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Red blood cell transfusions (RBCT) remain the cornerstone of supportive care in lower-risk myelodysplastic syndrome (LRMDS) [1]. Transfusion dependency in LRMDS patients is associated with inferior outcomes, mainly attributed to severe bone marrow failure [2]. However, iron toxicity, due to frequent RBCT or ineffective erythropoiesis, may be an additional negative prognostic factor [3,4,5,6]. Recently, much progress has been made in unraveling the iron metabolism. The peptide hormone hepcidin is the key regulator by inhibiting iron uptake through degradation of ferroportin, a cellular iron exporter [7]. Erythroferrone and GDF15, produced by erythroblasts, inhibit hepcidin production, which leads to increased uptake and cellular release of iron for the purpose of erythropoiesis [8]. The pathophysiology of iron metabolism in MDS is still not completely understood. Exceedingly high reactive oxygen species (ROS) levels are associated with iron toxicity, disease development, and progression in MDS patients [9,10,11,12]. Malondialdehyde (MDA), resulting from lipid peroxidation of polyunsaturated fatty acids, is a biomarker of oxidative stress [10, 12]. Currently, little is known about the prognostic impact of ROS in MDS patients. The aim of this study is twofold: (1) describe iron and oxidative stress parameters over time in LRMDS patients and (2) to assess their effect on overall and progression-free survival. The EUMDS registry prospectively collects observational data on newly diagnosed LRMDS patients from 148 centers in 16 countries in Europe and Israel as of January 2008. All patients provided informed consent. Clinical data were collected at baseline and at each six-monthly follow-up visit. Serum samples were collected prospectively at each visit from 256 patients included in six participating countries. Conventional iron parameters were measured with routine assays. We additionally analyzed hepcidin, growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), non-transferrin bound iron (NTBI), labile plasma iron (LPI), and MDA. Subjects were prospectively followed until death, loss to follow-up, or withdrawal of consent. All iron parameters were measured centrally at the department of Laboratory Medicine of the Radboudumc, Nijmegen, The Netherlands. Serum samples were collected just prior to transfusion in transfusion-dependent patients and stored at −80 °C. Details on the assays and reference ranges of hepcidin, GDF15, sTfR, NTBI, LPI, and MDA are provided in the supplement. The Spearman rank test was used to evaluate correlations between iron parameters. We stratified the results by transfusion dependency per visit and the presence of ring sideroblasts. When evaluating temporal changes in iron parameters, with linear quantile mixed models, we excluded patients from the timepoint they received iron chelation therapy. Overall survival (OS) was defined as the time from MDS diagnosis to death or, in case of progression-free survival, to date of progression or death; patients still alive at the end of follow-up were censored. Time-dependent Kaplan–Meier curves and cox proportional hazards models were used. In total, 256 consecutive patients, were included in this study. Over five six-monthly visits, 1040 samples were collected. Table 1 describes the patient characteristics. Most patients without ring sideroblasts were transfusion-independent at diagnosis (nonRS-TI; 55.9%), 18.8% with ring sideroblasts were transfusion-independent (RS-TI), 18.4% without ring sideroblasts were transfusion-dependent (nonRS-TD), and 7% with ring sideroblasts were transfusion-dependent patients (RS-TD). The median follow-up time was 6.6 years (95% CI 5.9–7.0). LPI was positively correlated with transferrin saturation (TSAT) (r = 0.15, p < 0.001, Fig. S1). LPI values increased exponentially at TSAT values above 80%. This effect was most pronounced in the transfusion-dependent groups, but also observed in the RS-TI group. MDA was weakly correlated with NTBI (r = 0.09, p = 0.069) and negatively correlated with hemoglobin level (r = −0.1, p = 0.033). GDF15 and hepcidin were negatively correlated in the RS-TI and nonRS-TD group and significantly negatively correlated in the RS-TD group (r = −0.34, p = 0.007, Fig. S2). Serum ferritin levels were elevated in all subgroups with a mean value of 858 µg/L at visit 5. The highest serum ferritin levels were observed in the RS-TD group (mean value at visit 5: 2092 µg/L, Table S1). Serum ferritin increased significantly per visit in the RS-TD group (beta 454.46 µg/L; 95% CI 334.65–574.27), but not in the other groups (Table S2). All subgroups, except for the nonRS-TI, had elevated TSAT levels. TSAT levels were most markedly increased in the RS-TD group with a mean TSAT of 88% at visit 5 (Table S1). In both transfusion-dependent groups the median increase per visit was significant (Table S2). LPI was elevated in the RS-TD group exclusively with a mean value of 0.59 µmol/L at visit 5 (Table S1). NTBI was elevated in all subgroups, with the highest values in the RS-TD group (Table S1). The increase in median NTBI level was significant in both transfusion-dependent groups (Table S2). Hepcidin levels were markedly elevated in the nonRS-TD group. Interestingly, hepcidin levels were lower in the RS-TD group, probably reflecting ineffective erythropoiesis, likewise supported by lower hepcidin/ferritin ratios in RS groups (Table S1). Median hepcidin levels increased over time in the transfusion-dependent subgroups only (Table S2). GDF15 levels, analyzed in the light of its potential role in hepcidin suppression, were increased in all subgroups (Table S1). The RS subgroups had higher GDF15 levels compared to the nonRS groups, reflecting increased erythropoiesis. Mean sTfR levels were within the reference range in all subgroups except for the RS-TI group, which showed elevated levels, reflecting...

As part of the UK NCRI AML17 trial, adult acute myeloid leukaemia patients in remission could be randomised to receive the mTOR inhibitor everolimus, sequentially with post-induction chemotherapy. Three hundred and thirty-nine patients were randomised (2:1) to receive everolimus or not for a maximum of 84 days between chemotherapy courses. The primary endpoint was relapse free survival (RFS). At 5 years there was no difference in Relapse Free Survival (29% vs 40%; OR 1.19 (0.9-1.59) p=0.2), cumulative incidence of relapse (60% vs 54%: OR 1.12( 0.82-1.52): p=0.5) or overall survival (45% vs 58%: OR 1.3 (0.94-1.81): p=0.11). The independent Data Monitoring Committee advised study termination after randomisation of 339 of the intended 600 patients due to an excess mortality in the everolimus arm without any evidence of beneficial disease control. Dose delivery of everolimus was variable, but there was no evidence of clinical benefit in patients with adequate dose delivery compared with no treatment. This study suggests that the addition of mTOR inhibition to chemotherapy provides no benefit. Clinical Trial: ISRCTN55675535

The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.

Objectives: It is widely assumed that, at matched transfusional iron-loading rates, responses to chelation therapy are similar, irrespective of the underlying condition. However, data are limited for rare transfusiondependent anaemias, and it remains to be elucidated if response differs, depending on whether the anaemia has a primary haemolytic or production mechanism. Methods: The efficacy and safety of deferasirox (Exjade) in rare transfusion-dependent anaemias were evaluated over 1 yr, with change in serum ferritin as the primary efficacy endpoint. Initial deferasirox doses were 10–30 mg⁄ kg ⁄ d, depending on transfusion requirements; 34 patients had production anaemias, and 23 had haemolytic anaemias. Results: Patients with production anaemias or haemolytic anaemias had comparable transfusional iron-loading rates (0.31 vs. 0.30 mL red blood cells ⁄ kg ⁄ d), mean deferasirox dosing (19.3 vs. 19.0 mg⁄ kg ⁄ d) and baseline median serum ferritin (2926 vs. 2682 ng ⁄ mL). Baseline labile plasma iron (LPI) levels correlated significantly with the transfusional iron-loading rates and with serum ferritin levels in both cohorts. Reductions in median serum ferritin levels were initially faster in the production than the haemolytic anaemias, but at 1 yr, similar significant reductions of 940 and 617 ng ⁄mL were attained, respectively ()26.0% overall). Mean LPI decreased significantly in patients with production (P < 0.0001) and haemolytic (P = 0.037) anaemias after the first dose and was maintained at normal mean levels (

The prospective 1-year Evaluation of Patients' Iron Chelation with Exjade (EPIC) study enrolled a large cohort of 116 patients with aplastic anemia; the present analyses evaluated the efficacy and safety of deferasirox in this patient population. After 1 year, median serum ferritin decreased significantly from 3254 ng/mL at baseline to 1854 ng/mL (P < .001). Decreases occurred in chelation-naive (3229-1520 ng/mL; P < .001, last-observation-carried-forward analysis), and previously chelated (3263-2585 ng/mL; P = .21, last-observation-carried-forward analysis) patients and were reflective of dose adjustments and ongoing iron intake. Baseline labile plasma iron levels were within normal range despite high serum ferritin levels. The most common drug-related adverse events were nausea (22%) and diarrhea (16%). Serum creatinine increases more than 33% above baseline and the upper limit of normal occurred in 29 patients (25%), but there were no progressive increases; concomitant use of cyclosporine had a significant impact on serum creatinine levels. The decrease in mean alanine aminotransferase levels at 1 year correlated significantly with reduction in serum ferritin (r = 0.40, P < .001). Mean absolute neutrophil and platelet counts remained stable during treatment, and there were no drug-related cytopenias. This prospective dataset confirms the efficacy and well characterizes the tolerability profile of deferasirox in a large population of patients with aplastic anemia. This study was registered at www.clinicaltrials.gov as #NCT00171821.

PubMedID: 20451251 The prospective 1-year EPIC study enrolled 341 patients with myelodysplastic syndromes (MDS); although baseline iron burden was >2500. ng/mL, ~50% were chelation-naïve. Overall median serum ferritin decreased significantly at 1 year (p=0.002). Decreases occurred irrespective of whether patients were chelation-naïve or previously chelated; changes were dependent on dose adjustments and ongoing iron intake. Sustained reductions in labile plasma iron were observed. Discontinuation rate (48.7%) and adverse event profile were consistent with previously reported deferasirox data in MDS. Alanine aminotransferase levels decreased significantly; change correlated significantly with reduction in serum ferritin (p

The prospective 1-year EPIC study enrolled 341 patients with myelodysplastic syndromes (MDS); although baseline iron burden was >2500. ng/mL, ∼50% were chelation-naïve. Overall median serum ferritin decreased significantly at 1 year (p=0.002). Decreases occurred irrespective of whether patients were chelation-naïve or previously chelated; changes were dependent on dose adjustments and ongoing iron intake. Sustained reductions in labile plasma iron were observed. Discontinuation rate (48.7%) and adverse event profile were consistent with previously reported deferasirox data in MDS. Alanine aminotransferase levels decreased significantly; change correlated significantly with reduction in serum ferritin (p

Background Following a clinical evaluation of deferasirox (Exjade) it was concluded that, in addition to baseline body iron burden, ongoing transfusional iron intake should be considered when selecting doses. The 1-year EPIC study, the largest ever investigation conducted for an iron chelator, is the first to evaluate whether fixed starting doses of deferasirox, based on transfusional iron intake, with dose titration guided by serum ferritin trends and safety markers, provides clinically acceptable chelation in patients (agedor=2 years) with transfusional hemosiderosis from various types of anemia.The recommended initial dose was 20 mg/kg/day for patients receiving 2-4 packed red blood cell units/month and 10 or 30 mg/kg/day was recommended for patients receiving less or more frequent transfusions, respectively. Dose adjustments were based on 3-month serum ferritin trends and continuous assessment of safety markers. The primary efficacy end-point was change in serum ferritin after 52 weeks compared with baseline.The 1744 patients enrolled had the following conditions; thalassemia (n=1115), myelodysplastic syndromes (n=341), aplastic anemia (n=116), sickle cell disease (n=80), rare anemias (n=43) and other transfused anemias (n=49). Overall, there was a significant reduction in serum ferritin from baseline (-264 ng/mL; P0.0001), reflecting dosage adjustments and ongoing iron intake. The most common (5%) adverse events were gastrointestinal disturbances (28%) and skin rash (10%). Conclusions Analysis of this large, prospectively collected data set confirms the response to chelation therapy across various anemias, supporting initial deferasirox doses based on transfusional iron intake, with subsequent dose titration guided by trends in serum ferritin and safety markers (clinicaltrials.gov identifier: NCT00171821).

The existence of separate gelatinase granules in human neutrophils has been a matter of debate in recent years. We have demonstrated that the 135-kD form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel 25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL) that, in addition to being complexed with part of the gelatinase, is localized in free form in peroxidase-negative specific granules. Because this association was not appreciated in earlier studies, we decided to reassess the ultrastructural localization of gelatinase using specific antibodies without immunoreactivity towards NGAL. Double-labeling immunogold electron microscopy was performed on frozen thin sections of human neutrophils. Twenty-four percent of all peroxidase-negative granules were labeled with antigelatinase antibody, but not with antilactoferrin antibody. These granules are defined as gelatinase granules. Sixteen percent reacted with antilactoferrin antibody but not with antigelatinase antibody. The rest (60%) reacted with both antibodies. All granules labeling for lactoferrin are defined as specific granules. Gelatinase granules were observed as round and oval forms of considerably smaller size than specific granules, and were less electron dense. Isolated granules obtained by subcellular fractionation were also examined by immunoelectron microscopy. This demonstrated that peroxidase-negative granules comprise a continuum from the most dense granules that contain lactoferrin but no gelatinase to the lightest that contain gelatinase but no lactoferrin. Thus, gelatinase granules do exist as a subpopulation of peroxidase-negative granules and may allow for exocytosis of gelatinase during neutrophil diapedesis without substantial mobilization of other peroxidase-negative granules, ie, specific granules.

A 25-kDa protein was found to be associated with purified human neutrophil gelatinase. Polyclonal antibodies raised against gelatinase not only recognized gelatinase but also this 25-kDa protein. Specific antibodies against the 25-kDa protein were obtained by affinity purification of the gelatinase antibodies. Immunoblotting and immunoprecipitation studies demonstrated the 135-kDa form of gelatinase to be a complex of 92-kDa gelatinase and the 25-kDa protein, and the 220-kDa form was demonstrated to be a homodimer of the 92-kDa protein, thus explaining the 220-, 135-, and 92-kDa forms characteristic of neutrophil gelatinase. The 25-kDa protein was purified to apparent homogeneity from exocytosed material from phorbol myristate acetate-stimulated neutrophils. The primary structure of the 25-kDa protein was determined as a 178-residue protein. It was susceptible to treatment with N-glycanase, and one N-glycosylation site was identified. The sequence did not match any known human protein, but showed a high degree of similarity with the deduced sequences of rat alpha 2-microglobulin-related protein and the mouse protein 24p3. It is thus a new member of the lipocalin family. The function of the 25-kDa protein, named neutrophil gelatinase-associated lipocalin (NGAL), remains to be determined.