ALBERT

Beschreibung: Key Points B-cell neogenesis is decreased independently by both aGVHD and cGVHD. B cells during GVHD undergo a higher number of cell divisions related, in the chronic form, to a higher BAFF/CD19 ratio.

Beschreibung: Background: Graft-versus-host disease (GVHD) is a major cause of mortality after unrelated hematopoietic stem cell transplantations (HSCT). Despite the development of modern immunosuppressive strategies, a nearly perfectly controlled compatibility of the classical HLA genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) and availability of numerous so-called minor histocompatibility antigens (e.g. HY or HA-1), its incidence remains largely unexplained to date. MIC genes (MHC class I chain-related) - a distinct lineage of MHC class I genes – are promising candidates to explain, at least partially, the incidence of GVHD in HLA-matched transplantations. MICA and MICB are highly polymorphic (100 alleles for MICA and 40 for MICB) and encode functional cell-surface glycoproteins up-regulated by cell stress. They interact with NKG2D, an activating receptor expressed on the surface of cytotoxic αβ CD8+ and γδ T lymphocytes and natural killer cells. MIC genes are already known to have a HLA-independent effect on solid graft outcomes and may play a similar role in HSCT by triggering GVHD. Objective: The objective of the present study was to determine the impact of donor/patient matching at the MICA and MICB loci on the incidence of GVHD in patients undergoing unrelated HSCT. Methods: We retrospectively analyzed a multicenter cohort of 1072 unrelated transplantations performed between 1996 and 2013. All donor-recipient pairs were fully typed at high resolution for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 and were matched for ten of ten HLA alleles (HLA 10/10 matched). High resolution genotyping of MICA and MICB was performed by sequenced-based typing in order to define matching grades between donors and patients. The endpoints of the study were acute and chronic GVHD. Apart from HLA-DPB1 matching, statistical models were adjusted for major clinical variables which have been shown to be associated with outcome (patient’s age, patient’s and donor’s sex, patient’s and donor’s serological status for cytomegalovirus, year of transplantation, time to transplantation, transplantation center, source of stem cells, conditioning regimen, GVHD prophylaxis, treatment with anti-thymocyte globulin, disease category and severity at transplantation). Results: Of the 1072 transplantations, 134 (12.5 %) and 380 (35.4 %) were mismatched at the MICA and MICB locus, respectively. Both MICA and MICB mismatches were significantly associated with an increased incidence of severe acute GVHD (grades III-IV) in univariate and multivariate models (multivariate model: HR = 2.32, 95 % CI = 1.84-2.92; p=0.0003 for MICA and HR = 1.49, 95 % CI = 1.24-1.79; p=0.03 for MICB). At day 100 post-HSCT severe acute GVHD incidences in mismatched vs. matched transplantations were 19.62 % vs. 15.08 % and 20.00 % vs. 14.84 % for MICA and MICB, respectively (Figure 1). Chronic GVHD was associated with MICA and MICB mismatches in univariate analysis (HR = 1.55, 95 % CI = 1.27-1.89; p=0.029 for MICA and HR=1.38, 95 % CI = 1.19-1.62; p=0.03 for MICB), but showed only a trend for association in multivariate models. Figure 1 Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Figure 1. Estimated cumulative incidence curves of grades III–IV acute GVHD according to MICA (panel A) and MICB (panel B) matching status. The solid and dashed lines represent MIC matched and mismatched grafts, respectively. The Fine and Gray model was used with relapse and death considered as competing risks. Conclusion: To date this is the largest reported MICA and MICB sequence analysis whether in HSCT or solid organ transplantation. Inclusion of MICA and MICB typing in the donor selection process may be a practical clinical strategy for lowering the risks of severe acute GVHD after unrelated HSCT. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only curative treatment for many patients with hematologic disease. Unfortunately, the transplanted immune system can reacts against the patient, inducing Graft-versus-Host Disease (GvHD). GvHD is the leading cause of treatment related mortality and morbidity following HSCT and is expected to increase in the forthcoming years. Pathophysiology and long-term determinants of GvHD are still largely unknown. The French Society for Stem Cell Transplantation and Cell Therapy (SFGM-TC) launched the CryoStem project in 2011 with the goal of establishing a national biobank on GvHD. CryoStem has been selected and funded to the extent of 5,5 millions € over a period of 9 years by the French Government’s Investissements d’Avenir program. As of today, CryoStem brings together 33 HSCT Units and 23 Biological Resources Centers that actively contribute to a networked, prospective, longitudinal and standardized peripheral blood samples collection with concomitant well documented clinical data. Patients sampling is scheduled before and after HSCT, depending on the occurrence or not of acute and/or chronic GvHD. Three types of pre-analytical products are processed and stored, according to standardized techniques: viable cells in DMSO, dried cell pellets and plasma. Samples and clinical data are anonymized and centralized in CryoStem-dedicated web-based database application. Recruitment started in July 2012: currently, about 1,590 patients and 690 related donors have been included. So far, 3,820 blood samples have been processed, corresponding to 48,400 frozen pre-analytical samples. In order to guarantee the quality of frozen samples, an ISO9001 certification of the network is ongoing. National projects are the only way to acquire the large number of samples needed for statistically significant results in a reasonable time. The collection is on embargo until further notice and the first call for scientific projects is expected to be launched in December 2014. CryoStem is a crucial step toward progress in human GvHD research: it will allow collaborative studies between leading French and international research organizations and foster technology transfer with potential industrial players whenever possible to eventually improve the translational research on GvHD. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background Donor lymphocyte infusions (DLI) can produce lasting remissions in patients with relapsed chronic myeloid leukemia (CML), but are less effective in non-CML diseases. Chemotherapy-induced lymphodepletion prior to DLI, achieved with cyclophosphamide (Cy) and fludarabine (Flu), has been shown to enhance activation of donor lymphocytes and cause significantly more acute graft-versus-host disease (GVHD) than DLI alone. To safely balance the toxic versus beneficial effects of activated donor lymphocytes, we combined lymphodepleting chemotherapy with the infusion of donor T cells engineered to carry a suicide gene to treat patients with aggressive hematologic malignancies. Methods Donor T cells were transduced with a retroviral vector expressing the Herpes simplex thymidine kinase (TK) suicide gene and the human Thy-1 selection marker and expanded in culture for 15 to 19 days prior to their infusion. In this phase I/II trial, the safety and efficacy of Cy/Flu/DLI-TK was studied between February and December 2011 in 10 adults with relapsed multiple myeloma (n=5) or myelodysplasic syndrome/acute leukemia (n=5). One had previously failed to respond to at least one standard DLI while others had not received any DLI before inclusion. All were free of immunosuppressive therapy at inclusion. Patients were infused with a mean cell-dose of 5 (range: 1-10) x106 CD3+ cells/kg of which 98% were TK+ cells (range: 97%-99%). DLI products contained a mean ratio of 0.6% (range: 0.1-1.6) CD4+FoxP3+Helios+ regulatory T-cells. This trial was registered at www.clinicaltrials.gov as NCT 01086735. Results The Cy/Flu regimen was profoundly lymphodepleting and led to a mean 3-day period of neutropenia below 500 PMN/µL. Three patients developed acute GVHD following TK+ cell infusion. One patient had a grade I cutaneous GVHD resolving with local steroids. In another patient with grade III cutaneous and digestive involvement, intra-venous administration of ganciclovir (GCV) led to the complete and lasting resolution of symptoms correlated with clearance of TK+ cells in peripheral blood. A third patient had a grade III liver GVHD. Due to only partially controlled leukemia at the time of DLI, GCV treatment was postponed for one week after GVHD onset in order to support a putative GVL effect. Treatment with GCV led to the rapid disappearance of TK+ cells in peripheral blood and liver (see Figure) but without clinical improvement after 2 weeks, so that an additional immunosuppressive therapy should be instituted. Six patients, including one of the 2 treated with GCV, experienced relapse/progression of their malignancy and received additional anti-cancer therapy. With a median follow-up of 22 months after Cy/Flu/DLI-TK, 6 patients are alive, 5 of them being disease-free. In patients not treated with GCV, TK+ cells could still be detected in peripheral blood till at least 12 months after their infusion. Conclusion Lymphodepletion followed by suicide-gene-transduced T-cell infusion may help to safely enhance the immune activity of DLI, which could be further improved by regulatory T-cell depletion (Maury et al, Science Translat Medicine 2010). Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Background: A recent prospective study (Socie & al Blood 2011) demonstrated that the addition of antithymocyte globulins (ATG) to prophylaxis for graft versus host disease (GVHD) in the setting of standard myeloablative conditioning (MAC) regimen matched unrelated donor (UD) allogeneic stem cell transplantation (HSCT) resulted in a decrease in incidence of chronic GVHD without an increase of relapse or non-relapse mortality. However, stem cell source was mostly peripheral blood stem cells (PBSC) and patients (pts) were compatible at HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 (eight out of eight alleles). A retrospective study using the same setting (Mohty & al Leukemia 2012) found very similar results with donor/recipient pairs matched at 10 loci (ten out of ten alleles). However, one third of the pts received PBSC as stem cells source and 20% of the pts were transplanted with a mismatch donor (9 out of 10 alleles). We thus conducted this study to assess the impact of ATG in pts with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) transplanted with MAC using bone marrow only (BM) from matched 10/10 UD. Patients and methods: We includedall consecutive pts older than 18 years who received a first HSCT in France for AML or MDS with BM from a matched 10/10 UD after a standard conditioning regimen (fractionated total body irradiation (TBI)-Cyclophosphamide(Cy), n=106 or Endoxan-Cy, n=91) between June 2000 and June 2012. Clinical data were prospectively collected using ProMISe (Project Manager Internet Server), an internet-based data registry system shared by all centers of the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC). This study was approved by the scientific committee of the SFGM-TC. Results: A total of 197 adult pts met the criteria and were analyzed (165 AML (84%) and 35 MDS). 54 (27%) pts received ATG in the conditioning regimen whereas 143 (73%) did not. Median follow up was of 20.3 months [0.4-156]. Both groups (with or without ATG) were well balanced except for the number of CD34+ cells infused (higher in the ATG group: 3.07x106/kg [0.7-13] vs 2.71x106/kg [0.6-89] for none ATG group (p=0.03)) and GvHD prophylaxis (more likely cyclosporine plus methotrexate with ATG (94%) vs 84% without, p=0.05). The probability of acute GvHD grade II-IV was similar between both groups: 41.3% with ATG vs 49.6% without, p=0.2. Incidence rate of severe acute GVHD grade III-IV at day 100 tended to be lower in the ATG group as compared with the none ATG group (20.3% vs 9.5%, respectively, p=0.052). The cumulative incidence (CI) of chronic GVHD at 2 years was 42% in the ATG group vs 28% in the none ATG group and 37% vs 44% at 4 years, respectively (p=0.18). The probability of extensive chronic GVHD at 2 years was identical between both groups (12% with ATG vs 16% without, p=0.95). CI of non-relapse related mortality (NRM), relapse as well as disease free survival (DFS) and overall survival (OS) were similar between the 2 groups (Table 1). In multivariate analysis, advanced disease status was the only parameter associated with decreased DFS (HR 0.37, 95% CI [0.19-0.70], p=0 .02) and OS (HR 0,41, 95% CI [0,24;0,71], p

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal stem cell disorder. Pregnancy in patients with PNH results in an increase in maternal and fetal complications and a consequent increase in maternal and fetal mortality. Maternal complications include thrombosis, cytopenias and infections while fetal complications relate to preterm delivery (de Guibert et al, 2011). Eculizumab is a humanized monoclonal antibody that blocks terminal complement activation, preventing intravascular hemolysis and its consequences. In PNH it reduces the need for transfusions, protects against thrombosis, and may improve long-term survival. We previously showed a benefit in using eculizumab in pregnancy for patients with PNH in a limited number of cases (Kelly et al, BJH 2011). This study was of 70 pregnancies in 57 women. Patients were identified through physician willingness to participate as well as through the Global PNH Registry. A specific questionnaire was sent to physicians. Local IRBs approved the study. Across the 70 pregnancies, 41 women were on eculizumab before conceiving and remained on the drug during pregnancy, 28 women started eculizumab in either the second or third trimester and 1 woman stopped eculizumab at 12 weeks gestation. The median age at PNH diagnosis was 23 years (range 13-36). The median age at the start of each pregnancy was 29 years (range 18-40). Nineteen women (33%) had a prior documented history of aplastic anemia and 8 (14%) had a prior thrombosis. The median PNH granulocyte clone around the start of pregnancy was assessed in 51 cases and was 94.3% (range 24.3-100). There were 62 live births, 2 stillbirths and 6 first trimester miscarriages. There were 3 twin pregnancies. In 1 of these, there was a fetal death due to intrauterine growth retardation (IUGR). Anticoagulation (AC) with low molecular weight heparin (LMWH) was used in 60 pregnancies (86%) and with fondaparinux in 1 case: therapeutic doses were used in 26 pregnancies, prophylactic doses in 28 pregnancies and an intermediate dose in a further 7. Aspirin was used in 4 pregnancies. Folic acid and oral iron supplements were used in 62 (89%) and 26 (41%) pregnancies, respectively. Transfusion requirements increased in pregnancy from a mean of 0.13 units per month in the 6 months before conception to a mean of 0.94 units per month whilst pregnant. This returned to pre-pregnancy levels after delivery. No platelet transfusions were needed in the 6 months before being pregnant compared with 99 platelet transfusions during the pregnancies. Higher doses, or more frequent dosing, of eculizumab was used to treat recurrent intravascular hemolysis in over half the pregnancies (52%) that progressed past the first trimester. Eculizumab was stopped in 10 instances after the postpartum period, 9 due to funding issues and 1 as the patient underwent a bone marrow transplant. Two patients who stopped eculizumab 12 weeks after delivery experienced a thrombosis. The first experienced a mesenteric thrombosis 4 weeks after stopping eculizumab whilst on therapeutic dose of LMWH and the second suffered a Budd-Chiari 8 weeks after stopping eculizumab. Both were recommenced on eculizumab immediately. The mean reduction in platelet count from the start of pregnancy to delivery was 37 x 109/l. There were 10 significant bleeding episodes: 1 recurrent epistaxis, 1 antepartum bleed and 8 postpartum bleeds. Two patients experienced a postpartum thrombosis whilst on eculizumab, 1 a deep vein thrombosis and the other a mesenteric thrombosis, likely precipitated by a plasma infusion. Nineteen births were premature (31%), mainly due to pre-eclampsia (6 cases) and IUGR (5 cases). Four babies had complications due to prematurity: 1 had toxic megacolon and required a temporary ileostomy, and the other 3 had initial growth retardation due to prematurity. Twenty-five babies were breast fed and in 10 cases, breast milk was tested for eculizumab but no drug was detected. Eculizumab was not detected in 13 cord blood samples and was found at very low levels in a further 7 samples (11.8-21.2µg/ml). In conclusion, eculizumab appears safe to use in pregnancy in PNH and does not appear to cross the placenta in significant quantities to block complement or to be excreted in breast milk. Higher doses may be required later in pregnancy to prevent hemolysis. Overall pregnancy outcomes in this group are better than historical controls with supportive therapies alone without eculizumab. Disclosures Kelly: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Höchsmann:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kulasekararaj:Alexion Pharmaceuticals: Speakers Bureau. Röth:Alexion Pharmaceuticals: Speakers Bureau. Weitz:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hill:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Risitano:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Patriquin:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Muus:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Turner:ICON Clinical Research: Employment. Schrezenmeier:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Szer:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Peffault de Latour:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Beschreibung: Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), −7/7q (17.2%), and 11q− (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

Beschreibung: In rodent graft-versus-host disease (GVHD) models, anti–IL-21 neutralizing mAb treatment ameliorates lethality and is associated with decreases in Th1 cytokine production and gastrointestinal tract injury. GVHD prevention was dependent on the in vivo generation of donor-inducible regulatory T cells (Tregs). To determine whether the IL-21 pathway might be targeted for GVHD prevention, skin and colon samples obtained from patients with no GVHD or grade 2 to 4 GVHD were analyzed for IL-21 protein expression. By immunohistochemistry staining, IL-21 protein-producing cells were present in all gastrointestinal tract samples and 54% of skin samples obtained from GVHD patients but not GVHD-free controls. In a human xenogeneic GVHD model, human IL-21–secreting cells were present in the colon of GVHD recipients and were associated with elevated serum IL-21 levels. A neutralizing anti–human IL-21 mAb given prophylactically significantly reduced GVHD-associated weight loss and mortality, resulting in a concomitant increase in Tregs and a decrease in T cells secreting IFN-γ or granzyme B. Based on these findings, anti–IL-21 mAb could be considered for GVHD prevention in the clinic.

Beschreibung: Abstract 1347 INTRODUCTION: Fanconi Anemia (FA) is an autosomal and X-linked recessive disease characterized by marrow failure, somatic malformations and cancer proneness primarily leading to AML and head and neck carcinomas. The disease is due to mutations in at least 15 genes responsible for a failure of DNA repair mechanisms that renders the cells sensitive to interstrand cross linkers leading to block of cell cycle in G2 phase. However there is evidence that FA proteins have multiple functions as they are also implicated in cytokine hypersensitivity, response to oxidative stress and immune response. Scanty information is available on immunological status in FA patients. We conducted a retrospective multi-centric analysis of the pre HSCT immunological status of 61 FA patients in aplastic phase by assessing peripheral blood immunophenotype and immunoglobulin serum level (46/61). RESULTS: Absolute lymphocyte number was within the normal range for age in 70% of patients (43/61). Only 10% of subjects (6/61) were severely lymphocytopenic (