ALBERT

Description: Background: The complex pathophysiology of Sickle Cell Disease (SCD) makes unlikely that a single therapeutic agent will prevent or reverse all SCD complications. Metabolomic analysis might help in the characterization of the endogenous and exogenous effects of potential new treatments. Metabolites are small molecules that are chemically transformed during metabolism and provide a functional readout of cellular state. Metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. The metabolome is typically defined as the collection of small molecules produced by cells and offers a window for investigating how mechanistic biochemistry relates to cellular phenotype. There are very few reports associated with SCD providing comprehensive measurements of metabolites present in blood. Low arginine bioavailability has been associated with a clinical phenotype of increased hemolytic rate, pulmonary hypertension risk and early mortality. Recently, the FDA approved the use of L-glutamine for the treatment of adults and children with SCD, on the basis of the results of randomized phase 3 clinical trials1, while L-arginine's involvement is under investigation2,3. In this context we aimed to quantify targeted metabolites' abnormalities in patients with Sickle Cell/beta thalassemia (HbS/βThal), to identify pathways that might be of interest to prevent disease complications. Patients and Methods: Thirty adult Caucasian patients with HbS/βThal aged 45.6±10.9y, (43% male), at steady-state were enrolled in the study, while 20 age-matched healthy individuals (45% male) served as controls. Along with measurements of hematologic and blood chemistry parameters, targeted metabolome analyses for 13 aminoacids and 2 aminoacid's derivatives were performed after extraction from dry blood spots (DBSs) on filter paper using LC/MS/MS, with derivatization (AB SCIEX 5500 triple quadrupole QTRAP® LC/MS/MS Systems, Framingham, MA, USA). Results: Multiple metabolite differences are identified in HbS/βThal vs. Controls (Figure1). From metabolites involved in the biosynthesis of glutathione, only L-glutamine's levels were lower in patients with HbS/βThal compared to controls, while 5-oxyproline levels, a catabolic product of glutathione metabolism, were markedly increased in patients with HbS/βThal compared to controls. Urea cycle amino acids, also involved in the production of nitric oxide, L-arginine and L-ornithine concentrations were significantly lower in patients with HbS/βThal compared to controls, with a trend towards lower L-citrulline in patients with HbS/βThal p=0.06). Finally, amino acids involved in catecholamines (dopamine, nor-epinephrine and epinephrine) biosynthesis, such as L-phenylalanine and L-tyrosine and its metabolite succinylacetone levels were significantly lower in patients with HbS/βThal compared to controls. No significant correlations were found between any metabolites and markers of hemolysis, HbF levels or iron burden. Conclusions: This study confirms prior observations concerning aberrations in multiple blood specific amino acid levels in patients with SCD compared to controls, but this is the first report on Caucasian patients with HbS/βThal and on whole blood from DBS. We also identified for the first time in Caucasian patients with HbS/βThal important metabolic abnormalities of glutathione and nitric oxide biosynthesis pathways associated with altered concentrations of the metabolites serving of substrates in these cycles. Importantly, we also demonstrate low levels of L-phenylanine and L-tyrosine, which are essential sources for multiple neurotransmitters biosynthesis and neurobehavioral health. The latter novel observation should be confirmed in larger studies, while measurements of urinary amino acid clearances are necessary to evaluate for potential etiologies of the deficiencies through urinary losses vs. low substrate availability, increased utilization or abnormal metabolism. Patients with SCD are in a precarious state with respect to many amino acid deficiencies, all of which may have clinical consequences warranting further investigation. 1N Engl J Med. 2018 Jul 19;379(3):226-235. 2Anesth Analg. 2017 Apr;124(4):1369-1370. 3Haematologica. 2013 Sep;98(9):1375-82. Figure 1. Figure 1. Disclosures Morris: Pfizer: Consultancy; Calithera: Consultancy; MAST Therapeutics: Research Funding; UCSF-Benioff Children's Hospital Oakland: Patents & Royalties: For nutritional supplement licensed to Lifetrients; FDA: Research Funding; NIH/NHLBI: Research Funding. Voskaridou:Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Hydroxycarbamide (HU) is a myelosuppressive drug marketed since 1968 for the treatment of hematological cancer, and authorized since 2007 in Europe as orphan medicinal product for the prevention of recurrent vaso-occlusive crises including acute chest syndrome in adults and children older than 2 years with sickle cell disease (SCD). ESCORT-HU (European Sickle Cell Disease Cohort – Hydroxyurea) is a multicenter prospective non interventional study implemented in Europe to collect more information about the safety profile of HU and morbi-mortality in SCD patients treated with HU. The study responds to EMA (European Medicines Agency) request and has been approved by the Ethical of Necker Enfants Malades Hospital (Paris, France).The ongoing study involves the largest number so far of patients with SCD treated with HU. Primary endpoints of ESCORT HU are to determine frequency of adverse events, and possible consequent changes of HU treatment. Secondary endpoints are to evaluate morbi-mortality of the disease although in the absence of control group. From June 2008 to June 2014, 483 patients (255 females; 228 males) were enrolled from 3 European countries, Greece (24%), Germany (19%), and France (56%). 67% patients were adults, median aged 37.35 yrs (17-83.5) and 33% were children, median aged 11.06 yrs (2.6-16.9). genotypes were HbS/HbS in 71.4% cases, and compound heterozygotous HbS/β-thalassemia in 22.8 % (Table 1). 137 (28.4%) patients experienced 421 events (Table 2). 132 (32.2%) of these events may be attributed to HU. The safety profile is roughly similar in children and adults. As expected the most frequent side effects were firstly blood disorders (n=86 events, 42.4%) such as neutropenia or thrombocytopenia. In all cases, these cytopenias were rapidly resolved with the transitory stop of HU. 71 events related to skin and subcutaneous tissue disorders were observed, mostly cutaneous dryness, skin reactions, alopecia and nails or skin pigmentation; 4 patients had a leg ulcer (34.8%). Most of these events are ongoing or stabilized despit the decrease of HU. No secondary cancer has been reported until now. Even if HU is an old drug with a relatively well-known safety profile, some uncertainties remain in terms of long-term safety as well as tolerance in the youngest people. The main interest of ESCORT HU is to offer the possibility of safety surveillance of hydroxycarbamide in European sickle cell patients. Table 1 Demographic data Adults Children 〈 17 years old Total Number of patients 322 (67%) 161 (33%) 483 Females/Males 183/139 72/89 255/228 Median age (yrs) (range) 37.35 (17-83.5) 11.06 (2.6-16.9) 28.58 Genotype SS 206 (64%) 139 (86.3%) 345 (71.4%) SC 1 (0.3%) 3 (1.86%) 4 (0.8%) Sβ0 51 (15.8%) 11 (6.8%) 62 (12.8%) Sβ+ 46 (14.2%) 2 (1.2%) 48 (9.9%) Other 18 (5.5%) 6 (3.7%) 24 (4.9%) Treatment with HU before enrollment in ESCORT HU No of pts 232 83 315 (65%) Median duration (range) of HU treatment before ESCORT HU 8.2 yrs (0.5 ans-24 yrs) 3. 1 yrs ( 71 days – 8.9 yrs) 6.85 (71 days-24 years)] HU ESCORT Daily mean dose (mg/kg/d) 16.11 ± 4.79 19.63 ± 4.69 17.32 ± 4.94 Abstract 564. Table 2 The most frequent events of hydroxycarbamide in the two populations of SCD patients ADULTS CHILDREN No ofGerman(%) No of adults No ofEpisodes(%) No of children Total(% /411) Events Related to HU treatment (Siklos®)(%**) Blood and lymphatic system disorders (%) 32 (17.7) 22 54 (31.03) 28 86 (20.9) 56 (65.1) Skin and subcutaneous tissue disorders (%) 42 (23.2) 28 29 (16.7) 19 71 (17.3) 46 (64.8) Nervous system disorders Headache (24), Dizziness/vertigo (14), 32 (17.7) 23 12(6.9) 10 44 (10.7) 11 (25) Gastrointestinal disorders Nausea (14), diarrhea (8), other (14) 20 (11) 17 23 (13.2) 16 43 (10.4) 7 (16.3) Metabolic and nutrition disorders: vit D deficiency (17), weight gain (5) 13 (7) 11 18 (18.3) 18 36 (8.75) 4 (11.1) Fever 11 (6) 10 12(6.9) 7 23 (5.6) 1 (4.3) Cardiac disorders (hypertension, bradycardia, chest pain, cardiomegaly) 4 4 2 2 6 1 (16.6) General disorders : fatigue 5 5 0 0 5 0 Hepatobiliary disorders 2 2 0 0 2 0 Neoplasms benign, malignant and unspecified (incl. cysts and polyps) Harmatoma, benign vulvar sebaceous cyst 2 2 0 0 2 0 Renal & urinary disorders 2 2 0 0 2 0 Reproductive system and breast disorders 3 3 0 0 3 0 Other 13 13 21 14 34 6 (17.1%) 181 80 /181(24.8%) 174 57 / 174(35.4%) 411 132/411 (32.2%) ** compared to the total number of “system organ class” events Disclosures No relevant conflicts of interest to declare.

Description: Abstract 806FN2 POEMS syndrome is defined by the presence of peripheral neuropathy (P), monoclonal plasma cell disorder (M), organomegaly (O), endocrinopathy (E) and skin changes (S). The majority of patients have sclerotic bone lesions, although the underlying mechanisms are still unclear. Increased circulating vascular endothelial growth factor (VEGF) is another feature of the disease. However, there is almost no information on the role of other angiogenic molecules in POEMS biology. The aim of the study was to evaluate bone metabolism and angiogenic cytokines in POEMS and compare the results with multiple myeloma (MM) and osteosclerotic patients of other etiology. We retrospectively studied 61 POEMS patients (40M/21F, median age 52 years) who were diagnosed between 1996 and 2010 and were treated and followed in Mayo Clinic Rochester (MN, USA), Hôpital Saint-Louis, Paris (France) and in Alexandra Hospital, Athens (Greece). We evaluated the following indices of bone remodeling and angiogenesis in all patients at diagnosis and in 22 of them one month after front-line treatment: i) osteoclast regulators [sRANKL and osteoprotegerin (OPG)]; ii) osteoblast inhibitor dickkopf-1 (Dkk-1); iii) bone resorption marker CTX; iv) bone formation markers [bone-specific alkaline phosphatase (bALP) and osteocalcin]; and v) angiogenic cytokines [VEGF, angiogenin (ang), angiopoietin (angp)-1 and -2]. These molecules were also measured in 60 newly diagnosed, untreated MM patients (34M/26F, median age: 54 years) and 44 healthy controls. Bone markers were also evaluated in 24 patients with HbS/beta-thalassemia (10M/14F, median age: 43 years) who presented with osteosclerosis, defined as osteosclerotic bone lesions in plain radiography and high T-score of lumbar spine DXA (median: +3.6, range: +2 to +7.9). All POEMS patients presented with P and M, while 90% had E, 83% had S, 61% had O and 32% had papilloedema. One or more documented sclerotic bone lesions in plain X-rays were observed in 78% of patients (diffuse lesions in 44%), while 43% of patients had also an osteolytic component. Seven patients had Castleman disease. At diagnosis, compared to controls, POEMS patients had increased levels of bALP (mean ± SD: 48.3 ± 22.9 IU/L vs. 21.6 ± 8.2 IU/L; p

Description: Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Bone metabolism is altered in thalassemia. Osteoclast function is elevated, while osteoblast activity seems to be reduced and thus the balance of bone remodeling is in favor of bone loss. The exact mechanisms of osteoblast dysfunction have not been fully clarified to-date. Wingless-type (Wnt) signaling is an important pathway for osteoblast differentiation. Dickkopf-1 (Dkk-1) protein is an inhibitor of Wnt pathway and is implicated in the pathogenesis of several bone disorders. Collagen type-I is the main structural protein of the bone. The collagen type-I alpha (COLIA)-1 specific protein (Sp)-1 polymorphism has been related to osteoporosis in thalassemia. The aim of this study was to evaluate the serum levels of Dkk-1 in patients with thalassemia-induced osteoporosis who receive therapy with zoledronic acid (ZOL) and evaluate possible correlations with clinical and laboratory data, including the COLIA-1 Sp1 polymorphism. Sixty-six patients (21M/45F; median age 35.5 years) with thalassemia and osteoporosis were studied. Patients were blindly randomized to receive ZOL at a dose of 4 mg, iv, in 15 min infusion, every 6 months (group A, n=23) or every 3 months (group B, n=21), or to receive placebo every 3 months (group C, n=22) for a period of one year. All patients received oral calcium (500 mg) during the treatment period. Dkk-1 was measured at baseline and after 12 months of therapy using ELISA methodology (Biomedica Medizinprodukte, Wien, Austria) along with a series of serum bone remodeling indices: bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], bone formation markers [bone-specific alkaline phosphatase (bALP), osteocalcin, and C-terminal propeptide of collagen type-I (CICP)], and osteoclast regulators [receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and osteopontin]. The above bone markers were also evaluated in 30, age- and gender-matched, healthy controls. The G--〉T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the COLIA-1 gene was detected by polymerase chain reaction using mutagenesis primers followed by restriction enzyme analysis in all patients. BMD of the lumbar spine (L1-L4), femoral neck (FN) and wrist (W) was determined using DEXA, before and 12 months after treatment. At baseline, all patients had increased serum levels of Dkk-1 (mean±SD: 39±17.1 pmol/L) compared to controls (27.4±9.7 pmol/L; p

Description: Deferasirox is an oral iron chelator approved for the management of iron overload in thalassemia major (TM). However, there are some concerns for its effect on renal function. Cystatin C (Cys-C) is a cysteine protease inhibitor, which is considered as a sensitive marker of GFR. Inflammation process has been recently implicated in TM pathophysiology. The aim of this study was to evaluate the effect of deferasirox on renal function and inflammatory cytokines in 52 TM patients. Deferasirox was administered at a dose between 10–30 mg/kg/day for a 12-month period. Serum Cys-C, serum creatinine (Cr), clearance of Cr (Ccr), albuminuria and inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), IL-1α, IL-1β, IL-4, IL-10 and transforming growth factor (TGF)-β1 and β2, were measured at baseline and then after 6 and 12 months post-deferasirox therapy. Standard hematology and biochemistry was evaluated monthly. Serum Cys-C was measured using a latex particle-enhanced nephelometric immunoassay (Dade Behring, Liederbach, Germany). Serum levels of the above cytokines were determined using ELISA (R&D Systems, Minneapolis, MN, USA, for ILs, and Diaclone, Bensancon, France for TNF-α, TGF-β1 and TGF-β2). Ten healthy blood donors were also evaluated as control group. At baseline, TM patients had elevated values of Cys-C (p1.4 mg/dl) and only 6 (11.5%) had low Ccr (

Description: Hydroxycarbamide (hydroxyurea; HU) is now considered as the main pharmacological agent capable to prevent the painful crises, to reduce the frequency and length of hospital admissions, and to improve the quality of life of patients with sickle-cell disease (SCD); whether HU can prevent the severe chronic complications or modify the mortality of these patients remains still an interesting unresolved question. The present study aims to evaluate the above effects of HU in a large number of SCD patients who received HU over long period of time and were followed in a single Center. To this effect, we evaluated the records of 330 patients with SCD who have been followed in the Thalassemia Center of Laikon Hospital over the last twenty years (136M/194F; median age: 42 years, range: 20- 76 years); 34 with homozygous HbS (SS) and 296 compound heterozygotes for HbS and beta-thalassemia. Of the latter, 131 patients had HbS/beta0-thal and 165 HbS/beta+-thal (107 with the IVSI-110 and 58 with the IVSI-6 thalassemic mutation). Administration of HU was started as early as 1991; progressively, the total number of patients who received HU reached 131 (“group A”); the remaining 199 were treated conventionally (“group B”). The mean age of the patients in the two groups was similar (mean ±SD: 43.3±9.9 years for group A and 44.1±12.6 years for group B) as well as the levels of HbF. The usual dosage of HU was 20 mg/kg/day, except for some patients, where toxicity or lack of effectiveness imposed modifications of the dosage in the range of 15 to 35 mg/kg/day. The median follow-up period was 8 years (range: 0.1–17 years) for the patients who received HU and 5 years (range: 0.1–18 years) for those who did not receive HU. Apart rare exceptions, patients reported every 4–8 weeks to the Outpatient Clinic, where they had a thorough clinical and laboratory evaluation, including FBC and reticulocytes, LDH, bilirubin, other basic biochemistries and HbF using conventional techniques. The percentage of HbS/HbS, HbS/beta0-thal, and HbS/IVSI-110 patients who were given HU was 70%, 49% and 40% respectively; in contrast, very few HbS/IVSI-6 compound heterozygotes had symptoms justifying use of the drug (3.6%). Patients receiving HU showed a dramatic reduction of the frequency of severe painful crises (from 7.4±6.5 episodes per year pre-HU to 0.2±0.4 episodes post-HU; p

Description: Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Patients with thalassaemia intermedia (TI) seem to have a more expanded bone marrow with pressure on cortical bone, which causes pain and bone loss in several cases. The measurement of soluble transferrin receptor (sTfR) and erythropoietin (Epo) in the serum is considered as accurate marker of erythropoietic activity in thalassemia. Bisphosphonates are potent inhibitors of osteoclast activity and have been used for the management of thalassemia-induced osteoporosis. The aim of this study was to evaluate the effect of zoledronic acid, the most potent aminobisphosphonate, on bone mineral density (BMD) of patients with TI and explore possible correlations with bone marrow expansion and erythropoietic activity. Thirty-five patients with TI and osteopenia or osteoporosis (13M/22F, median age 45 years) were evaluated. Twenty-three were randomized to receive zoledronic acid, 4 mg, IV, every 3 months (n=12) or every 6 months (n=11) for one year, while 12 patients received placebo every 3 months. There was no difference in terms of the presence of gonadal dysfunction between the three studied groups. BMD of the lumbar spine (L), femoral neck and forearm was determined in all patients, using DEXA, before and 12 months after treatment. Bone marrow expansion was assessed by the measurement of sTfR and Epo serum levels, using an ELISA methodology (R&D Systems, Minneapolis, MN, USA), before and 12 months post zoledronic acid or placebo administration. In all patients markers of bone remodelling, such as C-telopeptide of collagen type-I (CTX) and bone specific alkaline phosphatase (bALP) were also measured by ELISA (Nordic Bioscience Diagnostics, Herlev, Denmark, and Quidel, San Diego, CA, USA, respectively). Patients were asked to quantify their degree of bone pain on Huskisson’s visual analogue scale and the McGill-Melzack scoring system before and 12 months post-therapy. All patients had increased values of sTfR, Epo, CTX, and bALP compared with 40 controls of similar age and gender (p

Description: Abstract 2018 Poster Board I-1040 Angiogenesis is a crucial process in the pathogenesis of several inflammatory, autoimmune and malignant diseases. Endothelial damage and inflammation make a significant contribution to the pathophysiology of sickle cell disease (SCD) and the beta-thalassemia syndromes. However, there are very limited data in the literature for the role of angiogenic cytokines in the pathogenesis of thalassemia major (TM) and of double heterozygocity of SCD and beta-thalassemia (HbS/beta-thal). Deferasirox (Exjade®) is a once-daily orally administered iron chelator approved for the treatment of transfusional iron overload in patients with transfusion-dependent anemia which seems to reduce markers of inflammation in TM. The aim of this prospective study was to evaluate the levels of angiogenic and inflammatory cytokines in patients with TM and HbS/beta-thal with iron overload who received chelation therapy with deferasirox. Forty-five patients (16M/29F) with TM and 20 patients (7M/13F) with HbS/beta-thal were evaluated. Deferasirox was administered at a dose between 10-30 mg/kg/day based on the number of blood transfusions received before the initiation of treatment. After 3 months, dose adjustments (increases) were allowed in increments of 5 mg/kg/day every 3 months as required to reduce markers of iron overload. Deferasirox was given for a period of 12 months. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin (Ang), angiopoietin (Angp)-1 and -2 and of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-á), IL-1á, IL-1β, IL-4, IL-10 and transforming growth factor (TGF)-β1 and β2, were measured at baseline (day 1 of deferasirox administration) and then after 12 months post-deferasirox therapy, using ELISA methodology (R&D Systems, Minneapolis, MN, USA, for angiogenin cytokines and ILs and Diaclone, Bensancon, France for TNF-á, TGF-β1 and TGF-β2). Twenty healthy blood donors of similar age and gender were also evaluated as control group. Standard hematology and biochemistry was evaluated monthly in all patients. Patients with both TM and HbS/beta-thal had increased levels of all studied angiogenic cytokines compared with healthy controls but reduced levels of Angp-1/Angp-2 ratio (p