ALBERT

Description: Hematopoietic Stem cell Transplantation (HSCT) is the definitive treatment for bone marrow failure in diskeratosis congenita (DC). Organ dysfunction (mainly lung and gastrointestinal tract) are often part of the disease and may be a limiting factor for or may affect the final outcome of HSCT. Scarce data or relatively small cohort studies are available in current literature on outcome of HSCT in this disease. We analyzed the outcome of 87 patients diagnosed with DC reported in the data base of the European Society for Blood an Bone Marrow Transplantation (EBMT) who underwent HSCT from 1979 to 2014. In particular we analyzed data on type of HSCT, characteristic of donors, source of cells, incidence of acute and chronic GvHD. Males were 76%. Median age at diagnosis of DC was 5.7 years (0-33 yrs), median age at HSCT was 11.2 years (range 0.56-41 yrs). Median time from diagnosis to HSCT was 19 months (0.62-304 mo). The cell source was bone marrow (BM) in 67%, peripheral blood (PB) in 22% and cord blood (CB) 11%. Twenty six percent of patients were engrafted from a matched related and 53% from a matched unrelated donor. Twenty one percent of subjects were engrafted from a mismatched (both related and unrelated) HSCT. Engraftment was documented in 95% of subjects: 4 % had primary graft failure and 10% lost the graft. Overall 35 patients (40%) died, and 52 were alive (60%) at last follow-up. Causes of death were: infections (43%), multi-organ failure (26%), rejection/oss of graft (14%), GvDH (8.5%) undefined (8.5%). In 32% of cases death was related to transplant. Lung injury was present in 17% of subjects who died. Acute GVDH (mainly grade 2-4) and chronic GvHD occurred in 39% and 30% of cases. Five year OS was 60 %. OS was not significantly different by calendar period (before and after year 2000: 50% vs 67% respectively, p=0.424), by age at transplant (below and above age of 12 years: 64% vs 55% respectively ; p= 0.564) and by source of cells (marrow 60%, cord blood 68%, and peripheral cells 46%; p= 0.687). Conversely, OS in HSCT from mismatched donor was inferior to that of transplants from matched donor (38% vs 65% respectively; p= 0.045). The use of radiotherapy or busulfan (any dose) in the conditioning vs other regimens did not impact on 5 y-OS ( 58% vs 62% respectively; p=0.898). HSCT from HLA matched donor can be considered a treatment option in DC. Transplant related mortality looks rater high. Multi organ failure and lung injury are amongst the main causes of death thus pointing to pre-transplant organ status as important determinant of HSCT outcome. Disclosures Dufour: Pfizer: Consultancy.

Description: Key Points The outcome of HSCT in this large SCN cohort is acceptable. Given the TRM, a careful selection of HSCT candidates should be undertaken.

Description: Abstract 4724 Documented Infection Load In Patients With Neutropenia; a survey from the Italian Registry of Neutropenias. Fioredda F, Calvillo M, Caviglia I, Tucci F, Pusiol A, Bonanomi S, Ghilardi R, Martire B, Mastrodicasa E, Barone A, Farruggia P, Caso M, Castagnola E, Dufour C on behalf of the marrow failure Study Group of the AIEOP (Associazione Italiana Emato-Oncologia Pediatrica). Background: Children with severe congenital (SCN), autoimmune (AN) and idiopathic (IN) neutropenia may have infectious complications with high rate of morbidity/mortality. In spite of this scarce data on the incidence and type of documented infections are available in the literature Aims: To describe incidence and type of documented infections in a large population of patients with different types of neutropenia. Patients and Methods: Patients affected with SCN, AN and IN followed from 2000 to 2009 in Centres of the Italian Registry of Neutropenia (IRN) entered the study. Anagraphics and disease–realated data were extracted from the IRN, while details about infections were collected through a dedicated CRF containing uniform criteria to define infection type that was sent to each centre. The occurrence of infections was calculated during a period “at risk” defined as person-days at risk (pdr). The infection rate (IR) was calculated by dividing the number of events by the pdr and expressed as episodes/1000 pdr (95% CI) and was use to compare infection load of neutropenic patients with that of other haematological diseases. Results: Seventy three patients (28 females and 45 males) of whom 12 (16%) with SCN, 38 (52%) with AN and 23 (32%) with IN were analyzed. At least one infectious episode was observed in 100% of SCN and in 30% of both AN and IN. Overall 2/73 patients died (2,7%). They were both SCN subjects: one deceased from haplo-identical HSCT related complication that was performed for loss of response to G-CSF and the other from sepsis after the parents decided to stop G-CSF treatment. In the overall study population from birth to last follow-up, a total of 108 infections occurred in 31/73 subjects (42%). Sixty-nine episodes were observed in 12/12 patients with SCN, 25 infections were concentrated in 12/38 AI patients, and 14 episodes in 7/23 IN subjects. Skin/subcutaneous infections (49%) and pneumonia (18%) were the most frequent localizations. Othomastoiditis, tonsillar phlegmons, osteomyelitis occurred in 10% and deep abdominal infections in 5% of subjects, who were almost exclusively SCN patients. Microbiological characterization was possible in only 25% of the episodes and showed a slight prevalence of Gram-negatives without differences according to the type of neutropenia. Fungal infections were diagnosed only in two different SCN subjects. G-CSF was used in all SCN patients, in 26% of AN and 4% of IN. The IR in SCN was significantly higher than in IN/AN along the entire period of observation (p

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

Description: Introduction Haematopoietic stem cell transplantation (HSCT) is indicated in patients affected with severe congenital neutropenia (SCN) who transformed into myelodysplasia/acute myeloid leukemia or in low responders to granulocyte colony stimulating factor (G-CSF) therapy. The outcome of HSCT in SCN is not well known because the experience is based on single case report or on small series. Aim of the study describe outcomes of HSCT in SCN patients in a large cohort using EBMT data base. Methods and Patients all patients registered in the EBMT data base affected with severe congenital neutropenia (severe neutropenia diagnosed early in life with bone marrow block at promyelocytes stage in the bone marrow and G-CSF dependency) were considered eligible for the study. Data regarding HSCT and outcome were extracted from the “general” EBMT registry, while data on the history of disease before HSCT, was collected through a specific CRF sent to all the participating centers. Here we report preliminary of this survey. Results A total of 119 patients from 19 participating countries were considered eligible for the study; 66% of the eligibile patients originated from Western Europe and 34% from Eastern Europe and the Eastern Mediterranean area (Iran, Israel, Saudi Arabia,Turkey and Russia). Females were 51% of the cohort. Median age at diagnosis of neutropenia was 0.35 years (0-35.4y), while median age at first transplant was 4.8 years (range 0.2-43y). Four patients were affected with MDS at time of transplant. The cell source was bone marrow (BM) in 56%, peripheral blood (PB) in 26% and cord blood (CB) 18%. Fiftypercent of patients were engrated from a related and 50% from an unrelated donor. Conditioning regimen was myeloablative in 86% and at reduced intensity (RIC) in 14% of the cohort Engraftment was documented in 91%; 5% has no engraftment while 4% lost the engraftment for a total of 10 patients. Chimerism was assessed only in 30% of the patients. Six of the 10 patients who had graft failure died after first transplant. Four underwent a second HSCT and 2 are alive2 died (2 alive and 2 death).Overall 22 patients (18.5%) died while the remaining 97 were alive (81.5%). Causes of death were GVDH 23%, Infection 23%, organ failure 18% and “other causes” 27%. Nine percent of patients died because of relapse/progression of the disease. Transplant related mortality was assessed at 17% on the whole cohort. Acute GVDH grade 1-2 was documented in 31% grade 3-4 in 14% and no GVDH in 54%. The 5-year OS and EFS rate was respectively 77% and 70 % in the whole cohort. The 5-year OS according to HLA identical donor and matched unrelated was respectively 83% and 79% (p=0.99, log-rank). Also the OS according to source of cells (5-year OS: CB 85%, PB 79% and BM 62% was not significant (p =0.13). Likewise no significant difference was found in EFS (CB 85% BM 75% and PB 52% p =0.08). OS by age of patients (5-year OS: 82 % for patients aged 0-2y, 83 % for age 2-5 y, 83% for age 5-10 and 60% for subjects aged above 10y) (p=0.07) and by period of HSCT: (90% between 2008-2012, 75% between 2001-2007 and 64% before 2000) were again not significant. No difference was also seen in OS and EFS according to myeloablative conditioning regimen and RIC. Conclusion this preliminary analysis indicate that the 5-year survival in transplanted SCN patients is close to 80% with no difference between matched related and unrelated donor. TRM is still a not negligible and close to 17%. There is a trend towards a more favourable survival in patients younger than 5 years at time of transplant, in those transplanted with cord blood and after the year 2000. Disclosures: Marsh: Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Dufour:Pfizer: Consultancy, Research Funding.

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.

Description: INTRODUCTION: Autoimmune lymphoproliferative syndrome (ALPS) is a rare congenital disorder characterized by an impaired FAS-mediated apoptosis that leads to chronic benign lymphoproliferation and autoimmunity. In most cases mutation on FAS gene are responsible of the disease and the phenotype of individuals carrying the same variants can vary from asymptomatic/mild forms to severe disease, due to incomplete penetrance of pathogenic mutations. More rarely, other genes involved in this pathway, such as CASP10, are mutated. Few clinical and molecular data have been reported on small numbers of patients carrying CASP10 mutation showing that different genetic variations can produce contrasting phenotypic effects. So far, 2 mutations have been recognized as pathogenic (I406L and L258F) and other have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l) AIMS: The aim of this study is to evaluate the FAS-mediated apoptosis function in patients with ALPS or ALPS-like symptoms carrying mutations on CASP10 gene. METHODS: We evaluated FAS-mediated apoptosis pathway in all patients presenting with an ALPS/ALPS-like phenotype that were found to carry a CASP10 mutation in our Institution. Molecular findings were obtained by NGS analysis of a panel of genes involved in the most common immune-dysregulation syndromes and immune-deficiencies and were confirmed by Sanger sequencing. Functional studies were performed by Western blot analysis of CASP10, CASP8, and PARP proteins after TRIAL-induced apoptosis stimulation. Healthy individuals were used as control. RESULTS: We identified 6 patients with ALPS (2) or ALPS-like (4) phenotype, carrying the following heterozygous CASP10 mutations: I406L (1), V410l (2), Y446C (1) L522l (2). Western blot analysis showed an impaired activation of CASP10, CASP8, and PARP proteins in all cases compared to healthy control (Fig. 1) CONCLUSIONS: In our symptomatic patients, the CASP10 polymorphic variant L522l and other mutations whose pathogenicity is controversial (V410l, Y446C) were associated with impaired CASP10, CASP8, PARP activity -and therefore with apoptosis dysfunction- as in the case of I406L pathogenic mutation. We can speculate that, in addition to the functional impairment of apoptosis, other genetic and epigenetic factors might be crucial for the development of clinical symptoms in CASP10 mutated patients, as already described in FAS mutations, suggesting that the search of other mutations in patients with ALPS/ALPS-like phenotype should be encouraged. Nonetheless, further studies on epigenetic factors potentially implicated in the expression of symptoms are needed to fully understand the heterogeneity of clinical phenotype of this disorder. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background Autoimmune neutropenia is a chronic reduction of absolute neutrophil below the threshold for age, due to peripheral destruction by specific antibodies (AaN). "Primary autoimmune neutropenia" (pAN) usually appears in early infancy and generally resolve within 18-24 months, while "Secondary autoimmune Neutropenia" (sAN) rises later in life and is usually accompanied by autoimmune features. Very few descriptions on the topic are available in the literature. Aim of the study: to describe from a clinical and immunological point of view a cohort of subjects affected with sAN included registered in the Italian Neutropenia Registry (INR) and to compare these data with those from subjects diagnosed with pAN still in the INR . Patient and methods Subjects with neutropenia and positivity of AaN lasting for more than 2 years in subjects older than 5 years of life ( up to 18) or patients affected with neutropenia (plus AaN) associated with autoimmune features registered in the INR from 2005 to 2018 were considered eligible for the present study. Antibodies against neutrophils were always detected throught indirect test . Results: Data from 40 patients affected with sAN (26 females, 65%) were collected. Median age at diagnosis was of 11.6 years (0-21.558mo) with a median follow up of 17 months (0-159mo). The degree of Neutropenia was mild in 12.5%(5/40), moderate in 35% (14/40) and severe in 52.5% (21/40) Neutropenia was diagnosed by chance in half of the cohort and in only 10% (4/40) (10%) definitively resolved . Neutropenia coexisted with leucopenia in almost all the of subjects (36/40, 88%), and moreover, autoimmune haemolytic anemia and thrombocytopenia was concomitant to neutropenia in 12% (5/40) and 32.5% in (13/40) of cases respectively. As for clinical history, 15% (6/40) subjects (15%) were completely asymptomatic , while the remaining 85% (36/40) (85%) had clinical signs of infections and/or autoimmunity features . Infections were documented in 60% (24/40) (60%) of patients. Severe infections namely sepsis , meningitis, pneumonia, osteomyielitis and broad absesses/flemmons were identified in 21% (5/24) of the group. Other type of infections are listed Figure 1A. Apparently there was no correlation between severity of infections and degree of neutropenia ( p=0.2 Fisher' exact test) Autoimmune features and/or autoimmunity markers were present in 30/40 (75% ) of the entire cohort . Thyroiditis and artralgia/arthritis were the most common signs which accounted both for 17,5% of the total episodes. Positivity of ANA and Direct antibodies test ( DAT) were showed in 42% and 29% of cases respectively. (Fig 1B, 1C) As for immunological characteristic 37% (12/32) of the studied subjects were frankly lymphocytopenic, 19% (6/32) had borderline values , while the remaining 44% (14/32) had normal lymphocytes subclasses. In terms of frequency of sub-classes depletion CD19 + and NK were the most frequently decreased subsets. ( Fig 1D) . Dosage of serum immunoglobulin (IgG, IgM and IgA) were shown for all three classes were abnormal (either increased or diminished) in 74% (28/38) of patients (Fig 1D). Mutation analysis performed by Next Generation Sequencing in 16/40 subjects and pathogenic variants of : TACI, TINF 2, CASP 8, CASP 10, PI3K, CARD 11 genes were identified in 4/16 (25%). As for treatment, in 25 % of cases needed Granulocyte-Colony Stimulating Factor to control infections. Rapamicin, micophenolate mofetil, steroids, intravenous immunoglobulin were necessary in 25% (10/40) of patients cases, being (4 of them were already treated on with G-CSF) Comparison with a group of 236 pAN patients of the INR showed that sAN were older at diagnosis (P