Autoimmune Cytopenias and Dysregulated Immunophenotype Act as Warning Signs of Inborn Errors of Immunity: Results From a Prospective Study

Inborn errors of immunity (IEI) are genetic disorders characterized by a wide spectrum of clinical manifestations, ranging from increased susceptibility to infections to significant immune dysregulation. Among these, primary immune regulatory disorders (PIRDs) are mainly presenting with autoimmune manifestations, and autoimmune cytopenias (AICs) can be the first clinical sign. Significantly, AICs in patients with IEI often fail to respond to first-line therapy. In pediatric patients, autoimmune cytopenias can be red flags for IEI. However, for these cases precise indicators or parameters useful to suspect and screen for a hidden congenital immune defect are lacking. Therefore, we focused on chronic/refractory AIC patients to perform an extensive clinical evaluation and multiparametric flow cytometry analysis to select patients in whom PIRD was strongly suspected as candidates for genetic analysis. Key IEI-associated alterations causative of STAT3 GOF disease, IKAROS haploinsufficiency, activated PI3Kδ syndrome (APDS), Kabuki syndrome and autoimmune lymphoproliferative syndrome (ALPS) were identified. In this scenario, a dysregulated immunophenotype acted as a potential screening tool for an early IEI diagnosis, pivotal for appropriate clinical management and for the identification of new therapeutic targets.

Efficacy and Safety of Cyclosporine Treatment in Autoimmune Cytopenias: The Experience of Two Italian Reference Centers

Background: Autoimmune cytopenias (immune thrombocytopenia ITP, autoimmune hemolytic anemia AIHA, and chronic idiopathic neutropenia CIN) are a heterogeneous group of disorders characterized by the presence of autoantibodies directed against platelets (PLT), erythrocytes, and neutrophils (ANC). Frontline steroids are the mainstay of treatment, although most patients relapse and require 2 nd line therapies which slightly differ according to disease subtype. Rituximab is mainly effective in AIHA, although only a fraction of cases would experience long-term relapse-free. Splenectomy may be contraindicated due to age and risk of thrombotic and infectious complications and thrombopoietin-receptor agonists (TPO-RA), effective in more than 70% of ITP patients, may result in great PLT fluctuations and increased bone marrow reticulin fibrosis. Cyclosporine (CyA) is an immunosuppressant used for over 30 years in the post-transplant setting and in the treatment of aplastic anemia. It is a manageable oral drug with known toxicities and its plasma concentrations may be monitored to optimize treatment. However, few data exist about its efficacy in autoimmune cytopenias either used as single drug or in combination with other treatments. Aim: The aim of this study was to evaluate the efficacy and safety of CyA in a cohort of patients with ITP, AIHA, and CIN, followed at two reference hematology centers in Milan, Italy. Methods: Medical charts of consecutive patients treated with CyA 3-5 mg/ kg day in the last 20 years were evaluated. Responses were assessed at 3, 6 and 12 months, and divided into partial (PR, for Hb> 10 g/dL; PLT> 30x 109/L and ANC> 0.8 x 109/L) and complete (CR, for Hb> 12 g/dL; PLT> 100x109/L; ANC>1 x 109/L). Adverse events were recorded according to CTCAE criteria. Results: 41 patients, 27 ITP (66%), 11 AIHA (27%) and 3 CIN (7%), were included, 16 men (39%) and 25 women (61%), with a median age of 60 year (21-81). The median time from diagnosis to CyA start was of 10 years (5-15), with a median of 3 (1-8) previous therapy lines. Most patients were receiving concomitant treatment including steroids (N=13), IVIG (3), or TPO-RA (14). Reasons to start CyA included no response to previous treatments (N=27), platelets fluctuations (N=5) or bone marrow fibrosis (N=5) on TPO-RA, and contraindication for splenectomy (N=4). Median duration of CyA therapy was 5 years (1-9) and 34 patients (83%) responded: 34% CR, 44% PR at month+3; 39% CR and 39% PR at month+6; and 26%CR and 43%PR at month+12 (Figure 1). Specifically, median PLTs increased by 32 x10^9/L at month+3, by 121 x10^9/L at month+6, and by 43 x10^9/L at month+12. Median Hb improved by 0.5 g/dL at month+3, by 1 g/dL at month+6, and by 2,6 g/dL at month +12. Median ANC augmented by 0.7 x10^9/L at month +3, by 1.7 x10^9/L at month +6, and by 0.07 x10^9/L at month +12. Importantly, 9 patients could stop steroids, and 10 subjects discontinued or tapered TPO-RA (5 each). Better responses were observed in ITP patients with baseline bone marrow hypocellularity (p = 0.01), absence of reticulin fibrosis (p=0.02), and in those not previously splenectomized (p=0.04). Among AIHA cases, those with IgG+C direct antiglobulin test positivity showed higher percentage of non-response (67 versus 22% in IgG+). Adverse events were mainly G1-2, occurring in 52% of patients, and included asthenia, dyspnea, myalgia, nausea, vomiting, diarrhea and abdominal pain, epistaxis, petechiae and an Escherichia Coli cystitis. Three patients on concomitant long-term steroids developed a G3 event (1 pulmonary embolism, 1 Aspergillus lung infection and 1 bronchitis), and 1 died for Pneumocystis jirovecii pneumonia. Conclusion: Cyclosporine was effective in about 80% of highly pretreated patients and allowed tapering/discontinuation of concomitant treatments in 63% of cases. Responses were higher in those with ITP, and hypocellular bone marrow without reticulin fibrosis. The occurrence of infectious episodes, including a fatal pneumonia, warrants careful surveillance in this heavily pretreated patient population. Figure 1 Figure 1. Disclosures Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Barcellini: Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Alexion Pharmaceuticals: Honoraria.

Autoimmune Cytopenias and Covid-19 Vaccination: Relapse and Suggested Treatment

Introduction: There are "de novo" and relapsed autoimmune diseases in patients with COVID-19 that includes autoimmune thrombocytopenia, Evans syndrome and autoimmune hemolytic anemia among others (Hematology 2021;26:225-239 and Curr Rheumatol Rev 2021;17:193-204). There is scanty material about relapse of autoimmune hematological diseases after vaccination for COVID-19 (Blood Adv 2021;13:2794-2798). Material and methods: Adult patients 18 years or older with autoimmune thrombocytopenia, Evans syndrome and autoimmune hemolytic anemia who completed SARS-Cov2 vaccination. Results: Between December 2020 and June 2021 there were identified 53 patients with autoimmune hematological disease that completed SARS-Cov2 vaccination. Thirty-six with autoimmune thrombocytopenia, all were preexisting. Twelve with autoimmune hemolytic anemia, 5 secondaries to previous COVID-19 and 7 preexisting. Five with Evans syndrome, all preexisting. Twenty-three patients with autoimmune thrombocytopenia did not develop any fall in the platelet count. Ten patients had a fall of 50 % from basal counts and recovered spontaneously. Three patients developed counts below 30,000 with purpuric symptoms and needed treatment that consisted in two courses of dexamethasone 40 mg daily for four days every three weeks; all patients reached complete remission without any further treatment. All patients with Evans syndrome developed hemolysis and low platelet counts. Two patients maintained Hb levels above 10 and platelet counts above 50,000; both patients had spontaneously recovery. Three patients developed Hb levels below 7 with anemic syndrome and platelets below 50,000 but without purpuric syndrome; they received the same treatment as patients with autoimmune thrombocytopenia and reached complete remission too. All five patients with autoimmune hemolytic anemia secondary to COVID-19 developed Hb levels below 7 with anemic symptoms and needed treatment as described. The remaining 7 patients with preexisting autoimmune anemia developed hemolysis; five with Hb levels above 7 and recovery without any treatment; two had Hb levels below 7 and received the same treatment with full recovery and complete remission. Conclusions: Autoimmune cytopenias can be trigger by vaccines and viral infections by involving molecular mimicry and circulating immune complexes, including SARS-Cov2. The viral protein spike from SARS-Cov2 has mimicry between the Ankyrin-1 in the erythrocyte surface, and has been linked as one of the pathogenesis pathways of autoimmune hemolytic anemia secondary to COVID-19 (Br J Haematol 2020;190:e92-e93 and Blood 2020;136:suppl8,138001). Relapse of autoimmune cytopenias after vaccination with SARS-Cov2 involves stimulation of autoantibodies production from preexisting B cells. Although relapses were observed in the three kinds of patients, all with hemolytic component developed a drop in the hemoglobin levels, most of them needed treatment. It is important to notice that patients with hemolytic autoimmune anemia secondary COVID-19 had severe relapse, event that support the mimicry mentioned lines above. It is important to follow up closely this kind of patients after SARS-CoV2 vaccination, we suggest weekly complete blood counts, and a short courses of high dose dexamethasone can induce curable responses if treatment is advised. Disclosures No relevant conflicts of interest to declare.

Pediatric Patients with Immune Thrombocytopenic Purpura Have a Dysbiotic Gut Microbiome at Time of Diagnosis

Background: Pediatric immune thrombocytopenia purpura (ITP) is the most common cause of autoimmune cytopenias in children and results in autoimmune destruction of platelets, leading to significantly decreased circulating platelets, increased bleeding risk and fatigue. The underlying mechanism of action is thought to be auto-antibody driven, though auto-reactive T cells have also been implicated in some cases. Given the proximity of many cases of ITP to either recent viral infection or vaccine administration, a leading hypothesis is that the environmental exposure mimics a platelet produced epitope causing platelet clearance. Despite the correlation of viral infection and/or inflammatory cascades in the propagation of ITP, the gut microbiome in newly diagnosed pediatric patients has not previously been interrogated. The gut microbiome has previously been shown to modulate the host inflammatory milieu and dysbiosis has been associated with other auto-immune disorders. Thus, we assessed the gut microbiome in newly diagnosed pediatric ITP patients to determine if a similar dysbiosis is present. Methods: Stool samples were collected from 32 pediatric patients (0-18 years) at the Children's Hospital of Philadelphia. 17 patients were newly diagnosed with ITP, 7 patients had ITP for greater than 3 months at time of stool collection and 8 patients had newly diagnosed severe acquired aplastic anemia. Stool samples were kept on ice until processing at our PENN/CHOP Microbiome Core (not longer than 24 hours/sample). Age matched healthy control samples were provided by the Microbiome Core. Shotgun libraries were generated from 0.5 ng DNA using the Nextera XT Library Prep kit and libraries were sequenced on an Illumina HiSeq 2500 in High Output mode to produce paired-end 125 bp sequence reads. Shotgun metagenomic data were analyzed using Sunbeam, a user-extendable bioinformatics pipeline that we developed for this purpose.Diversity within samples were assessed by the number of OTUs at a rarefaction level of 1,000 sequences and the Shannon index. Sample similarity were assessed by Bray-Curtis and Jaccard distances, which were then visualized using principle coordinates analysis. Results: Subjects included 17 patients with newly diagnosed ITP (i.e. stool sample collected < 3 months from diagnosis), 7 patients with ITP > 3 months and 8 patients with newly diagnosed severe acquired aplastic anemia. Patient samples were assessed for gut microbial diversity and richness. As seen in figure 1a, gut microbial diversity in newly diagnosed pediatric patients with ITP was significantly decreased (p = 0.024) while gut microbial richness trended to a concomitant decrease as well (p = 0.093). Interestingly, healthy controls had similar gut microbiomes to one another than newly diagnosed ITP patients had to one another (Fig 1b). These alterations in the gut microbiome were not seen in pediatric patients with newly diagnosed aplastic anemia or with ITP diagnosed > 3 months prior to collection (data not shown). These findings indicate that while there is a dysbiotic gut microbiome in patients with newly diagnosed ITP, there is no dysbiosis present in other pediatric autoimmune cytopenias. Furthermore, the dysbiosis corrects by 3 months post-ITP diagnosis. When we assess specific bacterial families and subgroups we do not find a predominant species that is increased or decreased but rather there are global changes amongst many bacterial species. We did find a trend of lower Alistipes species and increased Eschrechia coli in newly diagnosed ITP patients which is similar to other autoimmune disorders with shifted gut microbiota. Functional pathways analyses showed similar global alterations in vital pathways such as decreased aminoacyl tRNA biosynthesis and homologous recombination. Conclusion: Pediatric patients with newly diagnosed ITP but not other autoimmune cytopenias have gut microbial dysbiosis with perturbations in many bacterial species that may be a cause of, or a result of the underlying mechanism of pathogenesis. Further studies will help determine the role of dysbiosis in the pathobiology of ITP and determine whether intervention alters the duration of disease. Figure 1 Figure 1. Disclosures Lambert: Bayer: Consultancy; Astra Zeneca: Research Funding; Shionogi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ClinGen, ISTH, ASH, GW University: Honoraria; PDSA: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dova: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Daratumumab As a Treatment for Adult Immune Thrombocytopenia: A Phase II Study with Safety Run-in (the DART Study)

Background: Immune thrombocytopenia (ITP) is characterized by antibody-mediated platelet destruction and impaired platelet production. Residual long-lived autoreactive plasma cells may be a source of treatment resistance in autoimmune cytopenias. Antiplatelet-specific plasma cells have been detected in the spleen of the rituximab refractory ITP patients. These cells can also migrate and reside in bone marrow as long-lived plasma cells. Daratumumab, an anti-CD38 antibody, targets plasma cells and is approved for the treatment of multiple myeloma. Daratumumab has been successfully used to treat refractory autoimmune cytopenias in children and a few cases of post-transplant autoimmune cytopenias and refractory SLE in adults. We hypothesized that long-lived autoreactive plasma cells may be the source of treatment failure in some ITP patients. Based on that, we initiated a multicenter, open-label, dose-escalating phase II study with a safety run-in to evaluate the safety and efficacy of daratumumab in patients with ITP (NCT04703621). The first 3 patients were included in the safety run-in during Jan - May 2021. Study design and methods: Main inclusion criteria include age ≥18, primary ITP with a platelet count of ≤30X109/L, failure of corticosteroid therapy, and at least one second-line therapy including rituximab and/or TPO-RA. Main exclusion criteria include active bleeding, secondary ITP, concomitant autoimmune hemolytic anemia. Twenty-one patients will be included in the study. The safety run-in phase includes 3 patients who receive 4 weekly subcutaneous daratumumab injections followed by a 4-week observational period. Enrollment of the next patient in this phase occurs after the previous patient has completed treatment and an observational period. In cohort 1, 9 patients will receive 8 weekly injections. If the response rate is <100% and no severe adverse events appear, the subsequent 9 patients will receive 8 weekly daratumumab injections followed by 2 injections administered every other week. Standard premedication before all daratumumab injections consists of antihistamine, corticosteroid (methylprednisolone 100 mg or equivalent before the 1 st daratumumab injection and 60 mg or equivalent before subsequent injections), and paracetamol. Rescue ITP medications are allowed during the first 8 weeks of the study. Steroid or TPO-RA (eltrombopag or romiplostim) dosing must remain stable during the 2 weeks preceding the inclusion. Dose escalation is not allowed during the study. The primary endpoint is a platelet count >50x10 9/L in 2 measurements 12 weeks after treatment initiation for cohort 1, and 16 weeks for cohort 2, without rescue therapy after week 8. Safety will be assessed by the incidence and severity of adverse events. Secondary endpoints will include the number of weeks with platelet count >50x10 9/L between the end of treatment and end of study without rescue therapy or dose increments of corticosteroids. Time to treatment failure (TTF) is defined as time to first platelet count <30x10 9/L or administration of any platelet elevating therapy after achieving response. Exploratory endpoints include: role of anti-GPIIb/IIIa and Ib antibodies; serial characterization of various subsets of immunocompetent cells in the bone marrow and blood; measurement of HRQoL and fatigue Statistics: The primary outcome (treatment response) will be reported separately for each cohort and the entire study population, expressed by absolute numbers and rates with the corresponding 95% confidence interval. Daratumumab treatment will be considered "successful" if we observe a response rate of 30% or higher. Current enrollment status: As of July 10, 2021, 2 sites are open. Two patients have completed the safety run-in; one is close. One/two responded to treatment at week 12. Platelet response in all 3 patients is shown in Figure 1. No serious or grade 3 adverse events were reported. Cohort 1 will start in August 2021. Figure 1 Figure 1. Disclosures Tsykunova: Ablynx: Consultancy; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sobi: Consultancy; Sanofi: Consultancy. Holme: bayer, Octapharma, Pfizer: Other: support to institution, Research Funding; Bayer, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda, Sobi: Consultancy, Honoraria. Tran: Astra Zeneca: Consultancy; Novartis, Janssen, Abbvie, Takeda, CSL Bering: Consultancy. Tvedt: Ablynx,Alexion, Novartis: Membership on an entity's Board of Directors or advisory committees. Michel: Amgen,Novartis,UCB,Argenx,Rigel: Honoraria. Frederiksen: Novartis: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Research Funding; Alexion: Research Funding; Gilead: Research Funding. Bussel: CSL: Other: DSMB; UCB: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Principia/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; UptoDate: Honoraria; RallyBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova/Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Momenta/Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuter: Rubius: Current equity holder in publicly-traded company; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BioCryst, Bristol Myers Squibb (BMS), Caremark, CRICO, Daiichi Sankyo, Dova, Genzyme, Immunovant, Incyte, Kyowa-Kirin, Merck Sharp Dohme, Momenta, Novartis, Pfizer, Principia, Protalex, Protalix, Rigel: Consultancy, Other: grant support and consulting fees; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, Bristol Myers Squibb (BMS), Immunovant, Kezar, Principia, Protalex, Rigel, Takeda (Bioverativ), UCB: Research Funding; Platelet Disorder Support Association: Membership on an entity's Board of Directors or advisory committees; Up-to-Date: Patents & Royalties: Up-To-Date. Ghanima: Amgen, Novartis, Pfizer, Bristol Myers Squibb, SOBI, Griffols, Sanofi: Honoraria; Bayer, BMS/Pfizer: Research Funding; Amgen, Novartis, Pfizer, Principia Biopharma Inc- a Sanofi Company, Sanofi, SOBI, Griffols, UCB, Argenx: Consultancy.

Monitoring of Patients with Autoimmune Cytopenias during Sars-Cov-2 Vaccination Campaign: The Experience of a Reference Center

SARS-CoV-2 infection and vaccination have raised concern in immune mediated diseases, including autoimmune cytopenias (AIC, i.e. autoimmune hemolytic anemia, AIHA; autoimmune thrombocytopenia, ITP; autoimmune neutropenia, AIN; aplastic anemia, AA; and their combination, termed Evans syndrome, ES). The latter are highly heterogeneous conditions with variable severity and a clinical course marked by several relapses often triggered by immune-activating events (infections, traumas, surgery, etc.) including vaccines. Some reports of ITP and AIHA post-SARS-CoV-2 vaccines have been described but no population studies have been conducted in AIC patients. Here we systematically studied a large series of 100 patients with AIC (44 AIHA, 38 ITP, 7 AIN, 6 ES, and 5 AA) prospectively followed at a reference center in Milan, Italy, who underwent SARS-CoV-2 vaccination from 24 th of March until the end of June 2021. Patients (median age 62 years, range 25-89, female/male ratio 1.7) were monitored with whole blood counts and LDH testing the week before and the week after each vaccination dose. Importantly, ongoing AIC therapy (38% of cases, including steroids, cyclosporine, eltrombopag, and complement inhibitor sutimlimab) were kept stable within the 2 weeks before vaccination. Patients mainly received Pfizer-BioNtech vaccine (N=88), followed by Moderna (N=10), and Astra-Zeneca (N=2). Table 1 summarizes hematologic trends and side effects observed after each dose in patients with ITP and AIHA. Regarding the former, a delta percentage reduction of 10% or higher was observed in up to 13% of cases after the first and the second dose, requiring therapy adjustment in 2 patients. They were two elderly male subjects on low dose eltrombopag treatment and experienced a severe/moderate relapse (platelets 28 and 21x10^9/L) with mucosal bleeding, after the 2 nd dose of Pfizer vaccine. Both had a concomitant trigger (1 hip fracture and 1 bronchitis reactivation) and were rescued by increasing eltrombopag dose and with the addition of prednisone 1 mg/Kg day. Regarding AIHA, 3 elderly patients experienced a clinically significant relapse (>10% Hb decrease): 1 female patient experienced an Hb reduction from 10.4 to 9.1 g/dL after the first dose of Pfizer vaccine, that required a slight increase of steroid dose (to 5 mg day prednisone) and remained stable after the second dose; 1 male subject had an Hb reduction from 13.9 to 9.1 g/d>L after the first dose of Moderna vaccine, requiring prednisone 0.5 mg/kg day; the third male patient experienced a severe relapse (Hb reduction by 47%, from 14 to 7.4 g/dL) with LDH increase to 2.3 x ULN after the second dose of Pfizer vaccine. The patient required high dose intravenous steroids and hemolysis improved in about 1 week. All patients had warm type AIHA and had complained no other triggers or non-hematologic adverse events. Patients with AIN, AA and ES had no significant changes in their hematologic values (1 AIN had a neutrophil decrease by 30% but was consistent with previous oscillations; 2 ES had a platelet or neutrophil decrease within the normal range) and required no treatment changes. Finally, the following non-hematologic adverse events were observed: fever (7%), pain at the injection site (15%) and arthralgia (<5%), without significant differences between the first and the second dose. These data show that SARS-CoV-2 vaccination may be associated with a mild decrease of hematologic values in about 10% of AIC cases. However, true ITP and AIHA relapses occurred in 5% of cases only, sometimes in the presence of a concomitant trigger, and were rapidly rescued with treatment adaptation. Overall, the hematologic monitoring of SARS-CoV-2 vaccine adopted in our survey appears appropriate to early detect and manage AIC reactivation, ensuring a safe vaccination campaign in this patient population. Figure 1 Figure 1. Disclosures Fattizzo: Annexon: Consultancy; Alexion: Speakers Bureau; Kira: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Barcellini: Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Honoraria; Agios: Honoraria, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees.

Genetic Screening of Patients with Evans Syndrome: A Single Centre Analysis

Background: Evans syndrome (ES) is a rare disorder defined as the simultaneous or sequential presence of autoimmune haemolytic anemia and immune thrombocytopenia but it can also be considered as the presence of at least two autoimmune cytopenias. Recent reports have shown that ES is often a manifestation of an underlying congenital immune dysregulation syndrome that, in some case, can benefit from specific treatments. Aims: The aim of this study is to investigate the clinical/immnunological characteristics and the underlying genetic background of a single centre cohort of patients with ES. Methods: Data were obtained from a retrospective charts' review of patients with a diagnosis of ES in our centre. Genetic studies were performed with NGS analysis of 315 genes related to both hematologic and immunological disorders as congenital bone marrow failure syndromes, primary immunodeficiencies, and primary immune regulatory disorders. Results: Fourteen patients (23 males, 17 females) with a median age at onset of 6 years (range 0-16) were studied. ES was concomitant and sequential in 18 (45%) and 22 (55%) patients, respectively. Nine/40 (8%) patients had a family history of autoimmunity. Other abnormal immunological features and signs of lymphoproliferation were present in 24/40 (60%) and 29/40 (72%) of cases, respectively. Seventeen out of 40 (42%) children fitted the ALPS diagnostic criteria. The remaining 15 (37%) and 9 (22%) were classified as having an ALPS-like phenotype and an isolated ES, respectively. Twenty patients (50%) were found to have an underlying genetic defect on TNFRSF13B, FAS, CTLA4, IKBGK, CARD11, LIG4, LRBA, STAT3, CASP10 and ADA2 genes. Table 1 shows the details of clinical/immunological characteristics of patients with or without a genetic diagnosis. No significant differences were noted between the two groups. Conclusions: This study shows that half of patients with ES have a genetic background, secondary to Primary Immunodeficiencies. Therefore, an immunological screening and an extended molecular evaluation should be offered to all patients, since specific genetic diagnosis may benefit from targeted treatments. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Serum Soluble Cytotoxic T-Lymphocyte-Associated Antigen 4 in Children and Adolescents with Autoimmune Cytopenia

Background Autoimmune cytopenias are characterized by the production of autoantibodies against differentiated hematopoietic cells because of defects in central and/or peripheral tolerance. It includes autoimmune hemolytic anemia (AIHA), immune thrombocytopenic purpura (ITP), autoimmune proliferative syndrome (ALPS) and Evans syndrome (EV). Aim of the Work to compare levels of sCTLA-4 in different types of immune cytopenias and their control. Patients and Methods Forty seven children and adolescents who have autoimmune cytopenias were recruited and assessed for eligibility in Pediatric Hematology Clinic, Ain Shams University Children’s Hospital and forming a patients group with age range 8 – 204 months old. An age and sex matched healthy control group were recruited including forty seven healthy participants with age range 6 – 156 months old. Results On initial examination in our study, the prevalence of hepatosplenomegaly and lymphadenopathy among the patients group was 27.7% and 2.1% respectively. Autoimmune cytopenic patients group have statistically significant higher serum sCTLA-4 levels (range 1 – 82 ng/ml) than control group (range 0 – 9 ng/ml) (where P < 0.01 HS). On the other hand, there was no significant difference when comparing levels of sCTLA-4 between sera of ITP, AIHA and ALPS patients (P > 0.05). Serum sCTLA-4 was inversely related to the age at diagnosis and positively related to disease duration. Our results demonstrated the presence of correlations between the levels of sCTLA4 and the severity of autoimmune cytopenias (negative correlation with Hemoglobin (R= -0.315; P = 0.031), mean Hemoglobin (last year) (R= -0.471; P = 0.001) and platelet (R= -0.324; P < 0.05)) and (positive correlation between levels of sCTLA-4 and Reticulocyte count (R = 0.413; P = 0.004), Indirect Bilirubin (R = 0.427; P = 0.003) and Lactate dehydrogenase (R = 0.357; P = 0.014)). There was a significant differences in the prevalence of hepatosplenomegaly among ITP, AIHA and ALPS patients (P < 0.05) with higher prevalence among ALPS patients. Conclusion Soluble form of CTLA4 (sCTLA4) presents in elevated levels in the sera of children and adolescents who have autoimmune cytopenia including AIHA/Evans, ITP and ALPS compared to healthy control group that suggests sCTLA4 could play a role in the pathogenesis of immune cytopenias.

Evans syndrome in adults: an observational multicentre study

Evans syndrome (ES) is a rare condition, defined as the presence of two autoimmune cytopenias, more frequently autoimmune hemolytic anemia and immune thrombocytopenia, and rarely autoimmune neutropenia. ES can be classified as primary or secondary to various conditions, including lymphoproliferative disorders, other systemic autoimmune diseases, and primary immunodeficiencies, particularly in children. In adult ES, little is known about clinical features, disease associations and outcome. In this retrospective international study, we analyzed 116 adult patients followed at 13 European tertiary centers, focusing on treatment requirement, occurrence of complications and death. ES was secondary to or associated with an underlying condition in 24 cases (21%), mainly other autoimmune diseases and hematologic neoplasms. Bleeding occurred in 42% of subjects, mainly low grade and at ITP onset. Almost all patients received first line treatment (steroids+/-IVIG), and 23% needed early additional therapy for primary refractoriness. Further therapy lines included rituximab, splenectomy, immunosuppressants, thrombopoietin receptor agonists, and others, with response rates greater than 80%. However, a remarkable number of relapses occurred, requiring ≥3 therapy lines in 54% of cases. Infections and thrombotic complications occurred in 33% and 21% of subjects, respectively, mainly grade ≥3, and correlated with the number of therapy lines. Besides age, other factors negatively impacting on survival were severe anemia at onset and occurrence of relapse, infections and thrombosis. These data show that adult ES is often severe and marked by a relapsing clinical course and potentially fatal complications, pinpointing the need for high clinical awareness, prompt therapy, and anti-infectious/anti-thrombotic prophylaxis.

Immune Phenomena in Myeloid Neoplasms: An “Egg or Chicken” Question

Immune phenomena are increasingly reported in myeloid neoplasms, and include autoimmune cytopenias/diseases and immunodeficiency, either preceding or complicating acute myeloid leukemia, myelodysplastic syndromes (MDS), chronic myeloproliferative neoplasms, and bone marrow failure (BMF) syndromes. Autoimmunity and immunodeficiency are the two faces of a dysregulated immune tolerance and surveillance and may result, along with contributing environmental and genetic factors, in an increased incidence of both tumors and infections. The latter may fuel both autoimmunity and immune activation, triggering a vicious circle among infections, tumors and autoimmune phenomena. Additionally, alterations of the microbiota and of mesenchymal stem cells (MSCs) pinpoint to the importance of a permissive or hostile microenvironment for tumor growth. Finally, several therapies of myeloid neoplasms are aimed at increasing host immunity against the tumor, but at the price of increased autoimmune phenomena. In this review we will examine the epidemiological association of myeloid neoplasms with autoimmune diseases and immunodeficiencies, and the pivotal role of autoimmunity in the pathogenesis of MDS and BMF syndromes, including the paroxysmal nocturnal hemoglobinuria conundrum. Furthermore, we will briefly examine autoimmune complications following therapy of myeloid neoplasms, as well as the role of MSCs and microbiota in these settings.