ALBERT

Description: Key Points ALPS DNT cells and their putative precursors reveal high proliferative activity in vivo, which is associated with hyperactive mTOR signaling. Rapamycin therapy controls mitotic activity and abnormal differentiation of ALPS DNT cells and reduces CD4+ or CD8+ precursor DNT cells.

Description: Background: Autoimmunity is a fundamental consequence of impaired tolerance checkpoints in patients with hypomorphic mutations in recombination activating genes (RAG1 and 2), the major player in V(D)J recombination of antigen receptors. We recently published a cohort of 63 patients with partial RAG deficiency with autoimmune and/or hyperinflammatory manifestations. Autoimmune hemolytic anemia (AIHA) was most common, occurring in (60.3%) of patients at a median age of 1.9 years. The episodes of AIHA were often severe and refractory to first or second-line therapy. Despite the severity of early-onset disease, a large fraction of patients ware diagnosed later for an underlying genetic cause. This may be due to deceased awareness and lack of routine clinical biomarkers for underlying RAG deficiency. The natural course of AIHA episodes, diversity of antibodies targeting red blood cells (RBC) and the underlying mechanisms that promote AIHA remain elusive. Our previous study on RAG-deficient patients has revealed that autoreactive B cells are present early in B cell development, and autoantibodies are produced to a broad selection of self-antigens, including those on RBCs. A prevailing theory for the development of anti-RBC antibodies involves dysregulation of somatic hypermutation (SHM). There are specific regions of the immunoglobulin (Ig) that are naturally reactive to self-antigen in the germline state, and mutation away from self-reactivity occurs through SHM. On example is the germline Ig VH4-34 AVY segment that has been known to bind to the i/I antigen on RBCs. Increased awareness for detection and mechanistic understanding of the generation of anti-RBC antibodies are needed to promote targeted therapy in this highly vulnerable young patient population with AIHA and immune deficiency. Methods: Through retrospective chart review, we collected detailed information on the episodes of AIHA. This included clinical evidence of hemolysis, recurrence and severity of episodes, specifics on red blood cell reactivity, treatment and outcome. In addition, We performed B-cell repertoire studies to assess rate of SHM and "mutation away" from RBC reactivity. Results In our 42 patients with RAG deficiency and AIHA, we accumulated data on 20 patients. Many of the patients experienced the onset of AIHA after viral infections and often recurred (up to 8 times in lifetime). Most of the patients had warm AIHA with broad reactivity (direct, indirect, IgG, IgM, C3). In parallel, patients developed specific antibodies to cytokines targeting IFNa, IFNw and IL12. Second or third-line therapy was required in 50% of cases. One patient underwent HSCT due to severe refractory AIHA. The B-cell repertoire in selected RAG patients with AIHA contained more unmutated Ig VH4-34 segments (including the AVY segment), and demonstrate less evidence of SHM when compared to healthy donors. Conclusions Patients with partial RAG deficiency may develop severe, recurrent and treatment-refractory AIHA at early ages. Diagnosis may be expedited with increased awareness and serological evidence of broadly reactive antibodies to RBC and cytokines. Mechanistically, decreased SHM may promote the survival of naturally occurring antibodies to RBC. Studies should continue to advance understanding for targeted approach to serve as bridge therapy before HSCT is considered or indicated. Disclosures No relevant conflicts of interest to declare.

Description: Background We previously reported (Styczynski et al, Blood, 2012) that hematopoietic stem cells (HSC) collection from bone marrow (BM) or peripheral blood (PB) in pediatric sibling donors is safe, however there is a risk of mild, short-term early side effects. No data are available so far on follow-up in children donating HSC. Objective To analyze donor safety and side effects related to HSC collection from BM or PB in pediatric sibling donors after 1-year. Donors 171 children: 90 male (M), 81 female (F), median age 9.2 (range: 0.7-17.3) yrs, including 45 children’s weight 40 kg; 31 donors were 8 yrs. There were 25 PB donors 9 (PBD) (18 M, 7 F; median age 11.0, range: 1.3-17.3 yrs ) and 146 BM donors (BMD) (72 M, 74 F; median age 8.3, range: 0.7-17.3 yrs). Methods Data from 14 participating EBMT Centers were registered on specific forms, sent to the PDWP office and statistically analyzed. Results During follow-up local pain was reported by 16/161 (9.9%) donors at catheter site (n=1) or collection site in 16 (14/137 BM; 2/24 PB, ns). 14 (8.1%) donors complained for pain persistence for 1-6 months (1mo-5, 2mo-5, 3mo-2, 5mo-1, 6mo-1). Late vomiting was observed in 7/163 (4.3%) (BMD only), and hematuria in 1 donor. Fever episodes were observed in 21/164 (12.8%) (21/140 BMD, p=0.045), lower respiratory tract infection in 3/164 (1.8%) (1/140 BMD, 2/25 PBD, p=0.056), upper respiratory tract infection in 21/171 (12.3%) (21/146 BM, p=0.046) and urinary tract infection in 5/171 (2.9%) (5/146 BMD, ns). No other infections (including gastroenterocolitis or hepatitis) were reported. Anemia was diagnosed in 6/164 (3.6%) (6/140 BMD, ns). At 1 year follow-up iron was supplemented in 24/164 (14.6%) (22/140 BMD, 2/24 PBD, ns) and folinic acid in 2/163 (1.2%) (BMD only). No other hematological problems (including thrombotic events) were reported. One donor was diagnosed for chronic disease (0.6%) (asthma), 4 (2.4%) had another hospital stay: physiotherapy (n=1), circumcision (n=1), plastic surgery due to an accident (n=1), and donation for MSC (n=1). Assessment of profile of psychological status consisted of a six item questionnaire. At 1-year follow-up, donors felt helpful in 132/154 (85.7%) cases (BMD 109/130, PBD 23/24, ns); happy in 117/149 (78.5%) cases (BMD 93/125, PBD 24/24, p=0.011); proud in 113/147 (76.8%) cases (BMD 91/124, PBD 22/23, p=0.039); depressed in 11/147 (7.5%) cases (BMD 11/124, PBD 0/23, ns) and responsible for recipient in 97/145 (66.9%) cases (BMD 83/122, PBD 14/23, ns). Willingness for subsequent HSC donation was reported by 113/147 (76.8%) children (BMD 94/125, PBD 19/22, ns). Conclusions At one-year follow-up after BM or PB HSC collection in pediatric sibling donors no major side effects were reported, however there is a risk of mild side effects. A donor registry should document and evaluate the course of minors who donate hematopoietic stem cells to better define possible risk factors of BM or PBSC harvest. Disclosures: Dreger: Riemser Pharma : Consultancy, Honoraria, Research Funding.

Description: ALPS is an inherited disorder characterized by lymphoproliferation and autoimmunity that mainly involves blood cells. In the last few years, new disorders other than ALPS have been found to cause lymphoproliferative syndromes, and many patients with similar clinical features but not fitting ALPS diagnostic criteria have been described. In this study we evaluate clinical features and response to the treatment of patients with ALPS and ALPS-related Syndromes (ARS) followed in a single Center. ALPS was defined according to the revised diagnostic criteria (first international Workshop, 2009). ARS was defined as the presence of cytopenia and/or lymphoproliferation and at least one absolute or primary additional criterion for ALPS. Medical records of patients followed in our Center between 2001 and 2014 for these conditions were evaluated. Sixty-five patients (35 males), with median age at diagnosis of 10 years (range 0-39) with ALPS (32) (definitive: 21, probable: 11), or ARS (33) entered this study. A positive history of lymphoproliferation was present in 8/32 (25%) and in 3/33 (9%) of ALPS and ARS subjects, respectively. Lymphoproliferation was present in 51/65 (78%) patients and consisted of isolated lymphoadenopathy (10/51, 19%), isolated splenomegaly (26/51, 51%), or both conditions (15/51, 29%). It was present in 32/32 (100%) and in 19/33 (57%) of patients with ALPS or ARS, respectively. In particular, lymphoadenopathy was present in 17/32 (53%) and in 8/33 (24%) of ALPS and ARS patients, respectively (p 0.04). Splenomegaly was found in 27/32 (84%) and in 14/33 (43%) of ALPS and ARS subjects respectively (p 〈 0.01) Cytopenia was present in 51/65 (78%) of patients and involved one (28/51, 55%) or more cell lineage in (23/51, 45%). Trilinear cytopenia was present in 7 ALPS patients and in no subjects with ARS (p

Description: INTRODUCTION Fanconi anemia (FA) is an inherited bone marrow failure syndrome that carries a high risk of transformation to myelodysplasia (MDS) and acute leukemia. Hematopoietic stem cell transplantation (HSCT) is used to treat FA patients in clonal evolution. The roles of chemotherapy before HSCT and the intensity of conditioning regimens in transformed FA patients are controversial, because of the high sensitivity of FA patients to DNA-damaging agents increases the risk of severe toxicities, the duration of aplasia, the risk of infective complications thus limiting the possibility to administer full dose cytoreductive treatments. The sequence chemotherapy-HSCT has been reported by some groups (MehtaPA et al Pediatr Blood Cancer 2007, Talbot A et al Haematologica 2014, Mitchell R et al Br J Haematol 2014, Ayas M et al J Clin Oncol 2013), but conclusive information is lacking because of small number of patients described without a risk factors' analysis. The aim of this retrospective study is to report the outcome of a large cohort of transformed FA who underwent allo-HSCT and to define the factors that may impact on its outcome. PATIENTS AND METHODS The study was conducted on behalf of the Severe Aplastic Anemia (SAAWP) and Chronic Malignancies Working Parties (CMWP) of the EBMT and was based on data of patients who underwent allo-HSCT between 1999-2016 for transformed FA, defined as a diagnosis of FA in presence of any hematological malignancies or cytogenetic abnormalities, registered in the EBMT Data Base. Clinical and biological information of the disease and details on transplant procedures and outcome were collected by a specific form distributed to Centres participating in the study. RESULTS Data of 71 patients (35 males-36 females) affected by transformed FA (42 MDS, 25 AL, 4 with cytogenetic abnormalities but without blasts) undergoing allo-HSCT were collected from 25 Centres . A matched related donor (MRD) was used in 31% of cases, an unrelated donor (UD) in 56.3% and a mismatched related donor (MMRD) in 12.7%. Bone marrow was the main source of cells (54.3%) followed by cord blood (22.9%), peripheral blood (21.4%) and bone marrow plus peripheral blood (1.4%). The median age at allo-HSCT was 12.7 years (range 9.3-23.4). Thirty seven (52.1%) patients received a chemotherapy before HSCT. Pre-HSCT status of malignancy in available patients was complete remission (CR) in 24% (n = 12/50) and an active disease (no-CR) in 76 % patients (n = 38/50). The conditioning regimen included total body irradiation (TBI) in 37 (52.1%) (radiation dose: ≤ 4.0 Gy in 30; 〉 4.0 Gy in 7), busulphan (BUS) in 16 (22.6%), no-TBI nor BUS in 18 (25.3%). Median follow-up was 93.7 months (71-110.6). GvHD prophylaxis and transplants' details are summarized in Table 1. All patients engrafted. Median time for neutrophils was 17 days (14-23) and it was 25 days (23-42) for platelets. The 2-and 5-year overall survival (OS) probability were 54% (41-66%) and 45% (32-57%) respectively; the 2- and 5-year event-free survival (EFS) (events being death, relapse and graft loss) 52% (40-65%) and 45% (32-58%). The cumulative incidence of relapse were 15% (7-24%) and 21% (11-31%), , of non-relapse mortality (NRM) were 37% (25-49%) and 39% (27-51%) respectively at 2 and 5-year. Most frequent causes of death were GvHD (33.3%), infections (23.3%) and relapse of malignancy (16.7%). Patients transplanted in CR, (neither blasts, nor major dysplastic features) and from matched related donor had a significantly better outcome (5-year OS: CR 83% (62-100%) vs no-CR 36% (19-52%) [p 0.01], MRD 60% (37-83%) vs UD 47% (31-64%) vs MMRD 12% (0-35%) [p 0.03]; 5-year EFS: CR 83% (62-100%) vs no-CR 34% (17-51%) [p 0.01], MRD 61% (38-83%) vs UD 48% (31-64%) vs MMRD 12% (0-35%) [p 0.02]; 5-year NRM: CR 0% vs no-CR 44% (27-61%) [p 0.007]) vs those engrafted in no-CR and from no-MRD. (Fig 1 a, b, c). No other tested variable (therapy before transplant and conditioning regimen) significantly affected the outcome. CONCLUSION This study on large cohort of FA patients transplanted because of transformation shows that allo-HSCT from MRD has a better outcome and that CR from malignancy before transplant appears to be a major determinant for a favorable outcome. Disclosures Peffault De Latour: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Risitano:Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amyndas Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Background: There is a significant unmet need for treatments to reduce vaso-occlusive crises (VOCs) in sickle cell disease (SCD). Until recently, the only pharmacological treatment approved for reduction of VOCs was hydroxyurea (hydroxycarbamide), and is recommended for children ≥9 months old with SCD. L-glutamine is approved for the reduction of acute complications of SCD in children ≥5 years old, although symptoms can start as early as 5 months of age. Inhibition of platelet activation has been proposed as a potential therapeutic option for SCD. The rationale for the use of antiplatelet therapies in SCD management is based on evidence that platelets participate in the vaso-occlusive process and that platelet activation correlates with the frequency of pain episodes (Ataga et al, 2012). Platelets are activated during the non-crisis steady state (Lee et al, 2006). The antiplatelet drug ticlopidine, which has a similar mechanism of action as ticagrelor (prevention of adenosine diphosphate [ADP]-mediated platelet activation), significantly reduced the frequency of VOCs in patients with SCD (Cabannes et al, 1984). In the phase III DOVE study in pediatric patients, the platelet inhibitor prasugrel was shown to result in a numerical reduction in VOC events with fewer painful crises in the prasugrel group (66%) versus placebo (72%); however, the differences versus placebo did not reach statistical significance (Heeney et al, 2016). Of note is that the prasugrel doses used in DOVE only resulted in a mean platelet inhibition of ~20% (Jakubowski et al, 2017). The low platelet inhibition may have contributed to the lack of efficacy in this study (Heeney et al, 2016). Ticagrelor is an oral, direct-acting, selective, reversibly-binding P2Y12 receptor antagonist that prevents ADP-mediated platelet activation and aggregation. Ticagrelor was approved in 2010 to reduce the rate of cardiovascular death, myocardial infarction, and stroke in adult patients with acute coronary syndromes, and is currently approved in 〉100 countries. A program is currently ongoing to assess the potential therapeutic benefits of ticagrelor in reducing the occurrence of VOCs in children with SCD. A phase III study (HESTIA3; NCT03615924) is underway to evaluate the efficacy of ticagrelor in reducing the rate of VOCs, as well as the safety and tolerability of ticagrelor versus placebo in SCD pediatric patients 2 to

Description: Severe Chronic Neutropenia (SChrN) is an heterogeneous group of disorders characterized by persistently low circulating neutrophils (

Description: INTRODUCTION Diamond-Blackfan Anemia (DBA) is a congenital pure red cell aplasia, secondary to ribosomal protein genetic defects that usually presents within the first year of age and can be associated with congenital abnormalities and increased risk of cancer. Some patients are successfully treated with steroids but most of them remain transfusion-dependent. Stem Cell Transplantation (SCT) represents the only curative option for this disease, however data from literature is scarce and limited to a very small number of cases. In this retrospective study we describe the outcome of SCT in patients with DBA reported in the EBMT data base. PATIENTS AND METHODS The study was conducted on behalf of Severe Aplastic Anemia Working Party of the EBMT and was based on data of patients affected with DBA who underwent SCT and registered in the EBMT Data Base. Clinical information of the disease and details on transplant procedures and follow-up were collected by a specific form distributed to Centres participating in the study. RESULTS Between 1985-2016, 106 patients (60 males-46 females) aged 6.8 yo (range 1-32) underwent SCT from matched sibling donor (58, 57%), unrelated donor (37, 36%) or from other donors (7, 7%) using bone marrow (73, 69%), peripheral blood (21, 20%) or cord blood (12, 11%) as cell source. 91 patients (86%) received a myeloblative regimen which included Busulfan in 66 cases. 86% (80-93%) of patients engrafted by day 28. Median days to neutrophils and platelets engraftment were 18 and 36, respectively. EFS and OS at 36 months were 81% (74-89%) and 84% (77-91%), respectively. OS was significantly higher (p=0.01) in patients

Description: Introduction. Refractory autoimmune cytopenia may be a manifestation of an another underlying disorder like an autoimmune disease including the autoimmune lymphoproliferative syndrome (ALPS) or an immunodeficiency. Management of complex disorders of which cytopenia is only an epiphenomenon, could benefit of clinical and therapeutic approaches different from those applied to isolated autoimmune cytopenias. Aim of the study. To design a scoring system to identify wider diseases underlying refractory autoimmune cytopenias and validate it on a patient population affected with this diseases referred to a national reference Pediatric Hematology Unit. Patients and methods. Biochemical and clinical data on patients affected by refractory cytopenias were collected within the database of our Unit.after informed consent from the patients. The symptoms/signs associated to cytopenia were arbitrarily grouped in 4 constellations: Lymphoprolypheration (LP), Immunodeficiency (IMM), Gastrointestinal (GI) and Reumathology (RHE). Each symptom/sign of the constellation was given a score (0, 1, 2 based on increasing the severity) whose sum constituted the final score of the single constellation (Table 1). If the score of each single constellation was up to 3, involvement was defined as mild. If the score was ≥4 the involvement was defined as severe.ALPS was defined according to NIH criteria. Results. From 2000 to 2016, 78 patients (37males, 47%), median age 13.19 years (1.5-50.6 year) at last follow up, affected with refractory autoimmune cytopenia seen at our centre, were considered eligible for the study. In details: isolated cytopenia was shown in 60% of the cohort (Thrombocytopenia- ITP 72 % , Neutropenia-NP 19% and Autoimmune Haemolytic Anemia -AIHA in 12% of cases), bilinear cytopenia in 28% (ITP+NP in 64% of cases, AIHA +ITP in 27% and AIHA+ NP in 9% of cases) and trilinear cytoenia (AIHA+NP+ ITP) in 12% of the cohort. In 85% of subjects cytopenia was associated to one or more constellation. In 77% of patients the association was with LP constellation; LP involvement was mild in 62% and severe in 38% of patients. In 69% of patients the association was positive for IMM constellation with mild and severe involvement in 70% and 30% of cases respectively. RHE constellation was positive in 58% (mild in 73% and severe in 27%) of patients. GI constellation was positive in 47% (mild in 89 % and severe in 11%). Patients positive for severe LP involvement were significantly associated to a diagnosis of ALPS (p=0.0003). ALPS was not associated with severe IMM constellation (p =ns). Patients with severe IMM score were significantly associated with severe RHE constellation (p=0.005). AIHA. either isolated or associated with other lineage cytopenia, was significantly associated with a severe IMM constellation (p=0.03). Conclusions. About three quarters of patients with autoimmune refractory cytopenia had concomitant signs of lymphoproliferation or of immunodeficiency. Diagnosis of ALPS was significantly associated to severe LP but not with IMM whereas the diagnosis of AIHA was associated with severe IMM constellation. This scoring system looks as an helpful tool to drive underlying diagnoses, not only at very young ages, beyond refractory autoimmune cytopenias and therefore to orient appropriate diagnostic and management strategies of these patients. Disclosures No relevant conflicts of interest to declare.

Description: Five genetic mutation on PI3KCD are known so far to cause the Activated-Phosphoinositide-3-kinaseδ-Syndrome (APDS) a Primary Immune-Deficiency characterized by lymphopenia/hypogammaglobulinemia, respiratory infections, bronchiectasias, gut/pulmonary infiltrates, splenomegaly, autoimmunity and predisposition to malignancies. We report 2 patients carrying 2 novel mutation of PI3KCD gene presenting with a singlelineage bone marrow failure. Case1: A9-months-old girl with splenomegaly,steroid-responsive autoimmune haemolytic anemia and immune thrombocytopenia,severe persistant neutropenia due to marrow myeloid precursors aplasiaresistant to multiple-agents immunosuppression,recurrent life-threatening infections was referred to our Unit. No myeloid precursors at any stage of maturation were found in the bone marrow aspiration. Growth of both homologous and heterologous myeloid progenitor cells was hampered by patient's marrow plasma.Most common causes of congenital neutropenias were excluded. Sirolimus partially and transiently normalized neutrophil count but after yet another life-threatening sepsis neutropenia relapsed. NGS analysis showed the presence of a novel mutation of PI3KCD gene (H273Y) affecting the enzyme's catalytic domain. She then received analpha-beta-CD19-depleted haplo-identical SCT from her mother (infused18.6x106/kgCD34+,2x104/kgAlpha/beta-T-cells) conditioned withATG-Fludarabine-Melphalan.Neutrophils and platelets recoveredat d+26and+10 respectively.Mixed-chimerismpersistedduring post-transplant andneutropenia relapsed on day +177 when full recipient chimerismwas documented witnessing for rejection.She then underwent anotherhaplo-SCTfrom the mother conditioned with Fludarabine-Melphalan-Campath(infused3x106/kgCD34+, 1.5x105/kgAlfa/beta-T-cells).Neutrophil and platelets recovered at d+18 and +13, respectively.Neither toxicity nor GvHD occurred after both transplants. Case 2: A 1 year-old girl presented with Pure Red Cell Aplasia (PRCA) associated with AHIA. No erythroid precursors were seen in the marrow and progenitor colony studies showed that the patient's plasma had an inhibitory effect on the growth of both homologous and control erythroid progenitors. At presentation she also had worsening seizures due to into acute disseminated encephalomyelitis that promptly recovered after few doses of intra-venous immunoglobulins. PRCA/AIHA subsided to combination therapy with Rituximab and Sirolimus and after 2 years of complete remission new episodes of seizures appeared requiring a change of the anti-comitial treatment which by interfering with Sirolimus serum levels caused a relapse of anemiathat was successfully rescued by the addition of monthly IVIG administration. Based on the low Ig levels at the onset of the disease, the response to both Sirolimus (suggesting a mTOR pathway involvement) and IVIG, , we performed Sanger PCR on PI3KCD gene and detected the S277M mutation. To confirm the pathogenetic role of this mutation, we studied the expression of AKT protein by Western blot analysis that showed an increased phosphorilation compared to a same age-healthy control. This report indicates that thesenew PI3KCD mutations may generate a novel APDS clinical phenotype, consisting of severe single-lineage marrow aplasia,and therefore widen the spectrum of diseases underlying single lineage marrow failure in children. Sirolimus and SCT represent valid options for the treatments of the disease. Disclosures No relevant conflicts of interest to declare.