Page 44 - Haematologica-April 2018

T. Lefebvre et al.
36% of Gaucher patients are anemic at diagnosis,3–5 but there is no direct correlation between anemia and the degree of splenomegaly or cytopenia, suggesting that other mechanisms may be involved. Accordingly, we have recently shown that red blood cells (RBCs) exhibit abnor- mal deformability and adhesion properties that may trig- ger ischemic events and phagocytosis in GD.6 Using ery- throid progenitor cultures derived from peripheral CD34+ cells, we also demonstrated that GD1 patients exhibit ineffective erythropoiesis with increased plasma levels of erythropoietin (EPO) and GDF15 factor.7 The treatment of GD is based on enzyme replacement therapy (ERT)8 using either imiglucerase (Cerezyme®, Genzyme, SANOFI company, Cambridge®, MA, USA) or velaglucerase alfa (VPRIV®, Shire HGT, Lexington, MA, USA). Substrate reduction therapy (SRT) using miglustat (Zavesca®, Actelion Pharmaceuticals, Allschwil, Switzerland) and the new molecule eliglustat (Cerdelga®, Sanofi-Genzyme) may also be used. However, ERT remains the current stan- dard of care for GD treatment, and once started, ERT is generally administered for life.
Several reports have documented the appearance of clinical hyperferritinemia in GD patients in whom the level of serum ferritin may exceed 2-3 times the normal level, suggesting an altered iron homeostasis pathogenesis in GD.4,9-11 Ferritin is an intracellular cytosolic protein that stores iron in almost all tissues, including the liver and spleen, but small amounts are secreted into the serum. The amount of this secreted fraction significantly increas- es when ferritin synthesis is exceeded due to iron accumu- lation in target tissues. However, serum ferritin may also increase in the case of an inflammatory stimulus.12
Iron homeostasis is mainly based on iron storage in the liver and macrophages of the spleen, which together con- tain the largest pool of iron (derived from the phagocyto- sis of senescent red cells and catabolism of heme) and con- trol its release according to the body’s needs (20-30 mg of iron daily). The intestine is the second compartment that provides 1-2 mg of iron, corresponding to the amount lost daily by the body. In macrophages, as well as in entero- cytes, iron is exported to the bloodstream through the iron exporter ferroportin (FPN). Hepcidin, secreted by hepato- cytes and acting as a hyposideremic factor, is the main reg- ulator of these iron fluxes.13,14 Hepcidin was first shown in HEK-293 cells transfected with GFP-tagged ferroportin to regulate iron efflux through a direct interaction with ferro- portin, leading to the internalization and lysosomal degra- dation of the exporter.15 This result was confirmed in pri- mary cultured macrophages expressing endogenous ferro- portin.16 In addition, hepcidin acts on the iron importer DMT1 (divalent iron transporter 1) at the apical side of the enterocytes, possibly leading to efficient inhibition of iron intestinal absorption.17,18 Hepcidin synthesis is regulated by iron through a complex of integral hemochromatosis pro- teins, i.e. HFE, HJV (hemojuvelin) and TfR2 (Transferrin Receptor 2). They tightly co-ordinate signaling through the BMP6/HJV/SMAD pathway and increase hepcidin gene (named Hamp) expression when serum or tissue iron levels increase. Inflammation with an accompanying pro- duction of cytokines such as IL-6 can also contribute to the elevation of hepcidin synthesis through the JAK/STAT3 signaling pathway.19 Thus, inflammatory conditions can raise the levels of hepcidin, leading to the anemia of inflammation characterized by a high plasma ferritin and low transferrin saturation (TS).20 Interestingly, besides
being predominantly produced by the liver, hepcidin was found to be expressed in other organs, although to a lesser extent (e.g. the kidney, gut, and retina).21-28 In addition, hepcidin is expressed in macrophages, where it is sup- posed to act through an autocrine/paracrine pathway to decrease iron release.29 Two previous studies have report- ed contradictory results concerning the serum hepcidin concentration in untreated patients with GD1. Thus, the question of the serum hepcidin concentration and its role in iron metabolism in GD requires more investigation.10,11
In this study, we sought to decipher the origin of hyper- ferritinemia in GD. We investigated iron metabolism parameters and measured the serum hepcidin levels in a large cohort of patients with GD1. In vitro studies, as well as the longitudinal follow up of 10 ERT-treated patients, allowed us to highlight iron retention in Gaucher cells and the improved redistribution of this iron after ERT in patients.
Methods
Patients
We conducted a retrospective observational study on a cohort of 90 patients with type 1 Gaucher Disease (GD1). Three centers participated in the study (Beaujon and Trousseau Hospital, Assistance Publique-Hôpitaux de Paris, and Saint Vincent Hospital, Lille, France). The patients were registered from 2009 to 2015 (for patients' characteristics see Online Supplementary Table S1). Patients with iron overload, defined by hyperferritinemia associated with a transferrin saturation exceeding 45%, and splenectomized patients were excluded from the study. Most of the patients (n=66) were treated with ERT using either imiglucerase or velaglucerase alfa. Twenty-four patients were untreated at the time of the study, and 10 of them had a longitu- dinal biological follow up with blood samples taken every 6-10 months. Only patients who received at least six months of ERT were considered as treated patients, and the longest time that a patient had been treated was 23 years. The cohort was explored according to sex and age. Patients aged 16 years or younger were considered as children (pediatric population). Hyperferritinemia was considered when the serum ferritin values were above 300 μg/L for men and above 150 μg/L for children and women. Cases with hyperferritinemia of known origin were excluded, including chronic or acute inflammatory syndrome, history of transfusion or iron supplementation, cellular lysis, dysmetabolic syndrome, and excessive alcohol consumption. Anemia was determined by hemoglobin levels below 13 g/dL for men, 12 g/dL for women and 11.5 g/dL for children.
The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the local ethics committee.
Hematologic and biochemical analysis
Serum iron, ferritin, transferrin, C-reactive protein (CRP) and soluble transferrin receptor (sTfR) were quantified using the Dimension RXL and Vista 1500 system (Siemens Healthcare, Saint-Denis, France). Transferrin saturation (TS) was calculated as the percent of [serum iron (mmol/L) / serum transferrin (g/L) × 25]. The hemoglobin level was measured on an automated counter (Sysmex, Roissy, France), and quantification of IL-6 was per- formed using a cytometric bead array (BD Bioscience, Le Pont de Cliax, France). Serum hepcidin was quantified by the previously published method of LC-MSMS.30 Biological markers for each subgroup were expressed in mean±Standard Error of Mean (SEM).
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