental stage at which ACE-011 acts. Methods Markers of IE and iron metabolism in 46 thalassemia patients (30 NTDT, 16 TDT) were taken before and during a median follow up of 722.5 days, (IQR 830.5, range 152-1427) of escalating doses of ACE-011 (3-weekly 0.1 to 1.0 mg/kg s.c. injections), depending on the protocol, as part of the approved study (Cappellini, et al. 2018 under review). Markers of erythropoiesis included Hb, Reticulocytes (Ret), soluble transferrin receptor 1 (sTfR), growth differentiation factor 11 and 15 (GDF11, GDF15), erythroferrone (ERFE). Markers of iron metabolism included plasma hepcidin, serum ferritin (SF), transferrin saturation (TSAT), and non-transferrin-bound iron (NTBI). Data were analyzed using longitudinal multilevel model for change (LMMC) on STATA (Version 14) as generalized linear mixed model with random and fixed effects to account for repeated measures and highly complex temporal data structure. Final LMMCs were built to explain the change in Hb from baseline and the behavior of key biomarkers. Independent variables were entered into the models based on the study design (predictors from design) and theoretical background (predictors of interest and control variables). P value Results Predictors of response. In NTDT, Hb response associated positively with dose and duration of exposure (both at p Biomarker changes with time In NTDT, significant changes with time on study were increases in Hb, sTfR, ERFE and Ret and decreases in hepcidin, bilirubin, and NTBI. GDF15 showed no change. In TDT, GDF15, sTfR and ERFE increased, whereas hepcidin decreased. The Hb change was insignificant in TDT (as expected per protocol) and was dose-independent, however the mean 41% reduction in transfusion iron load rate (ILR) was dose dependent, implying an equivalent net production of Hb in TDT (Table 1). Other significant relationships Absolute hepcidin level in NTDT and TDT was negatively predicted by ERFE (p Interpretation and conclusions NTDT patients allow a cleaner interpretation of biomarker changes as these are confounded in TDT by increased bone marrow stress from less transfusion. In NTDT, sTfR and ERFE (total erythropoiesis) increase on study while GDF15 (IE) and EPO do not. Thus Hb gain may result from increased effectiveness of late stage erythropoiesis (sTfR+ and ERFE+) possibly from decreased apoptosis and more rapid maturation (Carrancio et al, BJH 2014) in this compartment. We speculate that increased survival of those erythroid progenitor cells expressing TfR (and hence sTfR) and ERFE (hence increased ERFE) also explains the observed hepcidin reduction. Despite exposure to lower hepcidin, there is no iron loading: NTBI falls against stable SF in NTDT, consistent with shunting of iron into the sink of effective erythropoiesis. The reduction in GDF11 in vivo, not previously reported for ACE-011, is also consistent with increased apoptosis of the early progenitor pool and with improved erythroblast differentiation relative to proliferation as suggested in murine β-thalassemia treated with ACE-011 (Dussiot, et al. Nat Med 2014). Disclosures Garbowski: Vifor: Consultancy. Hermine:Erythec: Research Funding; AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene Corporation: Research Funding; Hybrigenics: Research Funding; Novartis: Research Funding. Cappellini:Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Vifor: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Origa:Bluebird Bio: Consultancy; Novartis: Honoraria; Cerus Corporation: Research Funding; Apopharma: Honoraria. Forni:Celgene: Research Funding; Novartis: Other: travel expenses, Research Funding; Shire: Research Funding; Roche: Consultancy; Apopharma: Other: DSM Board. Voskaridou:Acceleron: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding. Galactéros:Addmedica: Other: grant; Novartis: Other: grant. Taher:Novartis: Consultancy, Honoraria, Research Funding; La Jolla Pharmaceutical: Research Funding; Protagonist Therapeutics: Consultancy; Celgene Corp.: Research Funding; Ionis Pharmaceuticals: Consultancy. Ribeil:bluebird bio: Consultancy; Vitalaire: Other: grant; Addmedica: Other: grant; Cydan: Consultancy; Novartis: Consultancy. Laadem:Celgene: Employment, Equity Ownership. Miteva:Celgene Corporation: Employment, Other: grants. Zou:Celgene Corporation: Employment, Equity Ownership. Zinger:Celgene Corporation: Employment. Schwickart:Celgene Corporation: Employment, Equity Ownership. Sung:Celgene Corporation: Employment, Equity Ownership. Porter:Novartis: Consultancy; Cerus: Honoraria; Agios: Honoraria.

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P

© 2015 John Wiley & Sons Ltd. Iron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as 'shuttle' and 'sink' molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine-based detection system for intracellular iron removal from the human hepatocyte cell line (HuH-7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a half-maximum effect can be reduced by 3·8-fold when only 1 μmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron-chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second 'sink' chelator.

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

The pathophysiologic consequences of transfusional iron overload (TIO) as well as the benefits of iron chelation therapy are best described in thalassemia major, although TIO is increasingly seen in other clinical settings. These consequences broadly reflect the levels and distribution of excess storage iron in the heart, endocrine tissues, and liver. TIO also increases the risk of infection, due to increased availability of labile iron to microorganisms. The authors suggest that extrahepatic iron distribution, and hence toxicity, is influenced by balance between generation of nontransferrin-bound iron from red cell catabolism and the utilization of transferrin iron by the erythron.

Background There is a need to standardise non-invasive measurements of liver iron concentrations (LIC) so clear inferences can be drawn about body iron levels that are associated with hepatic and extra-hepatic complications of iron overload. Since the first demonstration of an inverse relationship between biopsy LIC and liver magnetic resonance (MR) using a proof-of-concept T2* sequence, MR technology has advanced dramatically with a shorter minimum echo-time, closer inter-echo spacing and constant repetition time. These important advances allow more accurate calculation of liver T2* especially in patients with high LIC. Methods Here, we used an optimised liver T2* sequence calibrated against 50 liver biopsy samples on 25 patients with transfusional haemosiderosis using ordinary least squares linear regression, and assessed the method reproducibility in 96 scans over an LIC range up to 42 mg/g dry weight (dw) using Bland-Altman plots. Using mixed model linear regression we compared the new T2*-LIC with R2-LIC (Ferriscan) on 92 scans in 54 patients with transfusional haemosiderosis and examined method agreement using Bland-Altman approach. Results Strong linear correlation between ln(T2*) and ln(LIC) led to the calibration equation LIC = 31.94(T2*)-1.014. This yielded LIC values approximately 2.2 times higher than the proof-of-concept T2* method. Comparing this new T2*-LIC with the R2-LIC (Ferriscan) technique in 92 scans, we observed a close relationship between the two methods for values up to 10 mg/g dw, however the method agreement was poor. Conclusions New calibration of T2* against liver biopsy estimates LIC in a reproducible way, correcting the proof-of-concept calibration by 2.2 times. Due to poor agreement, both methods should be used separately to diagnose or rule out liver iron overload in patients with increased ferritin.

Introduction In transfusion dependent thalassemias (TDT), plasma risk factors predicting myocardial hemosiderosis (MH) are needed. High labile plasma iron (LPI) and non-transferrin bound iron (NTBI, in its various species) may increase MH risk, so could be blood biomarkers, but the clinical utility seems unclear (Garbowski et al. 2015 Blood). This is partly due to the characteristics of the LPI assay and the two NTBI assays: the NTA-assay and the bead assay. Firstly, the former two assays are inhibited in the presence of iron free ligands (Aydinok et al. 2012 Haematologica; Walter et al. 2008 Haematologica), the NTBI bead assay less so (Ma et al. 2014 Biochem J; Garbowski et al. 2016 Transl Res). Secondly, the assays capture iron-chelate complexes to a variable extent: the NTA assay detects both deferasirox (DFX2Fe) and deferiprone complexes (DFP3Fe), the NTBI bead assay only detects the latter (Garbowski et al. 2016 Transl Res) - meaning that clinical utility is confounded without with-holding chelator (impractical) (Aydinok et al. 2012 Haematologica). However, sampling on the day of chelation could be useful: iron-chelator complexes could reflect chelatable body iron (Aydinok et al. 2012 Haematologica), another potential MH risk biomarker. We therefore determined whether using the assays on the day of chelation, capturing NTBI and iron-chelator complexes together could identify patients with the greatest MH risk. Patients and Methods 29 TDT patients included balanced proportions with and without MH (measured by CMR). Blood samples were taken on the day of CMR, without withholding chelation. All chelation modalities were accepted: DFX (n=13), DFO (n=7), DFP (n=1), DFO+DFP (n=4), DFO+DFX (n=1), DFX+DFP (n=1) or no chelation (n=2). NTBI was determined by both the NTA and bead-NTBI method (Ma et al. 2014 Biochem J). LPI was measured as described (Esposito et al. 2003 Blood). Regression of MH against NTBI was analysed in patients receiving same day chelation (n=16) or last chelation prior to this (n=13). Results 14/29 had MH (T2* Discussion These findings suggest a relationship between MH and chelatable iron pools. These are detected as plasma iron complexes by the NTA-NTBI method, which has not been previously reported. This is only clear where chelation is given on the day of blood sampling and reflects iron chelated from both plasma and hepatic iron turnover. This holds for patients receiving DFP and DFX but not DFO, where iron complexes are not visible to the NTA assay. This relationship is also not seen with the bead or LPI assays because iron complexes are less uniformly detected. These findings implicate the magnitude of chelatable iron pools to MH risk. Conclusion MH in TDT correlates with plasma levels of chelate complexes of DFP and DFX, detected by the NTA-NTBI assay, measured on the day of chelator administration suggesting that blood iron-chelator complexes could be a biomarker for myocardial iron hemosiderosis. Disclosures Porter: Novartis: Consultancy, Honoraria, Research Funding; Celegene: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Bluebird Bio: Consultancy.

Introduction Myocardial iron loading frequently complicates TDT but factors affecting the highly variable risk are not fully understood and current available predictors do not explain this variability. Chronic blood transfusion in TDT, by correcting anemia, is life saving but also aims to reduce ineffective erythropoiesis (IE) and its complications. Such transfusion will also decrease utilisation of transferrin (Tf) iron by the erythron and increase Tf saturation (TfSat) (Porter et al, Brit J Haematol 2014). Here we examine for the first time, how variable levels of residual erythropoiesis in TDT (marked by soluble transferrin receptors, sTfR), and the consequent presence of ApoTf, affect myocardial iron loading. We then examine how the difference between the transfusional iron loading rate (ILR) and Tf iron utilisation (calculated from sTfR levels) impact on myocardial iron loading. We finally test the hypotheses generated by these findings, using in vitro models of cellular uptake of NTBI and its redox active component LPI, the main conduits of myocardial iron uptake, in the presence and absence of ApoTf. Methods A cohort of 73 TDT patients on deferasirox (DFX) had cardiac MRI and blood biomarkers measured at baseline and at 1.5 years later, over which time the ILR was calculated. Patients were divided into those with cardiac iron (24 cases, cardiac T2*50s-1) and those without (59 controls, T2*>20ms, R2* Results and Discussion Tf iron utilisation and myocardial iron in TDT. Of all biomarkers analysed, sTfR was most significantly associated with heart iron (p3x higher in controls than cases. NTBI, LPI, TfSat and ILR were not significantly different in cases and controls, but LIC and ferritin were higher in cases (p=0.003 and 0.002). With sTfR expressed as Tf iron utilisation rate (or erythroid Tf uptake rate, ETUR) after Beguin et al, Blood 1993 and compared to ILR, myocardial iron was present only when the net ILR (ILR-ETUR) exceeded 0.21 mg/kg/d (p Effect of ApoTf and Fe:cit ratio on NTBI uptake into cultured cardiomyocytes. ApoTf or TfSat Conclusion Taken together these data suggest that the risk of myocardial siderosis in TDT is critically dependent on the degree of residual erythropoiesis and the net balance between the transfusional iron delivery (ILR) and the iron utilisation rate by the erythron (ETUR). Myocardial siderosis occurs when the net ILR-ETUR exceeds 0.21 mg/kg/day, the same threshold at which LPI is increased. The likely mechanism is that higher ILR-ETUR values decrease TfSat Disclosures Porter: Celgene: Consultancy; Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Background Beta (β)-thalassemia is characterized by ineffective erythropoiesis leading to anemia, bone marrow erythroid hyperplasia, iron overload, and organ failure. Sotatercept (ACE-011) is a novel and first-in-class activin type IIA receptor (ActRIIA) fusion protein that increases the release of mature erythrocytes into circulation by acting mainly on late-stage erythropoiesis (Carrancio S, et al. Blood. 2012;120:abstract 372). Clinical data in healthy volunteers have shown that treatment with sotatercept results in increased red blood cell (RBC) parameters, including hemoglobin level (Ruckle J, et al. J Bone Miner Res. 2009;24:744-52). RAP-011, a murine ortholog of sotatercept, was efficacious in a mouse model of β-thalassemia intermedia, reducing ineffective erythropoiesis as well as significantly improving anemia and decreasing bilirubin levels, supporting the clinical development of sotatercept (Dussiot M et al. Blood 2012;120:abstract 247). Methods This is an ongoing phase 2a, multicenter, open-label, dose-finding study to determine a safe and active dose level of sotatercept in adult patients with RBC-transfusion dependent (TD) β-thalassemia major or patients with β-thalassemia intermedia who are either TD or non-transfusion dependent (NTD). The dose levels of sotatercept studied to date are 0.1, 0.3, and 0.5 mg/kg, given subcutaneously once every 3 weeks. Safety is assessed according to NCI-CTC grading. Efficacy is assessed by hemoglobin increase from baseline and/or reduction in transfusion burden. Secondary endpoints include assessment of biomarkers for erythropoiesis, hemolysis, iron metabolism, and bone metabolism, as well as in vitro dyserythropoiesis. Dose escalation to higher dose levels is planned contingent on data review and favorable safety profile as determined by the Steering Committee. Results Patient demographics. A total of 25 patients have been enrolled as of July 26, 2013; 8 in the 0.1 mg/kg cohort, 9 in the 0.3 mg/kg cohort, and 8 in the 0.5 mg/kg cohort. Treatment and analysis for the 0.5 mg/kg cohort is underway and will be updated and presented. In the 0.1 and 0.3 mg/kg cohorts, 3 (18%) patients had β-thalassemia major and 14 (82%) had β-thalassemia intermedia (12 of whom were NTD and 2 of whom were TD). Of the 12 NTD β-thalassemia intermedia patients, 6 were treated at the 0.1 mg/kg dose level and 6 at the 0.3 mg/kg dose level. Median baseline hemoglobin for these NTD patients was 8.6 g/dL (range 5.8 to 10.7 g/dL). Median number of sotatercept doses administered was 4 (range 2 to 7) in the 0.1 mg/kg cohort and 8 (range 3 to 9) in the 0.3 mg/kg cohort; 13/17 (76%) patients remained on treatment. Safety.Sotatercept was generally well tolerated. There were no dose-limiting toxicities reported. Two serious adverse events were reported in the 0.1 mg/kg cohort: a grade 2 phlebitis in an NTD patient with a history of high D-dimer at baseline, and a worsening grade 3 bone pain in a TD β-thalassemia major patient with a history of osteoporosis; both were considered possibly study drug-related. Hemoglobin levels/transfusion requirements. Among NTD patients, preliminary data showed that 1 (17%) patient in the 0.1 mg/kg cohort and all 6 (100%) patients in the 0.3 mg/kg cohort had at least a 1 g/dL increase in hemoglobin level from baseline; among these, 1 patient treated with sotatercept 0.3 mg/kg showed a 2 g/dL hemoglobin level increase from baseline as well as a decrease in total bilirubin level from 2.7 × upper limit of normal (ULN) at baseline to 1.8 × ULN. No other relevant decrease in total bilirubin level was reported at the lower dose levels (0.1 mg/kg or 0.3 mg/kg). Three TD patients were still receiving treatment (2 β-thalassemia intermedia and 1 β-thalassemia major). There had been no appreciable reduction in transfusion burden in the 0.1 and 0.3 mg/kg cohorts to date; however further follow up is warranted and an update will be presented. Conclusion Based on these preliminary data, sotatercept administered subcutaneously every 3 weeks may improve anemia via a novel mechanism of action with a favorable safety profile, thereby addressing a significant unmet medical need for patients with NTD β-thalassemia intermedia. The current data suggest a dose-dependent response that supports further evaluation of the exposure–effect relationship of sotatercept in patients with NTD β-thalassemia intermedia. The first, second, and last authors contributed equally to this abstract. Disclosures: Cappellini: Genzyme: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Off Label Use: Sotatercept is an investigational agent that is being assessed for efficacy and safety in beta-thalassemia. Porter:Novartis: Consultancy, Research Funding; Shire: Consultancy; Celgene: Honoraria. Forni:Celgene: Research Funding; Shire: Research Funding; Novartis Pharma: Research Funding. Laadem:Celgene Corp.: Employment, Equity Ownership. Galacteros:Celgene: Consultancy. Miteva:Celgene Corp.: Employment. Sung:Celgene Corp.: Employment, Equity Ownership. Chopra:Celgene Corp.: Employment, Equity Ownership. Klesczewski:Celgene Corp.: Employment. Attie:Acceleron Pharma: Employment. Hermine:Celgene Corporation: Consultancy, Research Funding.

Abstract 1011 Background. CMR was introduced in London to assess myocardial iron loading in 1999 and some of these patients now have 10 years of follow-up, most with contemporary CMR determinations. The impact of long-term monitoring of myocardial iron loading in thalassemia major (TM) on the proportion of patients with increased myocardial iron (cT2* Patients and Methods. All patients regularly attending two London thalassemia centres, who received their first CMR Jan 1999 - Dec 2000 were analyzed as a cohort. Patients underwent initial CMR at the Royal Brompton Hospital and received CMR follow up (FU) either there or at the Heart Hospital (UCLH). 132 patients were identified as having received a CMR in 1999–2000. A minimum 7 years CMR FU was required for inclusion in the long-term CMR analysis. 109 patients had at least 7 years of CMR follow up (range 7.0–10.6 years, median 9.2). The median age at 1st CMR was 27.9 years (range 7.7 – 49.5 years). At baseline, patients were receiving chelation with deferoxamine (DFO) monotherapy (70%), deferiprone (DFP) monotherapy (21%), or a combination of these agents (9%). At latest FU, patients were receiving DFO (32%), deferasirox (DFX) (28%), DFP (22%), or combined DFP and DFO therapy (18%). Results: Improvement in cardiac iron: In 1999–2000, 60% of TM patients had cT2* values ≤20ms and 17% had cT2* values Changes to chelation therapy: 31% of patients stayed on the same chelator; 33% had 1 chelator switch, 26% 2 switches and 11% 3 or more switches. 18 switches in chelation therapy were due to side-effects (12 DFP, 5 DFX, 1 DFO). There were 9 breaks in chelation therapy during pregnancy in 8 different women. The proportions of patients with T2* < 20ms fell significantly for those who remained on DFO or DFP monotherapies throughout, or who changed chelation modalities on only one (p=0.002) or two (p=0.02) occasions. Patients who received had 3 or more switches did not show a improvement in this respect. The latter group was also the only subset that showed significant deterioration in myocardial iron (p Mortality rates: the overall mortality rate for the initial cohort was 1.65 per 1000 patient years (95% CI 0.71 – 3.24); median age at death 35.6 years (range 27.3–48.4). This is a substantial improvement in the mortality index compared with the UK thalassemia registry data, of 4.3 per 1000 patient years during the period 2000–2003 (Modell et al, JCMR, 2008). The incidence rate ratio is 0.387 (95% CI 0.11–0.961), p Causes of death: there were 8 deaths during the FU period: 3 with complications of hepatitis C (all with cT2* > 20ms), 3 with sepsis (2 with cT2* 20ms). Thus in only 2 patients could excessive cardiac iron loading be considered a causal/contributory factor. There was no significant difference in the baseline cT2* between those who died and those currently still alive (p= 0.2), meaning that death as a drop-out cause does not explain iron loading trends over FU. Chelators at death: DFO (4), DFP (2), DFX (1), combination (1). Conclusions: Over a decade we have seen an almost 3 fold fall in the proportion of patients with myocardial iron overload. Mortality has become substantially lower and cardiac iron overload is no longer the leading cause of mortality. In addition to CMR, this decade has seen the advent of two new oral iron chelators and many patients switched chelation regimen, sometimes several times, during the follow up period. Whilst the contribution of the individual components of this practice to the improved outcome cannot be concluded without randomized studies, it is clear that this modern management of TM is associated with reduced TM mortality. Disclosures: Off Label Use: Deferiprone is off label in the USA but licensed in Europe. Shah:Novartis: Honoraria, Speakers Bureau; Apotex/ Swedish Orphan: Honoraria. Pennell:Siemens: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Cardiovascular Imaging Solutions: Director of CVIS, Equity Ownership. Porter:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

A 30-year-old man was referred for cardiac and hepatic iron quantification by cardiovascular magnetic resonance. He was known to have β-thalassemia major and was heavily transfusion dependent, requiring intensive intravenous chelation therapy. However, after nonadherence to anticoagulation and parenteral therapy, his Portacath had thrombosed, necessitating removal. Cardiovascular magnetic …

Abstract 2005 Poster Board I-1027 Background Although the benefits to iron balance of supplementing deferoxamine therapy with ascorbic acid (AA, 2-3mg/kg/day) are well established, the role of this approach with deferasirox (DFX) chelation is unknown. We have previously reported that fasting plasma ascorbate (FPA) was significantly lower in patients on long-term DFX without AA supplementation (30.3 ± 20.8 mM) than in healthy controls (60.3 ± 12.6 mM, P2 SDs from the adult control mean) present in 72% of patients not receiving AA supplementation (Sarantos et all, Blood 112, Abstract 1858, 2008). It is therefore important to establish prospectively the relevance of ascorbate status to iron balance and also to myocardial iron loading in DFX-treated patients. Here we examine changes in FPA and markers of iron overload in the same patients over at least one year follow-up and examine the impact of AA supplementation in selected patients. Patients and Methods: 21 adult patients with a range of transfusional iron overload conditions excluding Sickle Cell Disease (17 with b-Thalassaemia Major, 2 with Congenital Syderoblastic Anaemia, 1 with Red Cell Aplasia, 1 with Diamond-Blackfan Anaemia) on long-term DFX chelation (duration of treatment 3-6 years, mean dose 27.1mg/kg/day) had measurements of FPA, ferritin, cardiac and hepatic T2* on two occasions, separated by a period of approximately one year (median 419 days). Of these, 10 received AA supplementation (2-3mg/kg/day) and 11 did not. The decision to prescribe AA was based on clinical criteria and patient preference rather than by randomisation. FPA was measured under controlled conditions: a 4 ml fasting heparinised blood sample was taken, placed immediately on ice and centrifuged at 4oC at 2000g for 10 minutes. An equal volume of 10% trichloroacetic acid (TCA) was added, the sample re-spun, and stored at —80oC until measurement of vitamin C fluorimetrically as previously described (Speek et al, 305, J Chromatogr, 1984). Results: In 10 patients who received AA supplementation for 1y (432 ± 96 (SD) days) , the FPA increased significantly from mean baseline of 35.99 ± 21.02 mM to 46.94 ± 14.69 mM at follow up (p=0.03) correcting the deficiency in 3 of the 5 scorbutic patients. FPA increased slightly but not significantly (p=0.3) in 11 patients without supplementation rising from 27.4 ± 15.8 mM to 34.0 ± 12.37 mM where deficiency was corrected in 2 of the 8 scorbutic patients. The increment in FPA levels in un-supplemented patients correlated with decrements in liver iron (LIC, r=-0.64, p=0.04), with mean liver R2* decreasing from 209.24 to 178.07 Hz (i.e. estimated LIC from 7.01 to 6.08 mg Fe/g dry weight (dw)), consistent with previous observations that iron overload accelerates oxidation of AA. Significant improvements in myocardial iron (shown as decreasing myocardial R2*) were seen in ascorbate replete patients at 1y (n=13, mean FPA 48.73 ± 10.61 mM) from a mean myocardial R2* of 61.4 to 52.5Hz p=0.008, i.e. T2* of 16.2ms to 19ms). By contrast, in patients who remained AA deficient (mean FPA 26.28 ± 8.44uM, n=8), the improvement in myocardial R2* was less marked (from 50.41 to 46.65Hz, i.e. T2* 19.8 to 21.4 ms) and did not reach significance. Conclusions: AA deficiency is a significant risk in transfusionally iron loaded patients on long term DFX without AA supplementation, particularly in patients with high levels of body iron loading. Our findings suggest that improvement in myocardial and liver iron with DFX therapy is more likely in AA replete than AA deficient patients. A prospective randomised study of the effects of AA supplementation on iron overload and myocardial iron in DFX treated patients is indicated but until such data are available, we recommend that patients on long term chelation with DFX have FPA levels assessed and consideration is given to supplementing deficient patients. Disclosures: Garbowski: Novartis: Research Funding. Evans:Novartis: Research Funding. Porter:Novartis: Consultancy, Research Funding.

Abstract 2004 Poster Board I-1026 Background and rationale Non-invasive estimation of tissue iron concentration is being used increasingly to follow responses to chelation therapy. Magnetic resonance (MR) T2* was originally developed to estimate myocardial iron but the first description of the myocardial T2* method also demonstrated a clear relationship between liver T2* and liver iron concentration (LIC) measured by biopsy (Anderson, Eur Heart J 2001). The inclusion of liver analysis at this time was intended mainly to show the relationship of T2* to tissue iron rather than as a standard method for LIC measurement, particularly as the echo times used were too long to measure the high iron levels commonly found in the liver. Since this work, other MR methods such as the R2 technique (St. Pierre, Blood 2004) have been specifically validated to measure LIC. However, if T2* could be validated and calibrated appropriately for LIC, it may be convenient to measure liver and heart iron at the same time using this rapid technique. Since the initial calibration of the T2* method, there have been major improvements in scanner technology and hardware. In the original paper, eight different echo times were acquired (TE 2.2- 20.1ms), each of which required a separate breath-hold with important implications for image registration regarding the measurement of T2*. Much shorter, more closely spaced echo times are now possible in a single breath-hold which makes the calculation of T2* more accurate, especially at higher liver iron concentrations where T2* is short. Methods: A large cohort of patients at UCLH have had liver biopsies as part of iron chelation studies on Deferasirox, while continuing to be monitored by T2* of both heart and liver. Data pairs for LIC calibration curve plots were chosen as nested values by shortest biopsy-to-MR time lapse criterion (±75 days) from all biopsy and hepatic T2* results. Liver biopsy iron was measured in a single central laboratory in Rennes, France (Clinique des Maladies du Foie [Clinic for Hepatic Illnesses], Center Hospitalier Universitaire) on paraffin embedded sections as previously described (Soriano-Cubells MJ, Atomic Spectrosc. 1984). All MR scans were performed on a 1.5T Sonata MR scanner (Siemens, Germany) using a 4-channel anterior phased array coil at Royal Brompton Hospital, London. A transverse slice through the centre of the liver was imaged using a multi-echo single breath-hold gradient echo T2* sequence with a range of echo times (TE 0.93-16.0ms). T2* decay was measured using Thalassaemia tools (Cardiovascular Imaging Solutions, London, UK) from a region of interest (ROI) in an area of homogeneous liver tissue, avoiding blood vessels and other sources of artefact. To account for background noise, a truncation method was used for curve-fitting (He, Magn Reson Med 2008). All T2* measurements were performed in triplicate by 2 independent observers choosing three separate ROIs to analyse. The ROIs were chosen to be as large as possible in three separate areas of the liver (anterior, mid/lateral and posterior). Spearman correlation method was used to estimate the degree of relationship between biopsy LIC and hepatic R2* (1/T2*) values, both of which showed positive skew. Results and conclusions: 61 patients had both liver biopsy and liver MR measurements undertaken. However, only 18 had biopsies within 75 days of MR T2* measurement and these are the patients included in the following analysis. There is a linear relationship between biopsy LIC and the reciprocal of T2* (R2*), given by: LIC = 0.03 x R2* + 0.74 (R2=0.96 p Disclosures: Pennell: Cardiovascular Imaging SOlutions, London, UK: Director.

Background: Under conditions of iron overload, ascorbic acid is oxidised at an increased rate leading to a risk of vitamin C deficiency. With deferoxamine (DFO) standard therapy, vitamin C is usually given at a dose of 2–3mg/kg on the days of DFO infusion as this increases iron excretion by up to 30%. With deferarisox (DFX) chelation treatment, although supplementation is permitted, there is currently no information about the effects of vitamin C supplementation on iron excretion and it is often left to patients or their clinician’s discretion as to whether supplementation is given. With long-term treatment, in the absence of supplementation there is a potential risk that vitamin C deficiency will develop and this could influence response to treatment. Patients and Methods: We have measured fasting plasma vitamin C in 41 patients who have been on long term deferasirox treatment for transfusional iron overload for between 1.5 and 5 years. 32 of these patients had received no supplementation and 9 patients had received 2–3 mg/kg/ day of supplementation. We have examined whether trends in serum ferritin, myocardial T2* and liver iron, during the final year of observation, relate to plasma levels of vitamin C. Results: Fasting plasma Vitamin C was significantly lower in the 41 patients (mean=30.3μmol/l, SD=20.8) than healthy control patients (mean=60.29μmol/l SD=12.6) (P36μmol/l) (r=0.88, p Conclusions: We conclude that with long-term deferasirox therapy without vitamin C supplementation, there is a significant risk of vitamin C deficiency with a potential for clinical scurvy. The risk of ascorbate deficiency is further increased at higher levels of body iron loading. These findings suggest that vitamin C supplementation (2–3mg/kg/day) should be recommended as standard for patients on long-term chelation therapy with deferasirox. It would also be of value to determine whether long term-response was improved by ascorbate supplementation.

Background: Previous reports on the effects of various iron chelation regimes, including deferasirox, have shown improvements in myocardial iron over a period of up to 1 year. However no longitudinal analysis of myocardial T2* (mT2*) trends with any chelator regimen beyond 1 year has been reported. Monitoring of mT2* has been part of standard management of transfusional iron overload at UCLH for over 8 years. In this study, we have analysed long-term trends in mT2* up to 4 years for patients entered into pre-registration and extension phases of deferasirox studies, 107 and 108, as well as factors that may determine such trends. Patients and Methods: 33 transfusion dependent patients with thalassaemia or rare anaemias, age range 7–51 years, had mT2*, and T2* LIC assessments repeated with a median interval of 14 months (3.6 occasions per patient) while receiving deferasirox. This allowed analysis of factors affecting trends in mT2* on a total of 104 observation periods (of a median of 371 days). Starting doses of 10–30mg/kg/day were predicated by baseline and 1 year biopsy LIC values according to study protocols. Subsequent dose adjustment depending on adverse events profile, trends of ferritin and serum creatinine, degree of proteinuria, or mT2* and T2* LIC was allowed at the investigators’ discretion. LIC was estimated from liver T2* with correction for liver R2. A maximum permissible chelator dose in the final two years of the study was 40mg/kg. Compliance was calculated from the % of intended doses that were missed over the period of observation, assessed from patient interviews conducted on monthly visits. 4 patients were excluded from analysis due to lack of an adequate baseline mT2* pre–treatment. At baseline, 13 patients had myocardial iron loading as evidenced by mT2* Results: Average baseline mT2* of 21.6ms improved significantly to 24.7ms (p= 0.046, n=29) at a median follow-up of 4 years (geometric mean 18.9ms and 20.7ms, respectively). The greatest improvement in mT2* was seen in the first year of treatment (0.16ms/month) and the least in the final year giving a median change over the 4 year period of 1.27ms/year. At 4 years, among patients with baseline T2* values 20ms all remained normal apart from one who received low dosing (10mg/kg) in the first months of the study. Analysis of factors associated with trends in mT2* revealed compliance, LIC, and ferritin as statistically significant. For each observation period, compliance 12 mg/gdw was associated with worsening of mT2* (p=0.02). Conclusions: With long-term administration of deferasirox, we conclude that compliance should be maintained above 90% to optimize the sustained control of mT2*. Longitudinal control of LIC and ferritin may also be important to reducing the risk of myocardial iron loading in patients receiving long-term deferasiox treatment.