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International audience; Given the lack of information about safety of the COVID-19 vaccines for sickle cell disease (SCD) patients, we sought to determine whether COVID-19 vaccine was associated with subsequent hospital admission for vaso-occlusive events (VOEs). We included 402 patients with SCD, including 88 regularly transfused. As of July 31, 2021, 213 (53.0%) of them had received a least one dose of COVID vaccine (Pfizer 93.0%). We showed similar risk of hospital admission for a VOE among vaccinated patients (whether transfused or not) and among a control group of non-vaccinated patients matched for age, sex and genotype.

International audience

Preoperative transfusion (PT) reduces acute postoperative vaso-occlusive events (VOE) in sickle cell disease (SCD), but exposes patients to alloimmunization, encouraging a recent trend towards transfusion sparing. The aim of this study was to investigate the benefit–risk ratio of PT before cholecystectomy on the occurrence of postoperative VOE. Adult SCD patients who underwent cholecystectomy between 2008 and 2019 in our center were included. Patients’ characteristics, collected retrospectively, were compared according to PT. A total of 79 patients were included, 66% of whom received PT. Gallbladder histopathology found chronic cholecystitis (97%) and gallstones (66%). Transfused patients underwent more urgent surgeries and had experienced more painful vaso-occlusive crises (VOC) in the month before surgery (p = 0.05). Four (8.5%) post-transfusion alloimmunizations occurred, and two of them caused a delayed hemolytic transfusion reaction (DHTR) (4.3%). The occurrence of postoperative VOE was similar between the groups (19.2% vs. 29.6%, p = 0.45). Though not statistically significant, a history of hospitalized VOC within 6 months prior to surgery seemed to be associated to postoperative VOE among non-transfused patients (75% vs. 31.6%, p = 0.10). PT before cholecystectomy exposes to risks of alloimmunization and DHTR that could be avoided in some patients. Recent VOCs appear to be associated with a higher risk of postoperative VOE and prompt the preemptive transfusion of these patients.

β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients’ HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

INTRODUCTION Therapeutic plasma exchange (TPE) is acknowledged to be an effective treatment in life-threatening pediatric disorders. Apheresis for pediatric diseases has been poorly investigated, and most studies to date featured small numbers of patients and lacked control groups. The objective of the present study was to evaluate the tolerance of TPE in pediatric patients. MATERIALS AND METHODS A retrospective cohort study via a web-based electronic case report form including pediatric patients referred for TPE between January 2005 and December 2014. RESULTS A total of 78 patients (median [range] age: 9.8 [0.53-17.93]) and 731 TPE procedures were analyzed. The indications were antibody-mediated rejection (n = 33; 42%) and desensitization therapy (n = 5; 6%) after solid organ or hematopoietic stem cell transplantation, thrombotic microangiopathy (n = 17; 22%), pediatric inflammatory diseases (n = 16; 21%), kidney diseases (n = 6; 8%), and hyperviscosity syndrome (n = 1; 1%). On average, each patient underwent six procedures during the first session [range: 1-19]. In the 2 weeks following the start of a session, 72 patients (92%) presented a total of 311 adverse events (AEs) potentially related to TPE. The risk of AEs was not related to the indication for TPE, the intensity of care, venous access, plasma substitute use, or body weight. None of the deaths was related to the TPE. CONCLUSION We studied one of the largest retrospective pediatric cohorts described to date. Our experience of TPE children's TPE feasibility concerned specific, life-threatening conditions and otherwise treatment-refractory diseases.

Several controlled studies have evidenced good efficacy and short- and mid-term safety profiles for hydroxyurea (HU), which has become the cornerstone for prevention of sickle-cell disease (SCD)-related vaso-occlusive crises. However, there are few large-scale reports on its long-term use and certain caregivers and patients have concerns about its safety. Following the licensing of HU in Europe for children and adults with severe forms of SCD, ESCORT-HU was designed as a Phase IV observational cohort study. It included 1,906 participants, of whom 55% were adults. The most common hemoglobin (Hb) genotypes were HbSS (84.7%) and HbSβ+ (7.0%). The median duration of follow-up was 45 months, for a total of 7,309 patient-years of observation. The dose of HU after one year was 20.6 mg/kg/d for children and 16.3 mg/kg/d for adults. There was a statistically significant decrease in the number of vaso-occlusive episodes lasting > 48 hours, acute chest syndrome episodes, hospitalizations, and the percentage of patients requiring blood transfusions within the first 12 months relative to the year before enrolment. Neutropenia and thrombocytopenia were the most commonly reported adverse effects. No new HU toxicity was identified. Overall, 125 pregnancies were reported in 101 women and no malformations were observed in the neonates. There were 12 pregnancies for partners of male patients treated with HU. One case of fatal myelodysplastic syndrome was reported, for which a causal association with HU could not be excluded. This cohort study of patients with SCD highlights the positive benefit-to-risk ratio of HU in children and adults. This article is protected by copyright. All rights reserved.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

Background Autologous and allogeneic hematopoietic stem cell transplantation of cytokine-mobilized peripheral blood stem cells (PBSCs) is increasingly used to treat patients with hematologic disorders. Different types of vascular access have been exploited for the apheresis procedure, including peripheral veins (PV) and central venous catheter (CVC). In some cases, PV access is unavailable. There are few published data on the efficiency and quality of harvesting with different types of vascular access. This study brings out complications and morbidity of this procedure linked to these different access. Methods We performed a comparative, retrospective, single-center study of hematopoietic stem cell collection using these two types of vascular access. We compared the efficiency and complication rate for 617 adults apheresis sessions in 401 patients and healthy donors, for PBSC collection via PV or CVC between 2010 and 2016. The quality of the HSC product was evaluated in terms of the total CD34 + count and neutrophil contamination. Results The PV and CVC groups did not differ significantly in terms of the quality of the apheresis product, mean ± SD CD34 + cells collected in PV group was 383.1 ± 402.7 × 10e6 and 298.8 ± 372.7 × 10e6 and the level of neutrophil contamination was 21.0 ± 17.8% in the PV group and 20.6 ± 18.4% in the CVC group. The complication rate did not differ between the two groups. Conclusion The type of vascular access for apheresis hematopoietic stem cell harvesting must be determined by trained staff. Successful harvesting can be performed via PV then CVC is not needed or not available.

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for sickle cell disease (SCD) allowing a SCD-free survival over 95% among children with matched HLA-identical donor (Gluckman E. Blood, 2017; Bernaudin F. Haematologica, 2020). In adults, myeloablative HSCT is associated with more graft-versus host disease (GVHD) and higher toxicity (Cappelli B. Haematologica, 2019). More recent approaches using non myeloablative (NMA) conditioning regimen (3 gray (Gy) total body irradiation (TBI) plus alemtuzumab) followed by HLA-identical peripheral blood stem cells (PBSC) and post-transplant sirolimus appear safe in adults (Hsieh MM. JAMA, 2014), but incidence of graft failure might be higher than the one reported after myeloablative conditioning (MAC) (Alzahrani M. Br J Haematol, 2021). Here, we compare outcomes after MAC or NMA conditioning regimens in patients over 15 years old transplanted from a matched related donor (MRD). Patients and methods All consecutive patients transplanted for SCD from a MRD from January 2015 to October 2020 were eligible if they were over 15 years old and received a busulfan-based MAC conditioning regimen or a NMA conditioning regimen. Chimerism was studied by analyzing various polymorphisms after polymerase chain reaction (PCR) amplification of DNA obtained from whole blood cells. Rejection was defined as donor chimerism Results Thirty-four patients were included: 20 in the MAC and 14 in the NMA groups. Median age at transplant was 17 years (range 15-46) without difference between groups. Forty four percent of patients had a history of cerebral vasculopathy, 79% of recurrent vaso-occlusive crises and 76% of acute chest syndrome. ABO major incompatibility was present in 15% of patients. There was no difference in patient characteristics according to the conditioning regimen group except for pre-transplant cerebral vasculopathy, more frequently reported in the MAC group: 70% versus 6% in the NMA group (p In the MAC group, conditioning regimen associated busulfan (12.8 mg/kg IV), cyclophosphamide (N=17) or fludarabine (N= 3) and anti-thymoglobulin (ATG, mostly 20mg/kg). Stem cell source was bone marrow and post-transplant immunosuppression combined cyclosporine and mycophenolate or methotrexate. In the NMA group, conditioning regimen associated 3Gy TBI and alemtuzumab (1mg/kg), followed by PBSC and post-transplant sirolimus. All patients engrafted and no secondary graft failure was observed. One MAC group patient died from GVHD. The 2-year overall and EFS were 95% (CI 95%: 85.9-100) in the MAC group (median follow-up of 39 months (range 8-63)) and 100% (CI 95%: 100-100) in the NMA group (median follow-up of 17 months (range 9-39)). Incidence of grade II-IV acute GVHD was 0% in the NMA group versus 20% in the MAC group (p=0.12). Incidence of chronic GVHD was 0% in the NMA group versus 25% in the MAC group (p=0.06). From the 27 patients with follow-up>12 months, 17/17 discontinued immunosuppressive therapy in the MAC group versus 8/10 in the NMA group. Hematopoietic recovery was faster in the NMA group with less platelet and red blood cell units transfused (Table). Throughout the follow-up, median donor chimerism was higher after MAC transplantation than after NMA transplantation: 98% (range 69-100) and 86 % (range 50-97) respectively at 1 year (p=0.017). Donor chimerism remained above 50% throughout the follow-up in all patients except 1 of the NMA group who displayed stable chimerism between 40 and 50%. All patients achieved HbS level close to the one of their donors. There was no difference between the 2 groups regarding occurrence of infections. MAC transplant was more often associated with hypertension, neurological complications, severe mucositis and need of enteral or parenteral nutrition. Moreover, duration of hospitalization was longer in MAC group: 54 days (range 39-192) versus 35 days in the NMA group (range 21-52) (p Conclusion In this series of adolescents and adults transplanted for SCD, the survival without SCD was excellent with a faster hematological recovery and a lower toxicity after NMA HSCT. Longer follow-up is required to confirm stable mixed donor chimerism and the cure of SCD after NMA approach. Figure 1 Figure 1. Disclosures Joseph: bluebird bio: Consultancy. Boissel: Servier: Consultancy, Honoraria; JAZZ Pharma: Honoraria, Research Funding; Incyte: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; SANOFI: Honoraria; CELGENE: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; PFIZER: Consultancy, Honoraria. Pondarré: ADDMEDICA: Honoraria.

The European Sickle Cell Disease Cohort - Hydroxyurea (ESCORT-HU) study was initiated when HU got an approval in SCD in Europe. This non-interventional prospective cohort study conducted in patients treated with HU according to current clinical practice with SCD was terminated in 2019 after enrolment of 1906 patients in 4 European countries. The main objective was to refine the safety profile of hydroxycarbamide, as well as to identify unexpected toxicities, especially after long-term treatment. However clinical effectiveness evaluated by recording painful crises lasting more than 48 hours, episodes of ACS, number of hospitalisations related to SCD, and biological parameters were also regularly recorded according to the frequency of the hospital visits. At inclusion, 926 (48.7%) patients had been previously treated off the label with HU (as HU was only approved for myeloproliferative disorders at the time), with mean duration of HU treatment of 5.74 ± 4.98 years. The mean age of this subgroup was 27.60 ± 14.81 years compared to 19.75 ± 15.79 years of the patients never been treated with HU before enrolment Of the 926 patients, 299 (32%) were younger than 18 years. As showed in the table 1, hemoglobin level remains unchanged during the first two years but the HbF% increased in the HU-pretreated subgroup. In the "HU-naïve group", despite lower baseline Hb level et HbF% at baseline , similar outcome were obtained after 12 or 24 months. In terms of clinical outcomes, after 1 year of treatment the number of vaso-occlusive crises (VOC), acute chest syndrome (ACS) or hospitalizations decreased dramatically (table 2) in both group. The median duration of follow-up in the cohort was 45 months (0-128). 123.7 ± 62.7 months was the mean total exposure to HU in the HU-pretreated subgroup. In term of safety, neutropenia, thrombocytopenia and dry skin were the most frequent HU- related adverse events reported but with comparable cumulated incidence between the HU-pretreated subgroup and HU-naïve subgroup (Table 3). Conclusion: In real life setting, a significant improvement of the vasoocclusive symptoms was observed. Improvement of the compliance thanks to a treatment dedicated to the disease is probably one reason for better effectiveness; as suggested bythe lower red blood cell MCV was lower than expected at baseline in patients previously treated with HU. It is also possible that the increase in HbF% observed during the treatment could be another reason for clinical benefit. Paule and al (2011) demonstrated that daily regimen of HU were superior to weekly regimen. The fine tuning of the daily dose possible with HU tablets might be another reason of clinical optimization. Disclosures Galactéros: Addmedica:Membership on an entity's Board of Directors or advisory committees.Voskaridou:BMS:Consultancy, Research Funding;ADDMEDICA Company:Consultancy, Research Funding;NOVARTIS Company:Research Funding;GENESIS Company:Consultancy, Research Funding;PROTAGONIST Company:Research Funding;ACCELERON Company:Consultancy, Research Funding.Habibi:Pfizer:Consultancy;Bluebird:Consultancy;Novartis:Consultancy;Addmedica:Consultancy.De Montalembert:bluebird bio:Honoraria, Membership on an entity's Board of Directors or advisory committees;Vertex:Honoraria, Membership on an entity's Board of Directors or advisory committees;Addmedica:Honoraria, Membership on an entity's Board of Directors or advisory committees;Novartis:Honoraria, Membership on an entity's Board of Directors or advisory committees.

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) prevalence has increased rapidly in Europe as a result of an increase in the life expectancy of these patients and the arrival of SCD migrants from Africa. The aim of our study was to compare the phenotypes of adult patients born in Sub-Saharan Africa (SSA) who migrated to France with those of patients with the same origin who were born in France. This single-center observational study compared the demographic, clinical and biological characteristics of SCD adult patients of SSA origin who were born in France or SSA. Data were collected from computerized medical charts. Groups were compared using multivariate logistic regression with adjustment for age, gender and type of SCD. Of the 323 SCD patients followed in our center, 235 were enrolled, including 111 patients born in France and 124 patients born in SSA. SCD genotypes were balanced between groups. Patients born in Africa were older (median age 32.1 (24.4&ndash, 39) vs. 25.6 (22.1&ndash, 30.5) years, p &lt, 0.001) and more often women (n = 75 (60.5%) vs. 48 (43.2%), p = 0.008). The median age at arrival in France was 18 years (13&ndash, 23). The median height was lower among patients born in SSA (169 (163&ndash, 175) vs. 174.5 cm (168&ndash, 179), p &lt, 0.001). Over their lifetimes, patients born in France had more acute chest syndromes (median number 2 (1&ndash, 4) vs. 1 (0&ndash, 3), p = 0.002), with the first episode occurring earlier (19 (11.6&ndash, 22.3) vs. 24 (18.4&ndash, 29.5) years, p &lt, 0.007), and were admitted to intensive care units more often (53.3% vs. 34.9%, p = 0.006). This difference was more pronounced in the SS/S&beta, 0 population. Conversely, patients born in SSA had more skin ulcers (19.4% vs. 6.3%, p = 0.03). No significant differences were found in social and occupational insertion or other complications between the two groups. Patients born in SSA had a less severe disease phenotype regardless of their age than those born in France. This difference could be related to a survival bias occurring in Africa during childhood and migration to Europe that selected the least severe phenotypes.

Sickle cell disease (SCD) in pregnancy can be associated with adverse maternal and perinatal outcomes. Furthermore, complications of SCD can be aggravated by pregnancy. Optimal prenatal care aims to decrease the occurrence of maternal and fetal complications. A retrospective, French, two-center study compared two care strategies for pregnant women with SCD over two time periods. In the first study period (2005-2010), the women were systematically offered prophylactic transfusions. In the second study period (2011-2014), a targeted transfusion strategy was applied whenever possible, and home-based prophylactic nocturnal oxygen therapy was offered to all the pregnant women. The two periods did not differ significantly in terms of the incidence of vaso-occlusive events. Maternal mortality, perinatal mortality, and obstetric complication rates were also similar in the two periods, as was the incidence of post-transfusion complications (6.1% in 2005-2010 and 1.3% in 2011-2014, P = .15), although no de novo alloimmunizations or delayed hemolysis transfusion reactions were observed in the second period. The results of this preliminary, retrospective study indicate that targeted transfusion plus home-based prophylactic nocturnal oxygen therapy is safe and may decrease transfusion requirements and transfusion-associated complications.

Introduction La prevalence de la drepanocytose augmente rapidement en Europe, du fait d’une augmentation de l’esperance de vie des patients drepanocytaires et de l’arrivee de migrants originaires d’Afrique sub-Saharienne (ASS). Le but de l’etude etait de comparer le phenotype de patients adultes nes en ASS, ayant migre en France a celui de patients originaires d’ASS nes en France. Patients et methodes Etude retrospective monocentrique comparant les caracteristiques demographiques, cliniques et biologiques de patients adultes drepanocytaires (tous genotypes) originaires d’ASS, nes en France ou en ASS. Tous les patients de notre cohorte dont les parents etaient originaires d’ASS etaient inclus. Les donnees etaient colligees a partir d’un dossier medical informatise specifique pour les patients drepanocytaires comprenant aussi des donnees sociologiques. Ces 2 groupes etaient compares par regression logistique multivariee, avec ajustement sur l’âge et le sexe. Un avis positif du CERES et de la CNIL etait obtenu. Resultats Parmi 323 patients suivis dans notre centre au 28 fevrier 2018, 235 ont ete inclus : 111 nes en France, 124 nes en ASS. Les genotypes drepanocytaires etaient repartis de facon identique. Les patients nes en ASS etaient plus âges (âge median 32,1 [24,4–39] vs 25,6 [22,1–30,5] ans, p Conclusion Les patients drepanocytaires migrants, nes en ASS, compares aux patients de meme origine geographique parentale nes en France, presentent une maladie moins severe, quel que soit l’âge. Cette observation peut sembler paradoxale etant donne les difficultes d’acces aux soins medicaux en Afrique (notamment a l’hydroxyuree et aux transfusions) mais pourrait resulter d’un biais de mortalite survenant durant l’enfance en Afrique et au cours de l’emigration vers l’Europe, selectionnant les phenotypes les plus favorables.

Therapeutic plasma exchange (TPE) is advocated as a treatment to several conditions and applications: solid organs transplantation, kidney and pediatric immune disorder. Apheresis for children diseases has been poorly investigated in mostly small, uncontrolled studies.The purpose was to report our experience, and provide uniform and relatable data, in order to improve disease management. We included 94 patients, aged under 18 years old, who underwent TPE in the pediatric center of Necker-Enfants-Malades hospital from January 2005 to December 2014. Data were retrospectively collected in an electronic case report form via a web-based data collection system. 78 patients were selected, including 36 females and 42 males with a median age of 9.78 years [range 0.53; 17.93]. They achieved a total number of 731 procedures. Indications were antibody-mediated rejection (n= 33; 42%) or desensitization therapy (n= 5; 6%) for solid organ or hematopoietic transplantations; microangiopathy (n= 17; 22%); renal diseases (n= 6; 8%) and pediatric inflammatory diseases (n= 16; 21%); or hyperviscosity syndrome (n = 1; 1%). Each patient had an average of 6 procedures for the first session [range 1; 19] with a median volume of 1834 ml [range 500; 5000 ml] corresponding to a median total plasma volume (TPV) equivalent of 1.39 l/m2[range: 0.58; 2.1 l/m2].Within 15 days in the beginning of the sessions, 72 patients (92%) presented a total of 311 Advers Events(AEs) potentially related to TPE. 94 AEs were not related to TPE sessions.There was a median of 5 AEs/patients [range: 0; 24]. There was no significant increased risk of AEs due to diseases, intensity of care, venous access, plasma substitute and body weight. Few of AEs were potentially life-threatening and concerned pediatric critical care situations. Allergic reactions represented only 20 AEs for 14 patients (grade I n= 18; grade II n= 1; grade III n= 1). At the endpoint of M12, 15 (19%) patients had died, no death had been related to the TPE process. Nine patients performed a second or a third session of procedures and 10 (13%) patients had severe persistent clinical disease. We describe one of the largest retrospective pediatric cohort to date, to the last international recommendations. Our experience on children TPE feasibility concern specific life‐threatening conditions and otherwise potential refractory diseases. TPE were generally well tolerated and the majority of the adverse effects were anticipated and could therefore be avoided. Based on this work, and in order to progress in the understanding of the TPE field, it would be interesting to increase pediatric data with prospective and multicenter cohort. Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Background LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV), encoding human β-globin with a T87Q substitution. This substitution confers anti-sickling properties to the gene therapy-derived hemoglobin (HbAT87Q) and allows for its quantification in transduced HSCs. The proof of concept for LentiGlobin gene therapy in patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) was established in the recently completed HGB-205 study (NCT02151526). Herein, we provide the safety and efficacy outcomes and long-term follow-up data for all 7 treated patients, 4 with TDT and 3 with SCD. Methods Patients 5−35 years old with TDT (≥ 100 mL/kg of packed red blood cells [pRBCs]/year) or severe SCD (e.g., ≥ 2 acute chest syndromes [ACS] or ≥ 2 vaso-occlusive crises in the preceding year or the year before regular transfusions) were enrolled. CD34+ HSCs were obtained by mobilization and apheresis in patients with TDT or by bone marrow harvest in patients with SCD. Following collection, cells were transduced with the BB305 LVV. Patients underwent busulfan myeloablative conditioning and were infused with transduced cells. Patients were monitored for engraftment, adverse events (AEs), HbAT87Q levels, and other hematologic and clinical parameters. After 2 years in HGB-205, patients transitioned into the long-term follow-up study, LTF-303 (NCT02633943). Summary statistics are shown as median (min-max). Results As of June 2019, patients with TDT (n=4) and SCD (n=3) had a median follow-up of 49.6 (40.5-60.6) and 28.5 (25.5-52.5) months, respectively. Table 1 shows patient and drug product characteristics and several key efficacy outcomes. All patients achieved HSC engraftment. LentiGlobin safety profile was consistent with busulfan myeloablative conditioning and, in case of SCD, with the underlying disease state. The most common non-hematologic Grade ≥ 3 AEs post-LentiGlobin gene therapy (≥ 2 patients) for patients with TDT were stomatitis (n=4) and increased aspartate aminotransferase (n=2), and for patients with SCD were ACS (n=2) and vaso-occlusive pain (n=2). In all 4 patients with TDT, total Hb and HbAT87Q levels remained generally stable up to 5 years post-LentiGlobin infusion. Three of 4 patients achieved transfusion independence (TI; defined as weighted average Hb ≥ 9g/dL without pRBC transfusions for ≥ 12 months), for an ongoing duration of 56.3 (38.2-57.6) months. Weighted average total Hb during TI was 11.4 (10.5-13.0) g/dL. One patient has been off transfusions for 37.5 months and had total Hb of 7.7 g/dL, which was below the ≥ 9 g/dL requirement to meet the protocol definition of TI. At last visit, HbAT87Q levels in these 4 patients ranged from 6.2-11.2 g/dL, which contributed 73.8-86.8% of the total Hb. The first patient treated with LentiGlobin for SCD experienced one vaso-occlusive pain episode, which developed at 30 months after LentiGlobin gene therapy following a case of acute gastroenteritis with fever and dehydration. The second SCD patient had 2 serious AEs (SAEs) of ACS approximately 6 and 8 months after LentiGlobin gene therapy. The patient resumed chronic pRBC transfusions and hydroxyurea treatment and subsequently experienced 2 SAEs of vaso-occlusive pain; no additional SAEs of vaso-occlusive pain or ACS were reported during the last 16 months of follow-up after LentiGlobin infusion. The third SCD patient had no episodes of vaso-occlusive pain or ACS during 25.5 months of follow-up post-LentiGlobin gene therapy as of the data cut-off. Two patients with SCD who have been off chronic pRBC transfusions, showed improvement in hemolysis markers post-LentiGlobin treatment and stabilization of HbAT87Q expression at approximately 6 months post-LentiGlobin infusion. Total Hb levels for patients with SCD at last visit were 13.0 g/dL (patient 1), 9.4 g/dL (patient 2), and 9.8 g/dL (patient 3), with corresponding HbAT87Q contributions of 47.9%, 7.9%, and 25.8%, respectively. Summary With up to 5 years of follow-up, treatment with LentiGlobin gene therapy was well tolerated and resulted in improvement in hematologic parameters and disease-related symptoms. Further results from the completed study will be presented. Disclosures Hermine: Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Brousse:bluebird bio, Inc: Consultancy; AddMedica: Consultancy. El Nemer:Hemanext: Other: Other. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Asmal:bluebird bio, Inc: Employment, Equity Ownership. Whitney:bluebird bio, Inc: Employment, Equity Ownership. Gayron:bluebird bio, Inc: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Cavazzana:SmartImmune: Other: Founder.

Background: Sperm parameters alteration is documented in untreated men with sickle cell disease (SCD). An aggravating effect of hydroxyurea (HU) on sperm parameters is also well established: HU, at current doses, causes significant, rapid, and unpredictable impairment of spermatogenesis. Reversal of its effect when treatment is stopped has also been documented, albeit in patients given very low doses of treatment (10 mg/kg/d). In France, sperm banking is free of charge and recommended whenever possible before initiation of HU treatment. While recent guidelines have broadened the indication of HU for asymptomatic infantssuch banking is impossible in younger boys before puberty. In addition, little is known on the effect of HU on sperm parameters when given at this specific period. Objectives and Methods: The main objective of this study was to compare sperm parameters after treatment resolution in young males treated by HU prior to puberty with sperm parameters of untreated males. Secondary objective was to analyze longitudinally sperm parameters during HU washout in those treated prior to puberty.Data regarding indication of HU, dosage, date of initiation and stop, clinical profile including vaso occlusive events (VOE) transfusion episodes and date of puberty was collected. Alternative treatment before or during semen analysis was also documented. A period of 3 months of HU wash out was required prior to semen analysis in treated patients. Results: A total of 26 patients (43 semen samples) were studied, with 16 patients treated with HU prior to puberty (HU-PP) and 10 untreated (HU-naive). Characteristics of patients are presented in Table 1. Indication of HU was cerebral vasculopathy (n=3), VOE (n=5), severe anemia (n=1) or combined (n= 7). The median stopping of the HU before CECOS is 4.5 years [0.5-11.0]. An alternative treatment based on a transfusion program was initiated in 14 patients (87.5%) at the time of sperm analysis in the HU-PP group versus 5 patients (50%) in the HU-naive group. Duration of transfusion program was 126 months [3-188] in HU-PP versus 13 [7-100] in the HU naive group. We compared the fraction of abnormal values in semen samples in both groups (25 samples in the HU-PP group and 18 in the HU-naive). No significant difference was observed regarding volume of ejaculate, spermatozoid concentration, total sperm count, spermatozoid motility, morphology and vitality, and sexual abstinence before sampling in both groups (Table 1). In the HU-PP group, there was a trend in improvement of sperm parameters with the duration of transfusion program. In addition, a kinetic analysis of sperm parameters during the period of HU wash out was performed in 3 patients exposed to HU prior to puberty, demonstrating reversibility of HU toxicity in all. All 26 patients were offered semen cryopreservation. For 22 of them, semen parameters after thawing indicated a possible use for assisted reproductive technologies, mostly in vitro fertilization with intracytoplasmic sperm injection. In the remaining 4 (3 in HU-PP group and 1 in HU naïve), there was serious concern about the possible use because of a very low initial sperm concentration and the absence of motile spermatozoids after thawing. Discussion/Conclusion: Toxicity of HU upon spermatogenesis has been well documented in animal studies and in adult males. In many settings, such issues may be a drawback for parents and/or caregivers to treat young boys before semen banking can be performed, given the lack of robust information on reversibility after treatment resolution. Abnormalities of sperm count are also common in non-treated males so that demonstrating the specific effect of HU may be complex. Oseggbe et al. and Berthaud et al. showed that 91% of untreated SCD males have at least one abnormal sperm parameter. Here, we show that after treatment resolution, there are no differences in sperm parameters in patients exposed to HU before puberty compared to untreated males. Because a majority of patients benefitted from transfusion therapy at the time of analysis, we were however unable to demonstrate whether reversal of toxicity occurs spontaneously or requires transfusion. Notwithstanding possible limitations due to small sample size, this study shows that in boys with severe disease requiring HU treatment before puberty, toxicity of HU on sperm parameters should not be a major drawback. Disclosures Bernaudin: AddMedica: Honoraria, Other: Help for travel; GBT: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Consultancy. De Montalembert:Novartis: Consultancy, Honoraria; Addmedica: Consultancy, Honoraria; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Brousse:bluebird bio: Consultancy; Add medica: Consultancy.

Hydroxyurea (HU) is approved in EU and USA for the prevention of vaso-occlusive crises (VOC) including acute chest syndromes (ACS) in patients over 2 years with sickle-cell disease (SCD). Patients on HU wishing to conceive should stop treatment 3 to 6 months before pregnancy if possible. Pregnancy in SCD female patients are considered at risk for the mother and the fetus. This condition is associated with increased pain, infections, thromboembolic events,[1] and vaso-occlusion in placenta can lead to adverse fetal outcomes.[2] A few cases of HU exposure during pregnancy in SCD patients previously published[3],[4] suggested that the risk of deleterious teratogenic effect of HU shown in animal species[5] may have been overestimated in humans.[6] Therefore, a much larger dataset of pregnancies with HU exposure was needed. ESCORT-HU study (European Sickle Cell Disease COhoRT - HydroxyUrea) is a multicentric, prospective, non-interventional European study initiated to collect information about long-term safety of HU when used in current practice. This is the first study in which all pregnancy courses were collected irrespectively of action taken with HU before or during pregnancy. Overall 1906 patients were enrolled from 63 centers in France, Germany, Greece and Italy, 854 men (45%) and 1052 women (55%) among them around 2/3 aged from 15 to 49 years during the follow-up. During the study, 125 pregnancies in 101 women and 12 pregnancies in 10 partners of male patients were collected in the study, regardless HU exposure. In pregnancies with HU paternal exposure, 10 live births and 2 miscarriages were reported. Durations of HU exposure and outcomes of pregnancies in females treated with HU are provided in Table 1. The mean age at the pregnancy was 30 years. The mean HU duration before pregnancy was nearly 5 years. In only 16 pregnancies with maternal exposure (15%) HU was stopped at least 15 days before conception. In 43 pregnancies (34%), transfusions were reported. Live births were reported in 73% of pregnancies with maternal HU exposure excluding voluntary abortions. There was no statistical difference (Chi-squared test) in the proportions of live birth in exposed and non-exposed females (p=0.577). VOC or ACS have been reported in 3 women after the stop of HU. Although women are advised to stop HU before pregnancy, in current practice continuation of HU may be required and HU remains the only alternative to protect the mother and the fetus from deleterious effects of VOC. In conclusion, the data on pregnancy outcome following HU exposure are reassuring when compared to those in general SCD population. Overall in ESCORT-HU, 61% of pregnancies resulted in live birth, which is more than the 42% of pregnancies in the MSH study participants.4 It is notable, without considering voluntary abortions, that the percentage of live birth in ESCORT-HU reached 71%. The rate of preterm delivery (13%) was similar to the one (16%) in HbSS patients from a French cohort.[7] The fertility in women treated with HU seems to be even better and no particular problem in newborns has been reported to date. Disclosures Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Cannas:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Voskaridou:Addmedica: Membership on an entity's Board of Directors or advisory committees; Acceleron: Consultancy, Research Funding; Genesis: Consultancy, Research Funding; Protagonist: Research Funding; Celgene Corporation: Consultancy, Research Funding.

Fanconi anemia (FA) is an inherited disorder, clinically characterized by congenital abnormalities, a fatal progressive bone marrow failure (BMF), and a predisposition to develop malignancies. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential alternative cure and to get around the problems of the Hematopoietic stem cell transplantation toxicity or the donor restriction. For gene therapy, an adequate number of HSC collected is a key point to a successful engraftment. However, the HSC collection in FA patients implies particular challenges because of their reduced BM stem cells numbers and implies a theorical risk of an inner depletion in stem cell reserve following collection.The main objective of this pilot study was to evaluate the feasibility and the safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and collection of peripheral HSC for potential use in a GT trial. We present the results of this open-label phase I/II trial (N°EUDRACT 2014-005264-14) from 4 selected FANCA mutated patients (FA-A) with a weight >10 Kg and an age between 2 to 18 years old. A systematic combination of G-CSF (12μg/kg twice a day) plus plerixafor (Mozobil® 0.240 mg/kg/d ) was used to maximise the CD34+ cells mobilization. CD34+ cells and white blood cells (WBC) blood counts were monitored tightly along the mobilization protocol. No short-term adverse events linked to the mobilization and the collection procedures were observed. The combination of G-CSF and Plerixafor allowed crossing the PB mobilization threshold (≥5 CD34+cells/μL) for 2 patients. Interestingly, CD34+cells were mobilized quickly but transitionally after plerixafor injection. One patient mobilization had more than 100 CD34+cells μ/L with a early peak 2h after injection. The peak disappeared 11 hours after injection. We adapted the time of collection to the C34+ cells mobilization. No CD34+ blood cell rebound was observed after the apheresis was stopped. Our new datas suggest that mobilization of FA patients with G-CSF and plerixafor is safe. However, the age of the patient, a potential cytopenia or the lack of bone marrow progenitor cell may heavely compromise the collection. Nevertheless, the datas show a stable cytopenia despite the stimulation and collection of stem cells during the following months. This study underlines that a very cautious collection of stem cell in the Fanconi anemia to consider gene therapy is a necessity. These results also confirm that the kinetic of CD34+ cells mobilization is one of the key point to a successful stem cell harvesting for gene therapy trial. Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by a profound reduction or alteration of T lymphocyte function. They arise from a variety of molecular defects which affect T lymphocytes development and function. The number of infections prior hematopietic stem cells tansplantaton (HSCT), genotype, and the type of donor are described as prognostic factors for stem cell transplants. In this retrospective study, we included 30 pediatric patients suffering from SCID who underwent to CD34+-selected grafts between January 2008 to December 2017 in our center. Diagnosis of reticular dysgenesis, ADA deficiency, or leaky SCIDs and intra thymic deficiency were excluded. A mechanistic mathematical model of all available data was performed and provided a dynamic appreciation of immune reconstitution, while removing bias. T-cell populations were maintained through proliferation and loss model and thymic output have been integrated to the production function. This joint modeling approach aimed to predict rate and extent of T cell immune reconstitution over time (mainly CD3+ T cells, CD3+CD4+ helper T cells, and the CD3+CD4+ CD45RA+ cells). With a median follow-up time of 97.28 months [range 0.85; 131.54], there were 345 points of T cell phenotyping concentrations in total with a median of 12 samples per patient (range, 0 -35 samples) taken post-transplantation. In this data-set, 13 % of the patients (n= 4) died from infections. Time to reach half of the maximal T cell concentrations was estimated to 3.4 months, 95%CI [2.5 - 4.6]. In covariate analysis, genetic diagnosis (p= 0.0047) and conditioning regimen (p= 0.01) were found to be significant pre transplant covariates which impacted the trajectory of T cell concentration with time. This modeling approach appeared to be the best method to learn about the dynamic T cell reconstitution after transplant in patients suffering from SCID. In the context of new cell therapy approach for T cell depletion and in vitro thymic maturation, this mechanistic joint model can be used for the design and analysis of incoming clinical trials. Figure Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

Hydroxyurea (HU) is approved in the EU and USA for prevention of vaso-occlusive crises (VOC) including acute chest syndromes (ACS) in patients over 2 years with sickle-cell disease (SCD). The major benefits of HU in SCD are directly related to its abilities to increase HbF, decrease sickling of red blood cells and hemolysis, leading to reduction of vaso-occlusive episodes, need for blood transfusions and consequently reduction of morbidity and mortality. Adherence to the treatment is paramount for effectiveness, but in spite of proven benefits, barriers to adherence persist.[1] ESCORT-HU study (European Sickle Cell Disease COhoRT - HydroxyUrea), is a multicentric, prospective, non-interventional European study designed to evaluate the safety profile of HU in real life. Patients were enrolled from January 2009 to June 2017 with a follow-up of up to 10 years. All interruptions and resumptions of HU treatment exceeding 15 days were recorded in this study. We hereby present the analysis of the group of patients who self-discontinued HU at least once during the study before informing their caregiver, with a view to identify potential barriers to long-term adherence. In total, 1906 patients were enrolled in ESCORT-HU from 63 centers in France, Germany, Greece and Italy. Of these, 619 patients (32%) stopped HU for over 15 days at least once, and around a third (11% of all patients) were due to patient's will. The mean duration of HU treatment before the first discontinuation was 4.8 ± 5.1 years. Data are summarized in table 1. Compared to the rest of the cohort, the 'treatment discontinuation' group had similar distribution by gender and indication for HU prescription, but a higher proportion of adults stopped HU more than 15 days. It is notable that the proportion of patients with SC genotype was higher in the 'treatment discontinuation' group (4.5% vs 1.7%). The patients in the 'treatment discontinuation' group had more frequent SCD symptoms before enrolment in the study (table 2). Hematological and clinical improvement compared to the baseline was observed in both groups. However, average mean Corpuscular Volume (MCV) and Fetal Hemoglobin percentage (HbF%) were lower and mean percentages of patients with SCD symptoms were higher over the three years of follow-up in the 'treatment discontinuation' group, suggesting that HU daily dose was insufficient (table 2). Sixty patients have no treatment resumption date reported which suggest a permanent interruption of their treatment. Among them 32% preferred to switch to another HU medicinal product and 13% have safety issue (table 3). Understanding and managing self-discontinuation of HU before taking medical advice is challenging for the physician. It is tempting to speculate that it may be due, at least in part, to lack of effectiveness potentially due to an underdosage of the treatment. Resistance to the treatment may also be suggested based on past literature data revealing a great variability in the response (determined by HbF%) to HU therapy. There is evidence that genetic modifiers affect individual response to HU.[2],[3] Finally, weariness from long-term use may also explain the patient's wish to discontinue HU. But treatment at optimal effective should be the primary goal of caregivers. [1]Smaldone A., Manwani D., Green NS, Greater number of perceived barriers to hydroxyurea associated with poorer health-related quality of life in youth with sickle cell disease, Pediatr Blood Cancer. 2019 [2] Steinberg MH, Voskaridou E, Kutlar A, Loukopoulos D, Koshy M, et al. (2003). Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease. Am J Hematol 72: 121-126 [3] Ware RE, Despotovic JM, Mortier NA, Flanagan JM, He J, et al. (2011) Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia. Blood 118: 4985-4991 Disclosures Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Voskaridou:Celgene Corporation: Consultancy, Research Funding; Protagonist: Research Funding; Genesis: Consultancy, Research Funding; Acceleron: Consultancy, Research Funding; Addmedica: Membership on an entity's Board of Directors or advisory committees. Cannas:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Les greffes de cellules-souches hematopoietiques (CSH) haplo-identiques, par leurs mismatchs HLA, predisposent a un risque accru immunologique. Elles connaissent cependant un essor important ces dernieres annees du fait d’une amelioration des procedures permettant une diminution de la reaction du greffon contre l’hote (GVH) et de la mortalite liee a la greffe (TRM). Ces progres ont en particulier ete observes avec des techniques de depletion T in vivo par cyclophosphamide ou depletion in vitro des lymphocytes T alpha-beta. Selon les series publiees, entre 11,3 % et 20,2 % des patients greffes en situation haploidentique presentent, avant transplantation, des anticorps diriges contre le donneur (DSA). Le titre de ces DSA est associe a un impact pejoratif sur la survie ainsi que le delai et l’incidence de prise de greffe. Il existe 4 types de strategies de desensibilisation pregreffes derivee de l’experience de la transplantation d’organes solides : – L’elimination des anticorps par plasmapherese ou immunoabsorption ; – l’inhibition de la production d’anticorps par anticorps monoclonaux diriges contre les lymphocytes CD20+ ou par un inhibiteur du proteasome inhibant les plasmocytes ; – la neutralisation d’anticorps a l’aide d’immunoglobuline intraveineuse (IgIV) ou d’antigenes HLA du donneur par transfusions de plaquettes ; – inhibition de la cascade du complement. Ces strategies sont associees les unes aux autres en fonction des techniques et de l’experience de chaque centre. Nous presentons ici 2 strategies d’utilisation de concentres de plaquettes d’apherese dans le cadre de desensibilisation pregreffe CSH haplo-identiques avec immunisation HLA.

Introduction: Gene therapy is a highly promising therapeutic strategy in sickle cell disease (SCD). The Phase 1/2 HGB-205 (NCT02151526) clinical study in France is evaluating the safety and efficacy of LentiGlobin gene therapy, which consists of autologous CD34+ cells transduced with a lentiviral vector encoding a human β-globin gene with a point mutation (T87Q) that confers anti-sickling properties. Data from the first successfully treated patient have been published (Ribeil et al, 2017 NEJM). In order to establish the effect of βAT87Q-globin production on red blood cell properties, we have analyzed membrane properties, hemolysis markers, morphology, hemoglobin content, and the extent of HbS polymerization. Methods: Whole blood samples were obtained from 3 patients with SCD (1204, 1207 and 1208) treated in HGB-205 during their clinical follow-up. HbS polymerization level was assessed by O2 dissociation and association curves and cell morphology. Membrane properties were evaluated by RBC density curves in phthalate gradient, deformability under increasing osmolality (LORRCA) and level of adherence to surfaces coated with thrombospondin (TSP), under increasing shear stress (from 0.5 to 5 dynes/cm²). Hemolytic level was determined by measurement of classical markers (LDH, bilirubin and haptoglobin). Hemoglobin contents of total RBCs and reticulocytes (CD71-positive cells sorted) were assessed by reverse-phase HPLC. Results were compared against untreated βSβS patients (n=11 for deformability assay, n=4 for adhesion assay) and healthy donors (n=10 for deformability assay, n=3 for adhesion assay). Results: As of May 29 2018, follow-up, total Hb and HbAT87Q contribution to total Hb for patients 1204, 1207 and 1208 were: 42, 18 and 15 months, 12.2, 8.4, and 10.4 g/dL, and 49.44, 7.77 and 26.99%, respectively. At approximately 30 months post-infusion patient 1204 developed vaso-occlusive pain following an episode of acute gastroenteritis, since then the patient has not had any vaso-occlusive episodes or acute chest syndrome (ACS). Patient 1207 had 2 episodes of ACS approximately 6 and 8 months after LentiGlobin gene therapy and has since been on chronic transfusions and hydroxyurea treatment; the patient subsequently experienced 1 vaso-occlusive pain episode. Patient 1208 has had no episodes of VOCs or ACS post LentiGlobin gene therapy. Dissociation and association of O2 curves for RBCs isolated from the 2 patients free of chronic transfusions (1204 and 1208) and performed 36 and 8 months post infusion, respectively, showed only a slight increase in P50 during re-oxygenation, indicating anti-sickling capability of transgenic HbAT87Q and low levels of HbS polymerization. Density curves showed an overall normal RBC hydration at multiple time points during follow-up, with dense cells contributing 0-4% compared to a mean (±SD) of 12.8% (±7.8) in untreated patients. The deformability of RBCs from the 2 patients (1204 and 1208) evaluated in HGB-205 study was lower than observed for healthy donors but higher than for untreated SCD patients. Under controlled shear stress, TSP adherence was consistently lower for RBCs isolated from the 2 patients (1204 and 1208) in HGB-205 compared to untreated patients with SCD. Slight intravascular hemolysis was observed for the 3 HGB-205 patients during follow-up, but the hemolytic levels improved compared to baseline. RP-HPLC analysis of total RBCs isolated at last visit showed an increase in βAT87Q and a decrease in βS in comparison to reticulocytes, indicating an improved survival of RBCs expressing more anti-sickling β-globin transgene (Table 1). Data on deformability, distribution of fetal Hb and additional adhesion markers will be presented. Conclusions: Our results suggest an improvement in RBC properties for 2 of 3 patients with SCD treated with LentiGlobin gene therapy in the HGB-205 clinical trial compared to non-treated patients with SCD, suggesting a promising potential of this treatment. Disclosures El Nemer: Imara: Research Funding. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Ribeil:Vitalaire: Research Funding; Bluebird Bio, inc.: Employment. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

Primary Amyloid Light-chain (AL) amyloidosis is a rare form of plasma cell dyscrasia characterized by tissue deposition of monoclonal immunoglobulin light chain. Kidney involvement is the most frequent manifestation, and patients usually present with glomerular disease. We report an exceptional case of bilateral kidney infarcts caused by AL amyloidosis. A 34-years-old man presented with progressive dyspnea, loin pain, recurrent macroscopic hematuria, and acute kidney injury. Computed tomography showed bilateral kidney infarcts. The diagnosis of AL amyloidosis was established on the kidney biopsy with the characterization of major vascular amyloid deposits that selectively stained with antilambda light chain antibody. An amyloid restrictive cardiomyopathy was also present, responsible for the life-threatening conduction disturbance, but without patent cardioembolic disease. The patient then underwent emergency heart transplantation, followed by a conventional chemotherapy with bortezomib, melphalan, and dexamethasone. More than 3 years later, the patient has subnormal renal function, a well-functioning heart transplant, and a sustained hematologic response. In addition to the very uncommon presentation, this case illustrates the tremendous progress that has occurred in the management of severe forms of AL amyloidosis.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

HU is a myelosuppressive drug marketed since 1968 for the treatment of hematological malignancies, and authorized since 2007 in Europe as an orphan medicinal product for the prevention of recurrent vaso-occlusive crises and 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 and morbidity-mortality in SCD patients treated with HU. The study responds to an EMA (European Medicines Agency) request and has been approved by the Ethical Committee of Necker Enfants Malades Hospital (Paris, France). Some teratogenic effects of HU have been observed in fetuses of treated pregnant rodents (Woo, Katayama et al. 2004;Yan and Hales 2005 ;Chahoud and Paumgartten 2009). Hydroxyurea has been demonstrated to be embryotoxic at doses over 150 mg/kg/day in rats and in monkeys (Liebelt, Balk et al. 2007). Embryotoxicity was characterized by decreased fetal viability, reduced live litter sizes, and developmental delays. On the other hand, a recent study on pregnant mice showed no evidence of teratogenic effects with intraperitoneal injection of HU (25mg/kg) on gestation day (GD) 7.5 (Zhen Guan et al. 2015). Due to the potential teratogenic effects in animal models, although no malformations have been reported in humans to date (Khushnooma et al. 2010,), the use of HU is not recommended during pregnancy. The pregnancy in SCD can be associated with adverse maternal and perinatal outcomes. The rate of spontaneous abortion is more than 30% in our cohort at Necker Hospital and in literature (Regina Amelia Lopes Pessoa Aguiar et al. 2014, Sophie Lanzkron et al. 2012). Women of childbearing potential receiving HU should be advised to use contraception to avoid becoming pregnant. If there is a desire for pregnancy, HU must be stopped 3 to 6 months before conception and switched for chronic transfusion until the delivery or the end of breast feeding with a significant risk of allo-immunization (Ngo et al. 2010, Howard et al. 1995). In our experience, we have noticed that as pregnancies started frequently under HU, it is necessary to improve our knowledge about the use of HU during pre-conception or organogenesis in SCD patients and eventually develop new therapeutic strategies. From June 2008 to July 2016, 1050 patients (496 children and 554 adults) were enrolled in ESCORT-HU from 3 European countries, Greece (11.7%), Germany (13.4%), and France (74.9%). Among the 315 women with childbearing potential (aged more than 15 to 49 years), 28 pregnancies in 27 women treated with HU have been reported despite the information to stop HU before conception (Table 1). Clinical data regarding the newborn are reported for 7 pregnancies with HU exposure and no malformations were observed. In women treated with HU: • HU was stopped in 15 (15 pregnancies) women during the first trimester of pregnancy (9 before 6 WA). The outcome of these pregnancies were 5 normal births, 5 premature births, 3 ongoing pregnancies, 1 voluntary abortion, 1 unknown • HU was used during the whole pregnancy in 1 woman (issue normal birth), • Information on treatment discontinuation was not reported in the database for 8 women. In our study, a large majority of pregnancies (89%) occurred while HU was not stopped which correlates with the high rates of unplanned pregnancy in SCD women (Smith-Withley et al 2014, Eissa et al 2014). The use of HU during organogenesis or pre-conception period in our cohort seems to have no impact on the materno-fetal and new-born morbi-mortality which is consistent with literature data (Khushnooma et al. 2010). Even if women of childbearing potential receiving HU should be advised to avoid becoming pregnant, in this series nested in the ESCORT HU cohort pregnancies occurred safely despite HU. These results are important because they could help to limit the indications and duration of transfusion and therefore the risk of allo-immunization and delayed hemolytic transfusion reaction in this at-risk population. Nevertheless this preliminary work needs to be confirmed by a comprehensive collection which could help us to evaluate the risk-benefit ratio, of HU versus blood transfusion during pregnancy in SCD women. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.

The palladium(0)-catalyzed Heck-type cyclization-β-alkoxy elimination reaction leading to enantiopure bicyclic compounds in the case of erythro 2,3-unsaturated glycosides has been successfully extended to the threo stereoisomer by only changing the aglycon moiety from an ethoxy group to an aryloxy moiety.