ALBERT

Description: RNA polymerase (Pol) III has a noncanonical role of viral DNA sensing in the innate immune system. This polymerase transcribes viral genomes to produce RNAs that lead to induction of type I interferons (IFNs). However, the genetic and functional links of Pol III to innate immunity in humans remain largely unknown. Here, we describe a rare homozygous mutation (D40H) in the POLR3E gene, coding for a protein subunit of Pol III, in a child with recurrent and systemic viral infections and Langerhans cell histiocytosis. Fibroblasts derived from the patient exhibit impaired induction of type I IFN and increased susceptibility to human cytomegalovirus (HCMV) infection. Cultured cell lines infected with HCMV show induction of POLR3E expression. However, induction is not restricted to DNA virus, as sindbis virus, an RNA virus, enhances the expression of this protein. Likewise, foreign nonviral DNA elevates the steady-state level of POLR3E and elicits promoter-dependent and -independent transcription by Pol III. Remarkably, the molecular mechanism underlying the D40H mutation of POLR3E involves the assembly of defective initiation complexes of Pol III. Our study links mutated POLR3E and Pol III to an innate immune deficiency state in humans.

Description: Key Points VPS45 is a new gene associated with severe infections and bone marrow failure in infancy that can be treated by bone marrow transplantation. The mutation affects intracellular storage and transport and results in increased programmed cell death in neutrophils and bone marrow.

Description: Key PointsSurvival was 100% for 18 patients with ADA-SCID treated with genetically modified CD34+ cells (2.3-13.4 years follow up; median, 6.9 years). Long-term engraftment, immune reconstitution, and fewer severe infections were observed in 15 out of 18 patients without leukemic transformation.

Description: Abstract 2249 Poster Board II-226 BACKGROUND. Allogeneic hematopoietic stem cell transplantation (HSCT) is being used to treat a range malignant and nonmalignant conditions. However, it often causes potentially lethal Graft-versus-Host Disease (GVHD). Several studies revealed that mesenchymal stromal cells (MSCs) from human bone marrow can down regulate GVHD after HSCT. METHODS. MSCs were obtained from BM, expanded and characterized by their morphology, immunophenotype, immunosuppressive potential for autologous, partially and fully mismatched lymphocytes. Twenty five patients (pts) got 39 (range, 1 to 4) MSCs infusions for 27 episodes of acute GVHD, which was defined as steroid resistant grade IV aGVHD in all but one patient. RESULTS. GvHD characteristics. Acute GVHD started from day +6 to d +46 from HSCT (median d+18) excluding single pt in whom it occurred on d+150. In 2 pts disease manifested in a hyper acute form before the engraftment (on d+6 and d+8, respectively). In all but one pt GI GVHD was defined as grade IV with full clinical picture of it. Skin GVHD accompanied GI symptoms in 17 pts and 13 was 〉 grade II, in 4 grade IV. Liver symptoms presented in 13 pts, one pt was determinate as VOD. In 3 pts liver involvement was defined as grade IV. In all 24 of 25 pts had GVHD grade IV 4 and one grade III. Previous conventional anti GVHD therapies included: MP in all pts, MTX, various calcineurin inhibitors, MMF, ECP, serotherapy. Only 3 pts showed temporary limited partial response. Treatment with MSCs. The 1st infusion with MSC was given on day +32.5 (range, 8 to 74); d+50 (range, +28 to +180) post diagnosis of aGVHD and HSCT HSCT, respectively. In 22 evaluable pts we treated 24 separate episodes of GVHD: 22 first episodes and in 2 pts relapse of GVHD. In 24 of 39 cases treatment was performed with frozen MSC and in 15 with fresh cultured cells. In 37/39 cases passages 1 to 3 were used. Median number of infused cells was 1 (range, 0.3 to 3.1) x10 6 per kg of pt body weight in each treatment. Initial response was seen in 17/24 episodes of aGVHD (70.8%), in 8 partial (PR) and in 9 complete (CR). Two pts experienced GVHD relapse after achieving CR and very good PR: one case was treated successfully while another was resistant to MSCs. The latest pt with grade IV aggressive liver GVHD received MSCs injections intra hepatic arteries under radiological control following IRB approval with no anti GVHD effect. The procedure was uneventful with no evidence of microembolisation, no changes of blood flow or deterioration of liver tests (cytolysis). We observed a trend for better effect with higher MSC cell dose: total and per first infusion MSCs dose (1.93±1.28 vs. 1.23±0.50, p=0.078; 1.28±0.79 vs. 0.88±0.28, p=0.086). In all cases effect was seen after the first procedure. No difference was noted in the anti GVHD activity between fresh vs. frozen MSCs. There were no immediate or late toxicity or side effects. Overall survival. 14/25 patients are alive up to 20 months follow up. 8/11 pts, died from GVHD (4 within 1-10 d from MSCs infusion), and 3 from other unrelated causes including TTP-1, DAH-1 and disease progression-1. CONCLUSION. Treatment with MSCs seems to be a new novel type of therapy for steroids resistant GVHD with promising preliminary results and low toxicity. Obviously multicenter 2 arm randomized study is in need to confirm these encouraging. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disease characterized by impairment of the phagocyte NADPH-oxidase complex, resulting in deficient microbial killing and life-threatening bacterial and fungal infections. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative approach, but it can be complicated by graft failure, graft versus-host disease (GvHD) and transplant-related mortality (TRM). In order to define prognostic risk factors in this setting, the IEWP of the EBMT performed a large retrospective registry study on 600 pediatric and adult patients with CGD undergoing allo-HSCT. Patients and Methods: We analyzed the outcome of patients with CGD who received allo-HSCT in EBMT centers between 1993 and 2017. The main end-points of the study were overall survival (OS) and event-free survival (EFS; events were death and primary or secondary engraftment failure) according to patient's age, donor type, stem cell source and conditioning regimen. One patient died before allo-HSCT and was excluded from analysis. Results: We studied 536 children (aged 〈 18 years) and 63 adults (aged ≥ 18 years) affected by CGD. The median follow-up was 45.37 months (IQR 15.8-81.8). Genetic results were available for 307 patients: inheritance was X-linked (75%) or autosomal recessive (25%). Median age at transplant was 7.2 years (range: 0.12-48.56). Conditioning regimen was Busulfan/Fludarabine (n=244; 41%), Busulfan/Cyclophosphamide (n=104; 17%), Treosulfan/Fludarabine (n=76; 13%), Treosulfan/Fludarabine/Thiotepa (n=52; 9%) or other drug combinations (n=123; 20%). Donors were human leukocyte antigen (HLA) matched related (MFD, 10/10; n=211, 40%), matched unrelated (MUD, 10/10 or 6/6 in UCB; n=201; 38%), mismatched related (MMFD, ≥ 9/10; n= 27; 5%) or mismatched unrelated (MMUD, ≥ 9/10 or 5/6 in UCB; n= 83; 16%). Stem cell source was bone marrow (BM; n=408; 69%), peripheral blood (PB; n=153; 26%) or umbilical cord blood (UCB; n=27; 5%). Donor engraftment occurred in 516 evaluable patients (88%), while primary or secondary engraftment failure occurred in 68 patients (12%). Seventy-nine patients (13%) died after allo-HSCT. The 2 year Kaplan-Meier estimate of OS and EFS were 87.1% (95% CI, 84.2-89.9) and 77.8% (95% CI, 74.2-81.4), respectively (Fig A). The 2-year cumulative incidence of grade II-IV acute GvHD, chronic GvHD and extensive chronic GvHD was 18.6% (95%, 15.1-22.2), 16.2 % (95%, 18.8-19.7) and 5.5% (95%, 3.4-7.7), respectively. A univariate cox model with spline term demonstrated that older age at transplant was associated with an increased risk of death (p=0.002). Children undergoing allo-HSCT had a superior 2y OS (88.1%; 95% CI 85.2-91.0), compared to adults (78.2%; 95% CI, 67.7-88.7), p=0.03 (Fig B). Patients undergoing allo-HSCT from a MFD had a superior EFS (86.5%; 95% CI 81.5-91.4) compared to MUD (73.3%; 95% CI 66.7-79.9), MMUD (78.2%; 95% CI 69-87.5) and MMFD (59.7; 95% CI 40.4-79.1), p〈 0.001 (Fig C). Patients receiving BM grafts had superior 2y EFS (81.0%; 95% CI 76.9-85.1) compared to PB (72.5%; 95% CI 64.7-80.4) and UCB (66.7%; 95% CI 48.9-84.4), p=0.04. The pattern of disease inheritance and the choice of conditioning regimen didn't have an impact on outcome (Fig D). Fifty-three patients with graft failure underwent a second allo-HSCT and the 2y OS in this group was 82.1% (95% CI, 71.5-92.7). Year of transplantation didn't have an influence on outcome. Conclusion: This is the largest study describing the outcome of allo-HSCT in children and adults affected by CGD. We demonstrate an excellent outcome, with a low incidence of graft failure, TRM and GvHD. Older patients with CGD have reduced survival after allo-HSCT, indicating that transplant should be considered at a younger age. The use of a MMFD is associated with poorer outcome; indication to transplant in this setting should be carefully evaluated by the treating physicians. Disclosures Chiesa: Bluebird Bio: Consultancy; Gilead: Consultancy. Kalwak:medac: Other: travel grants; Sanofi: Other: travel grants. Sykora:Aventis-Behring: Research Funding; medac: Research Funding. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wynn:Orchard SAB: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Equity Ownership; Chimerix: Research Funding; Genzyme: Honoraria; Bluebird Bio: Consultancy; Orchard Therapeutics: Consultancy; Chimerix: Consultancy. Zecca:Chimerix: Honoraria. Veys:Pfizer: Honoraria; Servier: Research Funding; Novartis: Honoraria. Slatter:Medac: Other: Travel assistance.

Description: Inherited bone marrow failures syndromes (IBMFS) are rare genetic disorders usually characterized by congenital anomalies, development of bone marrow failure and a tendency to develop malignant diseases. Although single disease registries for Fanconi anemia (FA), Diamond-Blackfan Anemia (DBA), Severe Congenital Neutropenia (SCN) and Dyskeratois Congenita (DC) have been established the true incidence of many of the IBMFS is still unknown. To investigate the prevalence of each of the IBMFS and to the types and frequencies of complications we set up a retrospective national registry of these disorders in Israel. We reviewed charts of 127 patients with IBMFS diagnosed between 1964 and 2005. This represents the majority of such patients in the country. Consanguinity was recorded in 50 (40%) patients. Median time since diagnosis was 6 years (range: 1 month-39 years). Genetic analysis was available in 60 (47%) patients. The number of patients within each disease category is presented in the Table. The majority of patients who succumbed to their disease had FA. Of the 29 patients who developed malignancies (hematological and solid) the majority 23 (79%) had FA. All of the 6 patients who developed solid tumors had FA. The solid tumors were squamous cell carcinoma of head and neck, esophagus, cervix and vulva. Table: Israeli Inherited Bone Marrow Failure Cohort - Diagnosis, Complications and Survival No. of pts. FA DBA SCN CAMT DC SDS TAR NOS All Total (%) 66 (52) 18 (14) 21 (16.5) 8 (6) 6 (5) 3 (2) 3 (2) 2 (1.5) 127 (100) Per 106 people 5.3 2.6 2.1 1.1 0.86 0.43 0.29 Deceased (%) 24 (36) - 6 (29) - 2 (33) 1 (33) - - 33 (26) Molecular diagnosis (%) 34 (51) 6 (33) 7 (33) 8 (100) 4 (67) 1 (33) - - 60 (47) MDS/AML/ALL (%) 19 (29) - 4 (19) 2 (25) - - - - 25 (20) Solid tumor (%) 6 (9) - - - - - - - 6 (5) CAMT, Congenital Amegakaryocytic Thrombocytopenia; SDS-Shwachman-Diamond Syndrome; TAR, Thrombocytopenia Absent Radii; NOS-not otherwise specified. This is the largest population-based study which has examined the relative frequency of each of the IBMFS. In this cohort, FA was by far the most common form of an IBMFS (52% of pts), followed by DBA (16.5%) and SCN (14%), while CAMT, DC, SDS and TAR were far less common. These findings agree with the frequencies of FA, DBA and SCN reported by individual disease registries, and contrast with the results from the Canadian inherited marrow failure registry (Pediatr Blood Cancer47:918, 2006), where among a smaller number (39) of patients with an IBMFS the frequencies of FA, SDS, DBA and SCN were similar (12% each). One caveat is that the Israeli cohort has a large proportion of consanguineous families, which differs from most cohorts in other countries. The number of undiagnosed patients with IBMFS in our study was very small. SDS and DC are either rare in our region or under-diagnosed. The implementation of new diagnostic tests will help to resolve this issue. These data provide a rational basis for longitudinal surveillance and prevention of complications in the severe forms of IBMFS.

Description: Key Points Deficiency of TPP2 is associated with Evans syndrome and viral infection susceptibility. TPP2 deficiency links premature immunosenescence of T and B cells with severe autoimmunity.

Description: Introduction Autoimmune lymphoproliferative syndrome (ALPS) is characterized by abnormal lymphocyte homeostasis caused by defective apoptosis. Mutations in genes involved in the Fas death receptor pathway (FAS, FASLG or CASP10 genes) are the cause for the pathogenesis of ALPS. However, in 20-30% of all ALPS cases, collectively classified as ALPS-U (undetermined), the genetic defect is still unknown. The objective of this study was to employ whole-exome sequencing to search for novel gene candidates underlying ALPS-U or ALPS-like disease. Resulting candidates should be validated and their impact on the Fas death receptor pathway studied. Methods Peripheral blood samples were collected from 26 patients diagnosed with ALPS (based on clinical phenotype and accumulation of DNT cells), relatives and healthy controls. PCR and Sanger sequencing were performed to check for germline and somatically acquired FAS, FASLG and CASP10 mutations. Whole-exome sequencing was carried out for all patients with unknown mutation and their parents. Candidate genes were identified by a KEGG-based protein interaction analysis interface of our in-house developed proprietary MySQL database driven workbench, termed SNuPy (Single Nucleotide Polymorphism Database). STRING 9.1 was used to identify high confidence (≥0.900) interaction partners of the Fas pathway. Single nucleotide variations (SNVs) were verified by PCR and Sanger sequencing. The impact of detected mutations on the candidate genes´ expression and functionality was analyzed by immunoblot. The candidate genes´ impact on Fas death receptor pathway and Fas/FasL expression was examined by flow cytometry, ELISA and qRT-PCR. Results Out of 26 analyzed ALPS cases 4 unrelated patients harbored heterozygous germline mutations in the death domain of the Fas receptor (p.Q282K, p.R249G, p.NVQ265-267KQT) or the extracellular domain (p.R191C), respectively. Two siblings with homozygous FASLGtruncating mutation (A69fs*138) and complete loss of FasL surface expression as a consequence were identified. Whole-exome sequencing also identified a homozygous R212* mutation in the IL12/IL23 receptor-component IL12RB1 (Interleukin 12 receptor, beta 1 subunit) in a patient who presented with classical ALPS symptoms (chronic non-malignant, noninfectious lymphadenopathy, splenomegaly, hepatomegaly, elevation of DNT cells, autoimmune cytopenias with polyclonal hypergammaglobulinemia, persistently increased vitamin B12 and IL10 levels). The p.R212* mutation in IL12RB1 leads to premature protein truncation by a stop codon gain, resulting in a complete loss of cell surface expression of IL12RB1 in the patient. IL12 is a factor known to regulate expression of both Fas and FasL. IL12 signaling was abrogated as demonstrated by deficient downstream STAT4 phosphorylation and IFNγ production. Low FasL expression on T-cells and low soluble FasL plasma levels were probably due to lack of IFNγ mediated transcriptional activation of FasL. In contrast to healthy controls, prolonged IL12 stimulation did not trigger upregulation of FasL nor apoptosis in the IL12RB1 deficient and FasL deficient (A69fs*138) patients, indicating that IL12 mediated apoptosis is FasL dependent. Heterozygous carriers of the IL12RB1 or the FASLG mutation showed an intermediate response but were asymptomatic and sub-clinically affected (showing e.g. moderate elevation of DNT cells). Conclusion Our data show that IL12 employs Fas signaling to achieve T-cell apoptosis and reveal IL12 signaling deficiency as a new cause of ALPS like disease via its impact on FasL expression. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Veno-occlusive disease (VOD) of the liver occurs after hematopoietic stem cell transplantation (HSCT) as a result of the toxic damage on sinusoidal and hepatic cells caused by the conditioning regimen. Diagnosis of VOD is usually based on clinical criteria. Intensity of preparative regimen, pre-existing liver disease, busulfan (Bu) as part of the conditioning, etc. have been proposed as significant risk factors with myeloablative conditioning (MAC). In the present study, we examined the incidence and the risk factors for VOD after allogeneic-HSCT using a reduced intensity conditioning regimen (RIC). Patients and Methods From April 1996 until November 2012, 271 consecutive patients with various hematological malignancies underwent allo-HSCT in our department with the same RIC regimen. The RIC regimen consisted of fludarabine 30 mg/m2/day x 6, oral Bu 4 mg/kg/day or i.v. Bu 3.2 mg/kg/day x 2, and ATG. Oral Bu was given to 138 patients, while 133 patients were treated with the intravenous formulation. Diagnosis of VOD was confirmed using the Seattle criteria. Clinical characteristics of patients are shown in Table 1. Cumulative incidence was calculated using a competitive risk model. In multivariate analysis the following variables were entered: age, sex, disease risk status, year of transplant, donor status, previous myeloablative conditioning, pre-existing liver disease, and route of Bu administration. Results Incidence of VOD and outcome: VOD was developed in 24 out of 271 patients. The cumulative incidence of VOD in the whole cohort of patients was 8.5% (95% CI, 5.8 - 12.6%). VOD onset occurred in a median of 3 days post graft infusion (range, 0 -24 days). Moderate and severe VOD was observed in 20 and 4 patients respectively. Risk factors for VOD: Using a univariate and multivariate analysis the only variable associated with VOD incidence was the route of Bu administration. The cumulative incidence of VOD was 11.7% (95% CI, 7.3 - 18.5%), and 5.3% (95% CI, 2.5 - 10.8%) in the groups of patients who received Bu per-os and intravenously respectively (p=0.02). (Figure 1). Severe VOD was observed in only 4 patients and none of the variables tested in multivariate analysis was significantly associated with the incidence of severe VOD. Outcome of patients with VOD: VOD resolved in most of the patients (20/24) in a median of 21 days (range, 8-82 days). The cumulative incidence of VOD resolution was 83%. All 4 patients with severe VOD died before day 100 and before VOD resolution. The overall cumulative incidence of VOD-related mortality was 1.8%. In multivariate analysis, none of the variables tested was significantly associated with VOD-related mortality. Discussion As compared with the reported incidence of VOD with MAC, RIC with low dose Bu is associated with reduced incidence of VOD, VOD severity and mortality. In our study, we observed that Bu administered intravenously was associated with significantly reduced incidence of VOD in comparison with Bu administered per-os. Disclosures: No relevant conflicts of interest to declare.

Description: Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)–8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression ofMEFV and C5a/IL-8–inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8–inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1β. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1β. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8–inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation.