Labile Plasma Iron Levels Research Articles

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of <0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p<0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

Labile plasma iron and echocardiographic parameters are associated with cardiac events in β-thalassemic patients.

Notwithstanding the improvement in therapies, patients affected by thalassemia major (TM) and intermedia (TI) are still at high risk of cardiac complications. This study aimed at evaluating the incidence and predictive factors for developing cardiac events in adult β-TM and TI patients. Data on diagnosis and clinical history were collected retrospectively; prospective data on new-onset cardiac failure and arrhythmias, echocardiographic parameters, biochemical variables including non-transferrin-bound iron (NTBI) and labile plasma iron (LPI), magnetic resonance imaging (MRI) T2* measurement of hepatic and cardiac iron deposits, and iron chelation therapy were recorded during a 6-year follow-up. Thirty-seven patients, 29 TM and 8 TI, were included. At baseline, 8 TM patients and 1 TI patient had previously experienced a cardiac event (mainly heart failure). All patients were on chelation therapy and only 3 TM patients had mild-to-severe cardiac siderosis. During follow-up, 11 patients (29.7%) experienced a new cardiac event. The occurrence of cardiac events was correlated to high LPI levels (OR 12.0, 95% CI 1.56-92.3, p .017), low mean pre-transfusion haemoglobin (OR 0.21, 95% C.I. 0.051-0.761, p .21) and echocardiographic parameters suggestive of myocardial hypertrophy. Multivariate analysis disclosed high LPI and left ventricle mass index (LVMI) as independent variables significantly associated with cardiac events. Cardiac iron deposits measured by MRI T2* failed to predict cardiac events. LPI, Hb levels and echocardiographic parameters assessing cardiac remodelling are associated with cardiac events in adult TM and TI patients. LPI might represent both a prognostic marker and a potential target for novel treatment strategies. Further studies are warranted to confirm our findings on larger populations.

Comparisons of serum non-transferrin-bound iron levels and fetal cardiac function between fetuses affected with hemoglobin Bart's disease and normal fetuses.

To compare the levels of Non-transferrin bound iron (NTBI) in fetuses with anemia, using Hb Bart's disease as a study model, and those in unaffected fetuses and to determine the association between fetal cardiac function and the levels of NTBI. A prospective study was conducted on pregnancies at risk of fetal Hb Bart's disease. All fetuses underwent standard ultrasound examination at 18-22 weeks of gestation for fetal biometry, anomaly screening and fetal cardiac function. After that, 2 ml of fetal blood was taken by cordocentesis to measure NTBI by Labile Plasma Iron (LPI), serum iron, hemoglobin and hematocrit. The NTBI levels of both groups were compared and the correlation between NTBI and fetal cardiac function was determined. A total of 50 fetuses, including 20 fetuses with Hb Bart's disease and 30 unaffected fetuses were recruited. There was a significant increase in the level of serum iron in the affected group (median: 22.7 vs. 9.7; p-value: 0.013) and also a significant increase in NTBI when compared with those of the unaffected fetuses (median 0.11 vs. 0.07; p-value: 0.046). In comparisons of fetal cardiac function, myocardial performance (Tei) index of both sides was significantly increased in the affected group (left Tei: p = 0.001, Right Tei: p = 0.008). Also, isovolumetric contraction time (ICT) was also significantly prolonged (left ICT: p = 0.00, right ICT: p = 0.000). Fetal LPI levels were significantly correlated inversely with fetal hemoglobin levels (p = 0.030) but not significantly correlated with the fetal serum iron levels (p = 0.138). Fetal LPI levels were also significantly correlated positively with myocardial performance index (Tei) of both sides (right Tei: R = 0.000, left Tei: R = 0.000) and right ICT (R = 0.013), but not significantly correlated with left ICT (R = 0.554). Anemia caused by fetal Hb Bart's disease in pre-hydropic stage is significantly associated with fetal cardiac dysfunction and increased fetal serum NTBI levels which are significantly correlated with worsening cardiac dysfunction. Nevertheless, based on the limitations of the present study, further studies including long-term data are required to support a role of fetal anemia as well as increased fetal serum NTBI levels in development of subsequent heart failure or cardiac compromise among the survivors, possibly predisposing to cardiovascular disease in adult life.

Unbalance in Iron Metabolism in Childhood Leukemia Converges with Treatment Intensity: Biochemical and Clinical Analysis.

Simple SummaryIn children undergoing therapy for acute leukemia or after hematopoietic cell transplantation, the following iron metabolism parameters were analyzed in the context of iron overload: (1) parameters measuring functional and storage iron pools: non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) levels, iron, transferrin, total iron-binding capacity, ferritin, ferritin heavy and light chains; (2) proteins regulating iron absorption and its release from tissue stores: hepcidin, soluble hemojuvelin, soluble ferroportin-1; (3) proteins regulating the erythropoietic activity of bone marrow: erythroferrone, erythropoietin, soluble transferrin receptor. It has been shown that the occurrence of NTBI and LPI in the circulation and the intensification of disturbances in iron metabolism were associated with the intensity of anti-leukemic treatment and were the highest in the transplant group followed by the acute leukemia after treatment and de novo groups. In patients after transplantation, the most significant changes were found in NTBI, LPI, iron, ferritin, hepcidin, and ferroportin-1 levels.Objective: The aim of this study was to evaluate non-transferrin-bound iron (NTBI) and labile plasma iron (LPI) levels and other parameters of iron metabolism in children undergoing therapy for acute leukemia or after hematopoietic cell transplantation (HCT), in the context of iron overload. Patients: A total number of 85 children were prospectively included into four groups: controls, acute leukemia de novo, acute leukemia after intensive treatment, and after HCT. Methods: The following iron metabolism parameters were analyzed: (1) parameters measuring functional and storage iron pools: NTBI, LPI, iron, transferrin, total iron-binding capacity, ferritin, ferritin heavy and light chains; (2) proteins regulating iron absorption and its release from tissue stores: hepcidin, soluble hemojuvelin, soluble ferroportin-1; (3) proteins regulating the erythropoietic activity of bone marrow: erythroferrone, erythropoietin, soluble transferrin receptor. Results: Intensive treatment of leukemia in children was associated with the presence of serum NTBI and LPI, which was the highest in the HCT group followed by the acute leukemia after treatment and de novo groups. In patients after HCT, the most significant changes were found in NTBI, LPI, iron, ferritin, hepcidin, and ferroportin-1 levels. Conclusions: The occurrence of NTBI and LPI in the circulation and the intensification of disturbances in iron metabolism were associated with the intensity of the anti-leukemic treatment.

Iron overload in the HCT patient: a review.

Iron overload (IO) is common in hematologic malignancies and hemoglobinopathies, largely due to red cell transfusion burden. End-organ damage from IO occurs via reactive oxygen species-mediated pathways. The impact of pretransplant IO on hematopoietic cell transplant (HCT) morbidity and mortality remains contentious; studies have shown mixed results, possibly due to variability in study population and design, as well as markers of IO. Ferritin has served as a traditional circulating marker of total body IO, but liver iron content by MRI appears to be a better marker of end-organ involvement. Novel surrogate markers including hepcidin, marrow Prussian blue staining, and labile plasma iron levels may prove to be more specific for HCT complications. Posttransplant phlebotomy, chelation, or both in combination remains the mainstays of treatment, though may ultimately be supplanted by pretransplant or peri-transplant use of bone marrow maturation agents or targeted chelation at time of highest IO risk. This review discusses the pathophysiology of IO in hematologic disease, the evidence supporting and refuting its negative impact on HCT outcomes, as well as current and future therapies.

Labile plasma iron levels in chronic hemodialysis patients treated by intravenous iron supplementation.

The increased usage of intravenous iron in hemodialysis patients during recent years has led to increasing concern over the potential development of iron overload. Current methods for detecting iron overload, transferrin saturation, and serum ferritin are neither sensitive nor specific. Labile plasma iron (LPI) represents a component of nontransferrin-bound iron and may be a more accurate indicator of impending iron overload. We studied whether LPI measured can serve as an early indicator of impending iron overload and mortality in hemodialysis patients. Chronic hemodialysis patients from two medical centers in Israel and Poland who received intravenous iron were included. Baseline clinical and laboratory parameters were recorded. LPI was measured before and 48 hours after a single IV administration. Correlation of positive LPI with laboratory parameters and 2-year mortality was evaluated. One hundred and one hemodialysis patients were included in the study. LPI became positive post-administration in 18 (17.8%) patients. Ferritin levels >526 ng/mL and monthly iron doses >250 mg were associated with positive LPI after intravenous iron. At a 2-year follow-up, higher mortality was observed in the positive LPI group (61.1% compared to 25.3%, P ≤ .05), although this effect was not statistically significant after multivariate adjustment. A substantial number of hemodialysis patients have positive LPI after intravenous iron administration. LPI positively correlates with laboratory parameters that are currently in routine clinical use for detecting iron overload and with higher intravenous iron dose. Further studies should be conducted to establish the clinical implications of LPI monitoring in hemodialysis patients.

Utility of Labile Plasma Iron Assay in Thalassemia Major Patients.

Labile plasma iron (LPI) levels are proposed as marker of iron overload in thalassemia patients and are also known to be the earliest parameter to indicate efficacy of chelation therapy. It was a prospective study in 35 patients of thalassemia major. Patients were recruited in two groups-group A (n = 13) patients not on chelation therapy and group B (n = 22) patients who were on regular oral chelation therapy. Ten age and gender matched healthy controls were also studied. For all patients, ferritin levels and LPI levels were measured at baseline, 6months and 12months. For group B patients paired samples for LPI were taken (before and 2h after chelator). LPI levels were found to be significantly higher in group B patients versus group A patients versus normal healthy controls at all time-points. (P value-< 0.0001, 0.001) In group A, both LPI levels and ferritin levels follow an upward trend and correlated well with each other (P value-< 0.0001). In group B, the serum ferritin trend was not significant over follow up period of 1year (P value 0.16), however LPI levels showed a significant decreasing trend on continued chelation (P value 0.0347) In patients on chelation therapy, the immediate change (2h) in LPI levels on administration of chelators was not found to be significant (P value 0.22). LPI assay appears potentially attractive alternate to serum ferritin and can serve to monitor the trend of iron overload during long-term follow up.

Elevated Labile Plasma Iron (LPI) Levels in Patients with Lower-Risk Myelodysplastic Syndromes (MDS) Are Associated with Decreased Quality of Life and Reduced Survival

Background: Patients with lower-risk MDS (LR-MDS) are prone to iron toxicity due to long-term iron accumulation either caused by RBC transfusions or ineffective erythropoiesis. Nontransferrin bound iron (NTBI), including labile plasma iron (LPI), are toxic iron species that may mediate cellular damage via oxidative stress. The EUMDS registry collects prospective observational data on newly diagnosed LR-MDS patients from 145 centers in 17 countries since 2008.Methods: We analyzed serum from 247 LR-MDS patients collected at six-month intervals for ferritin, transferrin saturation (TSAT), hepcidin-25, soluble transferin receptor (sTfR) and toxic iron species (NTBI and LPI) in order to evaluate temporal changes in iron metabolism, the presence of potentially toxic forms of iron and their impact on survival, and quality of life. In addition, we measured the impact of iron chelation on the iron species levels and its impact on the outcome parameters.Results: The median age was 73 years (range: 37 to 95 years) and 66% were males. WHO2001 MDS-subtypes were RCMD (45%), RARS (22%), RA (18%), RAEB-1 (7%), 5q-syndrome (4%) and RCMD-RS (4%). The IPSS-R categories were: (very) low risk: 66%; intermediate risk: 11%; (very) high risk: 2% and unknown: 20%. The median EQ5D index score was 0.80 (p10 to p90: 0.52 to 1.00). The table shows iron parameters at registration, 1 and 2 years follow-up both in transfusion-dependent (TD) and transfusion-independent (TI) patients and according to: MDS-RS (RARS/RCMD-RS) or MDS Other (RA/RCMD/RAEB/5q-syndrome). Mean serum ferritin levels were increased in TD patients, compared to TI patients (Table). Increase of ferritin levels over time was high in both TD groups, but the increase was more pronounced in the RS subgroup. Elevated CRP levels (> 10mg/L) were observed in TD nonRS patients, especially during the first year after diagnosis. Markedly increased mean TSAT levels (>75%) occurred in the subgroup of TD-RS patients throughout the observation period. Hepcidin levels were most markedly elevated in TD nonRS patients at registration and remained elevated during follow-up (Table). Hepcidin levels were low in MDS-RS TI patients at all time points compared to nonRS MDS patients and decreased over time as a result of an increased (ineffective) erythropoiesis (Table). This is supported by the highest levels of STfR also noted in this patient category (data not shown). The highest NTBI and LPI levels were observed in TD-RS patients compared to the other 3 subgroups (Table). Both NTBI and LPI levels had a strong correlation (p <0.001) with TSAT. Elevated LPI levels in combination with high TSAT levels (>80%) occurred almost exclusively in patients with MDS-RS and/or previous transfusions. Both the EQ5D index score and EQ-VAS showed a negative correlation (r) with LPI levels with r = -0.09 (p = 0.028) and r = -0.07 (p=0.046) respectively. This negative effect of elevated LPI levels was most pronounced in the TD RS subgroup with a negative correlation of -0.17 for the EQ5D index score and -0.2 for the VAS score. In total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 4 patients desferioxamine and 1 patient unknown). LPI levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferoxamine treatment.The Kaplan-Meier curves (Figure) demonstrate the prognostic impact of elevated LPI levels by transfusion status as a time dependent variable; once a subject had an elevated LPI level, they remained in this group. Patients were censored at time of starting iron chelation (16 patients). In a multivariate analysis comparing elevated LPI levels and transfusion dependency to the control group with undetectable LPI and no transfusion showed a significantly decreased survival in all 3 risk groups after adjustment for age at diagnosis, baseline IPSS-R category and ESA treatment status; for details, see supplementary table.Conclusion: This study illustrates labile plasma iron species as a clinically relevant assay for identification of the toxic fraction of overt iron overload and its negative impact on HRQoL and overall survival in transfusion dependent and transfusion independent patients. [Display omitted] DisclosuresCulligan:Merck Sharp & Dohme (MSD): Honoraria; Abbvie: Other: Support to attend conferences; Takeda: Honoraria, Other: Support to attend conferences; Pfizer: Honoraria; Celgene: Other: Support to attend conferences; JAZZ: Honoraria; Daiichi-Sankyo: Other: Support to attend conferences. Garelius:novartis: Honoraria. de Witte:Celgene: Honoraria, Research Funding; Novartis: Research Funding; Amgen: Consultancy, Research Funding.

Labile plasma iron levels predict survival in patients with lower-risk myelodysplastic syndromes

Red blood cell transfusions remain one of the cornerstones in supportive care of lower-risk patients with myelodysplastic syndromes. We hypothesized that patients develop oxidant-mediated tissue injury through the formation of toxic iron species, caused either by red blood cell transfusions or by ineffective erythropoiesis. We analyzed serum samples from 100 lower-risk patients with myelodysplastic syndromes at six-month intervals for transferrin saturation, hepcidin-25, growth differentiation factor 15, soluble transferrin receptor, non-transferrin bound iron and labile plasma iron in order to evaluate temporal changes in iron metabolism and the presence of potentially toxic iron species and their impact on survival. Hepcidin levels were low in 34 patients with ringed sideroblasts compared to 66 patients without. Increases of hepcidin and non-transferrin bound iron levels were visible early in follow-up of all transfusion-dependent patient groups. Hepcidin levels significantly decreased over time in transfusion-independent patients with ringed sideroblasts. Increased soluble transferrin receptor levels in transfusion-independent patients with ringed sideroblasts confirmed the presence of ineffective erythropoiesis and suppression of hepcidin production in these patients. Detectable labile plasma iron levels in combination with high transferrin saturation levels occurred almost exclusively in patients with ringed sideroblasts and all transfusion-dependent patient groups. Detectable labile plasma iron levels in transfusion-dependent patients without ringed sideroblasts were associated with decreased survival. In conclusion, toxic iron species occurred in all transfusion-dependent patients and in transfusion-independent patients with ringed sideroblasts. Labile plasma iron appeared to be a clinically relevant measure for potential iron toxicity and a prognostic factor for survival in transfusion-dependent patients. clinicaltrials.gov Identifier: 00600860.

Implications of Low Zinc and Copper Levels As Well As Altered Iron Trafficking Proteins on Oxidant Stress in Patients with Transfusion Dependant Thalassemia

Introduction:Patients with transfusion dependent thalassemia (TDT) have a genetic anemia that causes incomplete erythropoiesis and iron overload. Plasma zinc deficiency is also seen in roughly 25% of patients with TDT. Iron overload is thought to be related to a number of secondary complications in TDT including cardiomyopathy and diabetes. However, in TDT the effects of altered Zn status are not as well characterized and the effects of low copper (Cu) are even less well known. One possible cause of these complications is oxidative damage to tissues. This oxidative stress can be caused by labile plasma iron (LPI), a component of the non-transferrin bound iron pool, which is often seen in individuals suffering from iron overload. LPI is both redox-active and chelatable and is the likely culprit distributing iron to extra-hepatic tissues. Through reactive Fenton chemistry, LPI, can also cause lipid peroxidation releasing malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which are damage-associated molecular patterns and markers of oxidative tissue damage that activate the immune system to induce inflammation. Incomplete erythropoiesis as well as transfusional iron overload are responsible for an increase in the amount of poorly handled, redox active LPI in TDT. Thus, we hypothesized that changes in the levels of key iron trafficking proteins (such as soluble transferrin receptor (sTfR) or hemopexin) would affect oxidative stress levels in TDT. We also hypothesized that metal dyshomeostasis, such as a functional Zn or Cu deficiency would affect oxidative stress.Aims: The purpose of this pilot project is to 1) Determine the state of circulating levels of oxidative stress markers and iron trafficking proteins in TDT patients and 2) Explore the relationship between the markers and proteins measured in (1)and the Zn and Cu status of TDT patients.Methods:39 subjects with informed consent were enrolled (29 patients with TDT and 10 controls). Liver iron concentration (LIC) was measured by a superconducting quantum interference device (SQUID™). LPI was measured using dihydrorhodamine 123. Both MDA and MDA + 4-HNE were measured using N-methyl-2-phenylindole. The iron trafficking proteins sTfR, transferrin, haptoglobin and hemopexin were measured by immunoassay isolation followed by multiplex multiple reaction monitoring mass spectrometry. Zn and Cu were assessed by inductively coupled plasma atomic emission spectroscopy. Fructosamine was measured by quantitative spectrophotometry.Results: Patients with TDT had elevated LIC levels of 2681 ± 2424 ug iron/g wet weight. Plasma levels of the iron trafficking proteins, transferrin, hemopexin and haptoglobin were all decreased in TDT patients (P<0.001) with a corresponding increase in soluble transferrin receptor (StfR) and the LPI (P<0.001). Serum Zn was significantly reduced in TDT patients (p = 0.028) and urinary Zn was significantly elevated (p =0.024). Serum Cu was also significantly reduced in TDT patients (p =0.026). Reduced Zn levels in TDT patients correlated with elevated MDA levels (p = 0.0195, R = -0.382) as were serum Cu levels (p = 0.0158, R = -0.394). Reduced levels of plasma iron trafficking proteins (haptoglobin, hemopexin, and transferrin) were correlated with elevated levels of MDA and LPI (all p-values < 0.05). Plasma MDA was also correlated with fructosamine levels (p < 0.001, R= 0.57).Conclusion: Metal dyshomeostasis involving Zn and Cu may be important contributors to oxidative stress and iron injury in TDT. We confirm previous findings in TDT of elevated levels of LPI as well as the oxidative stress markers MDA and 4-HNE. We expand previous findings of reduced transferrin levels in TDT to show a similar reduction in both haptoglobin and hemopexin. StfR levels were elevated in TDT patients, possibly due to a strong erythropoietic drive. Both reduced haptoglobin and hemopexin as well as decreased Zn and Cu levels and increased Zn excretion appear to be present in TDT. These preliminary findings suggest that low levels of Zn, Cu, haptoglobin and hemopexin may be related to increased oxidative stress and LPI in TDT, which could be important contributors to secondary complications of TDT. DisclosuresWalter:Apopharma: Research Funding.

Lower Levels of LPI in Thalassemia Major Patients Treated with Deferasirox

▪Background In living systems iron appears predominantly associated with proteins (including transferrin in the plasma, and ferritin in the cells). However, when the binding capacity of these proteins is exceeded, the iron in the plasma begins to appear in the form of low molecular weight complexes called NTBI (Not-Transferrin Bound Iron), whose chemical composition is heterogeneous and thought to consist of several circulating isoforms, that is Fe (III) bound to albumin and citrate and potentially to acetate, malate and phosphate. The fraction of redox-active and chelatable NTBI is designated Labile Plasma Iron (LPI). It has been shown that these complexes, in particular LPI, are able to selectively penetrate cardiomyocytes and pancreatic islet cells promoting the formation of Reactive Oxygen Species (ROS) overriding the cellular antioxidant machineries and causing oxidative damage. The aim of our study was to quantify NTBI and LPI fractions on sera from 55 Thalassemia Transfusion Dependent patients (29 females and 26 males) under chelation treatment as monotherapy or in different combinations referred to our Center.Material and Methods Blood samples were drawn after a short period of washout that is 12 hours for Deferiprone (DFP) and 24 hours for Desferoxiammine (DFO) and/or Deferasirox (DFX).In each patient LPI was detected in 500 ml of serum using the FeROS™ kit (kindly provided by Aferrix Ltd., Israel) that measures the iron-specific redox activity. A reducing agent (ascorbic acid) and an oxidizing agent (atmospheric O2) cause labile iron in the tested sample to generate ROS via the Fenton reaction. The ROS are detected by an oxidation-sensitive probe (DHR) that becomes fluorescent when oxidized. With this assay an index of eLPI (enhanced/extractable LPI), that reflects the total NTBI levels, is provided by the use of a mobilizing agent.One-way analysis of variance (ANOVA) was performed to compare means of LPI for different types of chelation treatment. Furthermore, data were studied in terms of correlation (Pearson’s r) and association (Odds Ratio) for different LPI levels. LPI was categorized into two classes: <0.2 Units equal to 0 and ≥0.2 Units equal to 1.Results A statistically significant difference was found for LPI means of different types of chelation treatment (p-value < 0.005, Table 1). Furthermore, no statistically significant correlation was found among eLPI and LPI with age and serum ferritin. Association among eLPI and LPI with cirrhosis, diabetes and cardiopathy was studied, and no statistical significance was found (OR 0.7, 95% CI 0.07-9.3, p-value 0.7; OR 1.2, 95% CI 0.2 -14.4, p-value 0.81; OR 2.6, 95% CI 0.3 - 132.8; p-value 0.37, respectively), even if, it was observed that cardiopathy is more common (83.3%) for high (≥ 0.2 Units) LPI values versus low (<0.2 Units) LPI values.Discussion The results of this preliminary study showed that patients treated with DFX have lower LPI levels than other chelation treatments. In addition, for future analyses, it is to take into consideration that data could be indicative of an association between higher LPI values and heart disease. LPI and eLPI parameters when used together with other markers of iron overload (transferrin, ferritin, MRI heart-liver) can provide a convenient and immediate measure of chelation efficacy and are useful tool for monitoring treatment and patient compliance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Elevated Labile Plasma Iron Levels (LPI) and Increased Oxidative Stress Are Associated with Red Blood Cell Transfusions in Patients with Lower-Risk Myelodysplastic Syndromes (MDS) Subtitle: from the European Leukemianet MDS Registry

Background: The EUMDS registry, collecting prospective observational data on lower-risk MDS (LR-MDS) patients from 142 centers in 17 countries, is performing a study on the hypothesized negative impact of red blood cell transfusions (RBCT) on outcome in LR-MDS. The aim of this study istoevaluate in transfusion dependent (TD) patients temporal changes in iron metabolism and presence of potentially toxic forms of iron, measured as labile plasma iron (LPI) and their impact on oxidative stress, using malondialdehyde (MDA), and their impact on survival.Methods: We analyzed serum samples from 100 LR-MDS patients at 6-month intervals fortransferrin saturation (TSAT), non-transferrin bound iron (NTBI) and LPI.Cox proportional hazards regression models and Kaplan-Meier (KM) survival curves were used in time-to-event analyses to assess the impact of LPI, NTBI and TSAT by transfusion status. All variables were treated as time-varying covariates in the model by assessing the levels of the parameters: LPI, NTBI (<LLOD vs elevated), TSAT (<80% vs ≥80%) and transfusion status (TD vs transfusion independent (TI)) at each visit.We also explored in 56 patients the temporal prevalence of oxidative stress and its relation with toxic iron species by means of the MDA assay in paired samples with at least 1-year interval.Results: Themedian age was 73 years.IPSS-R risk groups of this cohort of LR-MDS, defined as IPSS low & intermediate-1 risk, were: very low 32%, low 41%, intermediate 8%, high 3% and unknown 16%. Patients were subdivided in ring sideroblastic MDS (RS-MDS) (34%) and non-RS-MDS (64%). Both groups were subdivided in TD and TI patients.Elevated LPI in combination with high TSAT (>80%) occurred almost exclusively in patients with TI RS-MDS and in all TD MDS patients.Time-dependent, multivariate analysis (MVA), adjusting for age and IPSS-R risk group, of elevated LPIconcentrations for overall survival (OS) resulted in a Hazard Ratio (HR) of 2.3 with 95%CI (0.9-5.8). Elevated NTBI was associated with HR of 0.7 and 95%CI: 0.3-1.8 and TSAT >80% with HR of 0.9 and 95%CI: 0.3-2.6.MVA of OS by LPI and transfusion status as time varying variable showed a significant impact of elevated LPI in the TD group only(HR=4.0, 95%CI: 1.02-15.9) (p = 0.05).MDA concentrations were measured in 28 patients with RS-MDS and 28 non-RS-MDS patients. 21 and 29 patients were TD at time of 1st and 2nd MDA measurement respectively. The overall median MDA concentrations in the 1st sample were elevated compared to controls: 0.9 (0.3 - 4.1) (range controls: 0.16 to 0.64 mM) and remained elevated in the 2nd sample. Median MDA increased from 0.8 to 0.9 mM in the MDS-RS group (p = 0.008 paired one-sided T-test). Significantly higher MDA concentrations were observed in TD patients at time of the 2nd measurement: a median MDA level of 0.7 mM (0.3 - 3.6) in TI and 1.3 (0.3 - 6.8) in TD patients resp. (p = 0.01). MDA levels did not increase in time in TI MDS, but showed a trend to higher MDA in 9 patients who became TD between 2 MDA measurements (p=0.11) and in the 21 patients TD before the 1st measurement (p = 0.12). Median MDA did not change in time in the majority of patients with LPI levels below the detection line (<LLOD). The correlation between elevated LPI and MDA concentrations was weak (r = 0.047, P = 0.82). Paired analysis of the 16 patients with LPI < LLOD at time of 1st measurement and elevated LPI at time of 2ndmeasurement showed a slight increase (p = 0.10) of the median MDA levels from 1.1 to 1.2mM/l. The impact on OS of lowest 50% of MDA levels at each measurement and the highest 50% of MDA levels, adjusted for MDS-RS status, TD and LPI>LLOD vs <LLOD was not evident with an unadjusted HR of 1.3 (0.6 - 3.1) and an adjusted HR of 0.6 (0.2 - 1.5).Interpretation:Iron toxicity and increased oxidative stress occurred in all types of TD MDS patients and in TI RS-MDS patients. This study illustrates LPIas a clinically relevant assay for identification of the toxic fraction of iron overload and its negative impact on overall survival. In contrast, high TSAT (>80%) and elevated NTBI concentrations did not predict survival.Oxidative stress, using MDA as a measurement of lipid peroxidation, is increased in the majority of lower-risk MDS patients, usually early after diagnosis. The highest MDA levels are measured in patients receiving RBCT, but the LPI and MDA were not correlated, which can be explained by the slower clearance of MDA after RBCT compared to the more rapid clearance of LPI. [Display omitted] [Display omitted] DisclosuresSymeonidis:Takeda: Consultancy, Honoraria; Amgen: Honoraria; Roche: Honoraria; Genesis: Honoraria. Smith:Jansen Cilag: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Celgene: Research Funding. de Witte:Incyte: Consultancy; Celgene: Consultancy; Novartis: Honoraria, Research Funding.

A Randomized Trial on the Safety and Efficacy of Early Start of Iron Chelation Therapy with Deferiprone in Newly Diagnosed Children with Transfusion Dependent Thalassemia

▪Iron toxicity is the main risk factor for morbidity and mortality in patients with transfusion-dependent thalassemia. Current practice is to start chelation therapy only after 10-20 transfusions, or when the serum ferritin (SF) level rises above 1,000 μg/L. This delay is aimed at minimizing the risk of chelation toxicity that was observed with the use of deferoxamine in children with low iron stores. Deferiprone has lower affinity for iron than deferoxamine and data from clinical trials on its use in patients without systemic iron overload indicate a safety profile for its use in those conditions. The current trial was designed to evaluate the safety of the early use of low-dose deferiprone in newly diagnosed pediatric thalassemia and to evaluate if it can postpone iron overload.Sixty-four children recently diagnosed with thalassemia major who had begun receiving blood transfusions every 3-4 weeks to keep pre-transfusion Hb above 10 gm/dl, had not yet started iron chelation therapy and had SF ≥ 400 μg/L or transferrin saturation (TSAT) ≥ 70% or labile plasma iron (LPI) ≥ 0.2 µM were randomized to start deferiprone (DFP) at a dose normally considered to be sub-therapeutic (50 mg/kg/day) or no chelation (NC). Age at 1st transfusion was 8.1 ± 1.7 for DFP-treated and 8.1 ± 1.6 months for NC children. The percentage of patients with LPI ≥ 0.6 µM, SF ≥ 1000 μg/L or TSAT ≥ 70% in each study arm was assessed at 6 months and 12 months. Patients with confirmed SF ≥1000 ng/mL were withdrawn from the study and placed on a standard chelation regimen.Results. Two patients (DFP) were lost to follow after baseline measurements, 1 patient (NC) withdrew consent at baseline, and 10 patients (5 DFP, 5 NC) have yet to complete all follow up visits. All NC patients had been removed from the trial prior to completing 7 months of follow-up 12 due to confirmed SF ≥ 1000 μg/L. Mean ± SD time of follow up was 10.4± 4.9 and 5.9 ± 2.5 months for DFP and NC, respectively. Most common adverse events in patients on DFP versus NC were diarrhoea (19% vs 13%, p= 0.73), vomiting (13% vs 13%, p=1.00), abdominal colic (13% vs 13%), increased liver enzymes (6% vs 3%, p=1.00) and neutropenia (neutrophil count between 1,000-1,500 x 109/L) (6% vs 6%). All adverse events were mild in severity and did not require interruption of DFP use. There were no cases of agranulocytosis or of moderate neutropenia, no arthralgia and no serious infections in DFP-treated patients. Preliminary efficacy results are presented in the table. DFP therapy was associated with a significant reduction in the rate of iron accumulation as measured by SF (P<0.0001) (Figure 1), LPI (P<0.001) (Figure 2) and TSAT (P<0.001). LPI ≥ 0.6 µM appeared as early as after 5 transfusions in NC children and was delayed to at least 10 transfusions with DFP therapy. TSAT ≥ 70% appeared after 10 transfusions in NC children and was delayed to at least 17 transfusions with DFP therapy.The results of this study show that LPI and TSAT may reach values ≥ 0.6 µM and ≥ 70%, respectively, after 5 -10 transfusions in children with TM and all NC children had SF ≥ 1000 μg/L after 8-9 transfusions. A sub-therapeutic dose regimen of deferiprone for a mean of 10 months in children with TM and low iron overload was not associated with safety concerns and able to significantly reduce the rate of iron accumulation as measured by SF and the appearance of high levels of LPI and of TSAT. [Display omitted] DisclosuresBerdoukas:ApoPharma Inc: Consultancy. Tricta:ApoPharma Inc: Employment.

Management of iron overload before, during, and after hematopoietic stem cell transplantation for thalassemia major.

Solid evidence has established the negative impact of high iron burden and related tissue damage on the outcome of hemopoietic stem cell transplantation for thalassemia major. Recent improvements in our knowledge of iron metabolism have been focused on elevated non-transferrin-bound iron and labile plasmairon levels in the peritransplantation period as potential contributors to tissue toxicity and subsequent adverse transplant outcome. As mouse models have shown,iron overloadcan injure bone marrow hematopoiesis by increasing reactive oxygen species. The Pesaro experience, conducted in the deferoxamine-only era, clearly defined three iron-related factors (liver fibrosis, hepatomegaly, and quality of lifelong chelation) as significantly affecting transplant outcome. The detrimental effect of iron has only been clarified in recent years. Active interventional strategies are ongoing. Although successful hematopoietic stem cell transplantation clinically resolves the thalassemia marrow defect, patients still remain carriers of iron overload and of all the clinical complications acquired during prior years of transfusion therapy. Therefore, adequate "iron diagnosis" and management is mandatory after hemopoietic stem cell transplantation. In transplanted thalassemia patients, body iron should be returned to within the normal range. Phlebotomy is the gold standard to reduce iron burden; though deferoxamine is a proven, acceptable alternative, clinical investigations on deferasirox are ongoing.

Markers of Oxidative Stress Do Not Correlate with Labile Plasma Iron Blood in Patients with Myelodysplastic Syndromes

▪Background: Modalities of monitoring and clinical impact of post transfusional iron overload in MDS patients remain controversial. Labile Plasma Iron (LPI) is a component of Non-Transferrin Bound Iron (NTBI) that is thought to contribute to oxidative stress in MDS. However, little data support this hypothesis and most studies on LPI have only addressed its ability to produce free radicals in vitro. The aim of this work was to assess LPI and to evaluate the correlation between LPI and plasma and blood markers of peroxidation in MDS patientsDesign and Methods: In 67 non-chelated MDS patients, we evaluated: hemoglobin (Hb), indices of iron overload, peroxidation status at inclusion and/or during follow up. The peroxidation status was evaluated by measuring blood Glutathione (GSH) and plasma Advanced Oxidation Protein Products (AOPP). LPI was measured using the Afferix kit. NTBI was measured by a flow cytometry- based assay (FeFlobead) in a subset of patients with high transferrin saturation.Results: Median age of the 67 patients was 79 years (range 61-93). 38 patients were included at diagnosis of MDS and 29 were included during the disease course (after a median of 50 months, range 3-168, for the latter group). WHO sub-type was: 14 refractory anemia, 22 refractory anemia with ring sideroblasts, 7 refractory cytopenia with mutlilineage dysplasia, 4 5q minus syndrome, 16 refractory anemia with excess of blasts ( RAEB) (type I n=6, Type II n=5, 5 RAEB-t/AML , 3 chronic myelo monocytic leukemia type I, one unclassifiable case. Prognostic score according IPSS was low in 43 patients, Int-1 in 13, Int-2 in 6 and high in 5. 36/67 patients had been transfused and had received a median of 22 packed RBCs (range 2-102). Transfused patients (N=36) were more heavily iron overloaded than non-transfused patients (N=31), as indicated by significantly higher median transferrin (Tf ) saturation (42% versus 62%) (P<0.005) and serum ferritin (SF) values 389microg/L versus 1494)(P<0.0001). A highly significant correlation (r2 =0.5, P<0.001) was seen between serum ferritin values and the number of PRBCs. 9/31 (29 %) non transfused and 26/36 (72 %) transfused patients had Tf saturation > 50 %. However, the mean value of LPI was very similar in transfused and non-transfused group (0.34 microM versus 0.38). 8/67pts had LPI levels above limit upper value (≥ 0.4 µM) and their SF was significantly higher than that of other patients(p=0.02). However no significant difference was found between patients with LPI < 0.4 µM and ≥ 0.4 µM, for Tf saturation, blood GSH or AOPP median values. No correlation between LPI and NTBI was seen. GSH and AOPP levels did not statistically differ according (IPSS) groups. 14 patients were studied during follow up, 7 of them after 3 months of iron chelation, and 7 after treatment with EPO. While chelation slightly improved markers of oxidative stress (MAO) (p= NS), EPO induced a very highly significant improvement of MAO-(p= 0.005).Conclusion: We found no evidence to support LPI pro-oxidant role in vivo. A few patients had an elevated LPI level in this large, moderately transfused, MDS cohort. Utility of LPI in monitoring of post transfusional iron overload remain to be demonstrated. Results obtained following EPO treatment suggest that ineffective erythropoiesis rather than iron overload may be responsible for RBC oxidative stress DisclosuresRose:novartis: Honoraria, Research Funding; celgene: Honoraria. Fenaux:Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Beaumont:novartis: Research Funding.

Labile Plasma Iron (LPI) Is a Clinical Indicator of Overt Iron Overload in Patients with Lower-Risk Myelodysplastic Syndromes (MDS) from the European Leukemianet MDS Registry

Background: The EUMDS registry collects prospective observational data on lower-risk MDS patients from 142 centers in 17 countries. Red blood cell (RBC) transfusions remain one of the cornerstones in supportive care. Patients are prone to iron overload due to long-term iron accumulation either caused by RBC transfusions or ineffective erythropoiesis and associated iron toxicity through the formation of toxic iron species.Methods: We analyzed serum from 100 lower-risk MDS patients at six-month intervals for ferritin, transferrin saturation (TSAT), hepcidin-25, and toxic iron species (non-transferrin bound iron (NTBI) and labile plasma iron (LPI)) in order to evaluate temporal changes in iron metabolism and presence of potentially toxic forms of ironand their impact on survival.Results: Themedian age was 73 years (range 44-95 years). WHO2001 MDS-subtypes were RCMD (37%), RARS (30%), RA (18%), RAEB (7%), 5q-syndrome (4%) and RCMD-RS (4%). The table shows iron parameters at registration, 1 and 2 years follow-up both in transfusion-dependent (TD) and transfusion-independent (TI) patients and according to: MDS-RS (RARS/RCMD-RS) or MDS Other (RA/RCMD/RAEB/5q-syndrome).Serum ferritin levelswere increased in TD patients, compared to TI patients. Ferritin correlated significantly with hepcidin-25 (r=0.57, p<0.001) as well as CRP (r=0.27, p<0.001). Hepcidin-25 levelswere elevated in TD patients at registration and remained elevated during follow-up (Table 1). Hepcidin levels were low in MDS-RS TI patients at all time points compared to nonRS MDS patients (Table 1).NTBI levelswere elevated in TD patients compared to TI patients at registration and during follow-up. Also TI MDS-RS patients had NTBI levels above the lower limit of detection (LLOD; 0.47 µmol/L). NTBI levels over time were highest in TD MDS-RS patients.LPI levelswere mainly increased above LLOD (0.21 µmol/L) in patients with high TSAT levels (>80%). Both NTBI and LPI had a stronger association with TSAT (Figure 1AC) compared to ferritin (Figure 1BD). Elevated LPI levels in combination with high TSAT levels (>80%) occurred almost exclusively in patients with MDS-RS and/or previous transfusions (Figure 1AC).Cox regression analysis for overall survival was adjusted for age at diagnosis, cumulative number of transfused units and TSAT, NTBI or LPI respectively as a time varying covariate. LPI showed a negative impact on overall survival (HR 1.97; 95%CI 0.39-9.92), independent of transfusion status (Figure 2), while NTBI and TSAT did not show relevant impact on overall survival (HR 1.04; 95% 0.91-1.20; HR 1.00; 95%CI 0.99-1.02, respectively).Conclusion: This is the first clinical outcome study which illustrates LPI as a clinically relevant assay format for identification of the toxic fraction of overt iron overload and its negative impact on overall survival.Table 1Ferritin, Hepcidin-25, NTBI, LPI during follow-upIron parameterRegistration1 yr follow-up2 yrs follow-upNMedian (p25-p75)NMedian (p25-p75)NMedian (p25-p75)Ferritin (µg/L)MDS Other: TI MDS Other: TD MDS-RS: TI MDS-RS: TD94 51 13 26 4274.0 (131.0 - 546.0) 188.0 (81.2 - 375.0) 408.0 (272.0 - 665.0) 271.5 (160.0 - 580.0) 947.5 (359.0 - 1333.5)82 34 22 20 6290.5 (149.0 - 845.0) 164.5 (78.3 - 293.0) 837.0 (550.0 - 1538.0) 257.0 (189.5 - 557.5) 1354.0 (859.0 - 2217.0)64 28 15 11 10346.5 (185.5 - 723.5) 204.5 (86.5 - 346.5) 642.0 (264.0 - 1970.0) 357.0 (222.0 - 597.0) 846.0 (590.0 - 1922.0)Hepcidin (nmol/L) MDS Other: TI MDS Other: TD MDS-RS: TI MDS-RS: TD93 51 13 25 44.5 (2.7 - 8.4) 4.4 (2.7 - 8.3) 9.6 (4.5 - 26.3) 3.5 (1.5 - 4.9) 10.4 (7.9 - 13.5)82 34 22 20 65.7 (2.7 - 12.7) 5.0 (2.3 - 8.1) 17.4 (10.2 - 23.7) 3.6 (1.7 - 5.0) 9.4 (8.8 - 14.4)64 28 15 11 105.1 (2.5 - 8.9) 4.6 (2.5 - 7.1) 8.1 (2.4 - 12.8) 2.9 (1.3 - 6.1) 5.3 (2.9 - 9.3)NTBI (µmol/L) 0 RBC units <=10 RBC units >10 RBC units91 78 12 10.65 (0.31 - 1.14) 0.58 (0.30 - 1.13) 0.94 (0.40 - 3.12) 0.81 (0.81 - 0.81)76 52 12 120.57 (0.20 - 1.43) 0.53 (0.19 - 0.84) 0.58 (0.16 - 1.32) 2.97 (0.50 - 3.77)60 39 14 70.62 (0.33 - 2.27) 0.52 (0.33 - 1.05) 1.64 (0.39 - 2.27) 3.31 (1.35 - 7.25)LPI (µmol/L) 0 RBC units <=10 RBC units >10 RBC units91 78 12 10.13 (0.06 - 0.18) 0.13 (0.06 - 0.18) 0.13 (0.04 - 0.18) 0.13 (0.13 - 0.13)76 52 12 120.12 (0.05 - 0.18) 0.11 (0.04 - 0.16) 0.07 (0.04 - 0.18) 0.20 (0.15 - 0.34)60 39 14 70.10 (0.06 - 0.17) 0.10 (0.06 - 0.16) 0.11 (0.08 - 0.16) 0.18 (0.04 - 1.08) [Display omitted] [Display omitted] Disclosuresde Witte:Novartis: Research Funding.

Efficacy and Safety of Deferasirox Estimated By Serum Ferritin and Labile Plasma Iron Levels in Patients with Aplastic Anemia, Myelodysplastic Syndrome, or Hematologic Malignancy with Transfusional Iron Overload

INTRODUCTIONPatients receiving red blood cell (RBC) transfusions are at risk of iron overload. Humans do not have a physiologic mechanism to excrete excess iron, and total body iron is regulated primarily by the rate of absorption. Transfusion induced Iron overload can cause significant organ damage and is an important cause of morbidity and mortality.METHODSThis study was an open-label, single-arm, prospective, phase 4, multicenter clinical study to evaluate the efficacy and safety of deferasirox (DFX) in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), or hematologic malignancy (HM). Eligibility criteria were serum ferritin (SF) levels ≥1000 ng/mL and ongoing transfusion requirements. For evaluation of the iron overload, SF and transferrin saturation (TFST) were measured every 4 weeks, and labile plasma iron (LPI) levels were regularly followed once every 6 months. Patients received DFX at an initial dose of 20 mg/kg/day for up to 1 year.RESULTSA total of 109 patients were enrolled. SF levels decreased significantly following treatment (from 2000 to 1650 ng/mL, p=0.003). The median absolute reduction in SF levels was -389 ng/mL (range -5428 to 3788) in AA (p=0.029), -567 ng/mL (range -3040 to 4969) in MDS (p=0.136), and -552 ng/mL (range -2899 to 5451) in HM (p=0.057). Median TFST reduction was -14.9% (range -69.4 to 71.0) in all patients (n = 65, p = 0.064). In the MDS and HM groups, TFST decreased significantly from baseline: -14.9% (range -57.4 to 52.2) in the MDS group (p = 0.040) and -16.3% (range -69.2 to 20.8) in the HM group (p = 0.005), while TFST reduction in the AA group was -7.4% (range -58.3 to 71.0) (p = 0.790). Baseline LPI levels were within normal laboratory ranges in all groups. Mean LPI levels decreased from 0.24 μmol/L at baseline to 0.03 μmol/L at 1 year in all patients (p=0.035). The mean LPI reduction in each group was -0.23±0.41 μmol/L (p=0.220) in AA, -0.26±0.51 μmol/L (p=0.110) in MDS, and -0.19±0.70 μmol/L (p=0.336) in HM. All of the AEs related with DFX were grade 1 or 2, and there were no severe AEs (grade ≥3) reported during the study period. Gastrointestinal disorders were commonly observed among groups (n=32, 29.4%), including diarrhea in 8.3%, nausea in 7.4%, and abdominal discomfort in 5.5% of patients. Overall differences in end organ function, including heart, pancreas, thyroid, and gonad, between baseline and 1-year follow up were not statistically significant. No significant differences in LVEF at 1-year after DFX treatment were seen (p = 0.103). Pancreatic dysfunction measured by FBS (p = 0.480) and C-peptide (p = 0.096) levels did not appear to be affected by iron overload during DFX treatment. The results of thyroid function tests (TFT) were not significantly different between the pre- and post-treatment periods in terms of TSH (p = 0.207), free T3 (p = 0.259), or free T4 (p = 0.654) levels. Gonadal dysfunction was not observed during the DFX treatment.DISCUSSIONICT may be an appropriate option for patients with HM or higher risk MDS. In the current study, DFX successfully reduced serum ferritin and LPI levels in HM from baseline to 1 year of treatment. The roles of ICT or DFX during treatment for HM on infection risk and survival benefits need to be elucidated in prospective studies. In conclusion, DFX reduced serum ferritin and LPI levels in patients with transfusional iron overload. Despite the relatively high percentage of gastrointestinal side effects, DFX was tolerable in all patients. DisclosuresNo relevant conflicts of interest to declare.

Impact of Labile Plasma Iron on Viability of Cultured Mononuclear Cells

Myeloablative conditioning for hematopoietic stem cell transplantation (HSCT) leads to a fast increase of non-transferrin bound iron, including the labile plasma iron (LPI) pool, mainly due to suppression of erythropoiesis. It has been suggested that increased LPI, the redox-active and toxic form of iron, may cause cell damage and ultimately lead to tissue toxicity and other complications commonly observed in the early post-HSCT period. However, this assumption has not been reported yet in this setting. In order to evaluate if increased LPI levels following conditioning can cause cell toxicity, we assessed its impact on viability of mononuclear cells obtained from eight patients undergoing autologous HSCT for multiple myeloma (n=4) and lymphoma (n=4). Firstly, LPI was measured in plasma samples obtained from each patient before (baseline) and after conditioning (on day 0), revealing normal levels (<0.5µM) in all baseline samples (mean: 0.2µM; range: 0-0.4µM) and increased levels in all day 0 samples (mean: 2.0µM; range: 1,1-4.0µM). Then, on day 0, an aliquot from peripheral blood stem cells collected by apheresis was obtained before infusion and mononuclear cells were isolated by Ficoll gradient and cultured in DMEM culture media in duplicates for five days in four experiments: i) without addition of plasma (control), ii) with addition of autologous plasma containing normal LPI levels (baseline sample), iii) with addition of autologous plasma containing increased LPI levels (day 0 sample) and iv) with autologous plasma containing increased LPI levels (day 0 sample) in addition of an iron chelator (50mM deferiprone). Mononuclear cells were cultured with autologous plasma on a 1:1 volume ratio. Viability of cultured mononuclear cells was determined by trypan blue method at 3, 24, 48, 72, 96 and 120 hours. Cell viability decreased over time in all experiments (p < 0.001; Figure). There was no difference in cell viability between cells cultured without addition of plasma (control) and cells cultured with plasma containing normal LPI levels. Cells cultured with plasma containing increased LPI levels presented the lowest viability in relation to the other three culture experiments at all time points (p<0,001). The viability of cells cultured with plasma containing increased LPI levels in addition of iron chelator was similar to that of cells cultured with normal LPI levels, also at all time points (p>0,05). In conclusion, these results indicate that increased LPI levels can decrease substantially the viability of mononuclear cells, an effect that can be prevented or at least attenuated by the use of iron chelators. [Display omitted] DisclosuresNaoum:Novartis Oncology: Research Funding.

Assessmet of Labile Plasma Iron and Hepcidin in Patients Who Undergo Hematopoietic Stem Cell Transplantation

Nontransferrin-bound iron (NTBI) has been reported shortly after myeloablative conditioning in patients undergoing hematopoietic stem cell transplantation (HSCT), and it is assumed that the appearance of unbound iron in this nontransfusion setting possibly reflects a major disturbance in body iron distribution, which is related mainly to its underutilization due to erythropoiesis suppression. There is a concern that labile plasma iron (LPI), the most toxic fraction of NTBI that includes the redox-active forms of iron, can be involved in the occurrence of toxicity and other complications commonly observed in the early post-HSCT period. Recently, we demonstrated that LPI levels, albeit normal at baseline measurements, increased substantially 48 hours after the start of conditioning in HSCT patients and remained increased until engraftment, when it returned to normal levels (Acta Haematol 2014;131:222-226). Here we provide a more detailed analysis of iron status in HSCT patients by adding determinations of hepcidin and standard iron parameters along with LPI in 25 adult patients undergoing first autologous HSCT following myeloablative conditioning. All iron parameters were determined before the start of conditioning (baseline), on day 0 (before stem cells infusion) and upon documented engraftment (Table). The fast and substantial increase in LPI levels on day 0, indeed reflected a disruption of iron homeostasis by conditioning, that was accompanied by a response in hepcidin and TfS levels. However, reutilization of iron by a restored erythropoiesis upon engraftment lead to a substantial drop in LPI, but not in hepcidin levels, possibly due to the fact that production of hepcidin by the liver is not only modulated by iron loading and erythropoietic activity, but also inflammation. Ferritin baseline levels were already increased and did not change throughout the study. Considering all determinations (baseline, day 0 and engraftment), hepcidin levels correlated positively with ferritin (r=0,43; p=0,01; Figure Panel A) and TfS (r=0,37; p=0,02; Figure Panel B); and LPI correlated positively with TfS (r=4,3; p=0,002; Figure Panel C), although increased LPI levels were observed with normal TfS levels in some patients. A tendency of correlation between hepcidin and LPI was found considering only baseline and day 0 levels (r=0,30; p=0,05; Figure Panel D). These results indicate that LPI reflected better the changes in iron status caused by cytotoxic chemotherapy in HSCT patients, and could serve as a target in the eventuality of chelation therapy in the early period of HSCT. The other iron parameters were probably influenced by inflammation, even upon engraftment, and would not behave as appropriate surrogate markers for increased LPI levels.*LPI levels <0.5μM are considered normal.† p<0,05 in relation to baseline levels. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of a novel oral active iron chelator: 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in iron-overloaded and non-overloaded mice

ObjectiveTo evaluate efficacy and toxicity of a novel orally active bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in mice under normal and iron overload conditions. MethodsWild type C57BL/6 mice were fed with normal and 0.2% (w/w) ferrocene-supplemented (Fe) diets, respectively for 240 d and orally given the CM1 (50, 100 and 200 mg/kg) for 180 d. Blood iron profiles, hematological indices, liver enzymes and histopathology were determined. ResultsCM1 treatment lowered plasma levels of labile plasma iron and non-transferrin bound iron, but not ferritin in the Fe-fed mice. However, the treatment did not impact blood hemoglobin level, white blood cell and platelet numbers in both normal diet and Fe diet-fed mice. Interestingly, CM1 treatment did not markedly elevate plasma aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase activities in the normal diet-fed mice but it tended to increase the levels of the liver enzymes slightly in the Fe-fed mice. Hematoxylin and eosin staining result showed no abnormal pathological changes in heart, liver and spleen tissues. ConclusionsIt is clear that CM1 would not be toxic to bone marrow and liver cells under normal and iron-overload conditions.