ALBERT

Description: Abstract 650 The Seattle group previously reported that the adapted Charlson CI (Sorror-CI) was useful for predicting NRM and survival in patients undergoing allo-SCT. However, the value of this CI index is still under considerable debate, since the cohort used by Sorror et al. (Blood 2005; 106:2912-9) included a large age range, a wide variety of diseases, with both myeloablative and RIC transplants. On the other hand, toxicities and NRM prediction is most likely needed in elderly and medically infirm patients who are increasingly offered RIC allo-SCT. Therefore, the current study was designed to test the performance of this CI and its association with outcomes among a cohort of 345 patients (i) aged 〉50 y.; (ii) diagnosed with a single disease entity, AML in CR1, and (iii) who underwent a RIC allo-SCT reported to the EBMT registry between 1999 and 2006. In this series, the median year of allo-SCT was 2004, and the median age was 58 y. (range, 50-76). 32% of patients needed more than one induction course to achieve CR1. A fludarabine-based RIC regimen was used in 64% of patients, while 31% of patients received low-dose TBI as part of their RIC, and 6% received other non-specified RIC regimens. 76% of the patients received allo-SCT from an HLA-matched sibling donor. Based on score calculated with hazard ratios (HR) estimated on the population studied, 161 patients (47%) had a CI score of 0, 96 patients (29%) had a score of 1, and 49 (14%) had a CI score of 2. The remaining 39 patients (11%) had CI scores of 3 or more. In this cohort, 2 years overall and leukemia-free survival rates were 64±3% and 54±3% and the 2 years relapse and -NRM cumulative incidences were 32±2%, and 15±2% respectively. The 2 years NRM incidences according to comorbidities score 0, 1, 2 and 3+ were 9±2%, 15±4%, 18±5% and 31±7%, respectively. In multivariate models (adjusted for recipient age, donor type, use of TBI or not, and cytogenetics risk group) comorbidities such as moderate active liver disease, obesity, prior history of renal dysfunction, and prior history of severe liver disease were associated with the highest HRs for 2 years NRM (varying from 2.11 to 2.76) and 2 years cumulative incidences of NRM varying from 22% to 44%, whereas previous solid tumor, diabetes, rheumatologic abnormalities, moderate pulmonary diseases, cardiac abnormalities (other than arrhythmia and valve disease) were associated with the lowest HRs (varying from 0.2 to 1.0) with 2 years cumulative incidences of NRM varying from 5 to 17%. Results from this large study performed in a single disease entity and homogeneous allo-SCT setting, suggest that the hematopoietic cell transplantation-specific CI is a simple, informative and useful tool for capturing pre-transplant comorbidities, and for predicting NRM after RIC allo-SCT for AML in CR1 in patients aged 〉50 y. Such index may be used for clinical trials and patient counselling before RIC allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3738 Graft-versus-host disease (GVHD) represents a major challenge and the main cause of morbidity and mortality after allogeneic transplantation. Using the standard GVHD prophylaxis based on a calcineurin inhibitor plus methotrexate (MTX). The incidence of acute GVHD is in the range of 30–60%, so that new strategies are required in order to decrease GVHD without hampering GVL. Sirolimus, an mTOR inhibitor, allows to decrease the risk of GVHD and increases the number of Treg after transplantation. Unfortunately, its use may increase the risk of microangiopathy and, moreover, its combination with calcineurin inhibitors blocks the development of Treg. Bortezomib is a proteosome inhibitor and blocks the nuclear translocation and activation of NF-kB. It induces depletion of alloreactive T and allows the expansion of T-cells with suppressive properties. Accordingly, both drugs could favour the development of a tolerogeneic immune response after transplantation. In the current study we have analyzed the synergistic effect of sirolimus together with bortezomib. Bortezomib 100 nM plus sirolimus 5nM synergistically inhibited T-cell activation as assessed by the expression of CD25, production of IFNg and expression of CD40L as well as proliferation assessed as expression of PKH. Remain vibility, these effects could not be attributed to a decreased viability of T-cells, as assessed by 7-AAD, at the concentrations evaluated. As compared to each drug alone, the combination significantly decreased the production of Th1 cytokines (IFNg, IL-2 and TNF) while regarding TH2 cytokines, only IL-6 significantly decreased upon combining both drugs. Concerning the mechanisms involved in this synergistic effect, the combination of both drugs resulted in an inhibition of the Akt and Erk ½ phophorylation, thus indicating that sirolimus inhibit pathways which could allow T-cells to escape from the effect of bortezomib at the doses used in the current experiment. In order to confirm in vivo the synergistic effect of sirolimus and bortezomib, a GVHD mouse model (C57/BL6-Balb/c) was carried out. Mice receiving both drugs (Bortezomib 1μg/day intravenous on days 0, +1, +2 postransplantat and sirolimus 0.25mg/Kg intra-peritoneal on days 0 to 12) has a significantly lower incidence of GVHD and longer survival as compared to each drug alone. We also wanted to evaluate whether the immunosuppressive effect of the combination was unspecific or, by contrast, it allowed induce specific immune tolerance against host but maintaining the immune response against other antigens. For this purpose a haematopoietic cells of Balb/c mice which were C57/BL6 complete chimeras were infused to NOD-SCID mice. While none of the donors had developed GVHD after transplantation plus sirolimus and bortezomib postransplant, the NOD-SCID mice succumbed due to GVHD. Furthermore, we infused WEHI cells to BALB/c after total body irradiation and we observed that, while all BALB/c mice receiving WEHI plus C57BL/6 donor BM cells died due to leukemic infiltration, none of those receiving WEHI cells plus C57BL/6 donor BM cells plus splenocytes (and GVHD prophylaxis with sirolimus and bortezomib) did develop leukemic infiltration, thus confirming that, using this approach, we were able to separate GVHD and GVL effect. In conclusion, the current study shows a potent synergistic effect between sirolimus and bortezomib in vitro and in vivo which prevent GVHD while maintaining GVL. Disclosures: Cañizo: CELGENE: Membership on an entity's Board of Directors or advisory committees. San Miguel:JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; MILLENNIUM: Membership on an entity's Board of Directors or advisory committees. Off Label Use: sirolimus and bortezomib are not approved for use in prophylaxis of graft versus host disease.

Description: Abstract 1457 Background Factors such as hypoxia, angiogenesis and increased cell proliferation have been described to the pathogenesis of myeloid neoplasms. In the current study we have analyzed the expression of different genes involved in such pathways and its correlation with clinical and biological parameters in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods and patients: A total of 83 bone marrow (BM) samples were analysed including: Lower-risk MDS (LR-MDS; defined as IPSS low/Int-1; n=37), higher-risk MDS (HR-MDS; IPSS Int-2/high risk; n=13) and AML (blasts 〉20%; n=33). All samples were collected at the time of diagnosis. Bone marrow samples from healthy controls were used as control group (n=11). Baseline characteristics are shown in table 1. Total RNA from BM was treated with DNase, and cDNA was synthesized using a cDNA Sinthesis kit. Gene expression was quantified by qRT-PCR in triplicate using §-actin gene as control. Relative gene expression was determined by 2 (ΔΔCt) method. Cells populations of blasts (CD45+,CD34+,CD117+) were sorted with MoFlo Cell Sorting and separated by positive selection. No differences in gene expression were noticed by sorting as compared to total RNA from whole BM samples. Genes analysed were: vascular endothelial growth factor (VEGF) for angiogenesis, cMYC, macrophage migration inhibitory factor (MIF) and glycogen synthase (GYS) for metabolism and cell-proliferation. Clinical and biological parameters evaluated were: peripheral counts, BM blast percentage, karyotype (good, int and poor by IPSS in MDS and favourable, Int-I, Int-II and adverse by the European Leukemia Net Prognostic System in AML), transfusion dependency, response to intensive chemotherapy (IC) and survival. Statistical significance was determined by t-test, Wilcoxon test and Kaplan Meier with SPSS software (v.16). P values

Description: Introduction The generation of the immune response requires the recognition of peptides presented by the major histocompatibility complex (MHC) through the T cell receptor (TCR). In the hematopoietic transplantation context, T cells (LT) from the donor recognize foreign MHC or own MHC bound to foreign peptides (pMHC), generating an alloimmune response. Currently, the molecular mechanisms of LT alloimmune activation are unknown. In order to analyze the molecular interactions between peptides, MHC and TCR, we have implemented Molecular Dynamics techniques. We have compared immunologically reactive complexes (HLA-A2/TAX/TCR-A6; HLA-A2/HUD/TCR-A6) to non/weakly reactive complexes (HLA-A2/V7R/TCR-A6; HLA-A2/P6A/TCR-A6; HLA-A2/Y8A/TCR-A6). Methods Starting structures of two reactive complexes were downloaded from the PDB database and used to model mutations known to lead to different degrees of immune reactivity. Dynamics simulations were performed and analyzed using the program AMBER version 9. The simulation time was approximately 10 ns. Further analysis was carried out using the script ARO (Díaz-Moreno et al. 2009) in the VMD Tk console. Results A total of 17 MD trajectories have been reckoned, to simulate the behavior of isolated components of the different MHC-TCR complexes. Analysis of the fluctuations shows that pMHC binding barely restrains TCR motions, affecting mostly to CDR3 loops. Opposite, pMHC displayed substantial changes in its dynamics upon comparing its free versus ternary form (pMHC-TCR). Furthermore, taking as reference the positions of the MHC´s helices in free binary structures (MHC-peptide) and comparing them to the positions in ternary structures (pMHC-TCR), we can observe that in reactive complexes MHC exhibits a higher variability than in non-reactive complexes, suggesting that the MHC must tighten and acquire a position further away from its position in free binary form. We also analyzed the position of the peptide in the groove of MHC and we found that at position 5 (aa aromatic) the peptide is diverted although its position in the groove of MHC seems to be unrelated to the reactivity of the interaction TCR-pMHC. Conclusions The MHC shows strong changes in its molecular dynamics upon binding TCR, decreasing its mobility. The structure of MHC is slightly perturbed in reactive complexes, but not in non-reactive ones. Financial support Spanish MINECO (BFU2009-07190/BMC, BFU2012-31670/BMC) the Andalusian Government (BIO198) and Instituto de Salud Carlos III (PFIS - FI12/00189 and FIS PI11/02366) Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Marginal Zone Lymphoma (MZL) is an indolent lymphoma in which the use of PET/CT is poorly explored and also controversial due to the heterogeneous 18F- FDG avidity described in these types of lymphoma. Our aim in the present study is to evaluate the role of 18F-FDG-PET/ CT (PET/CT) in comparison to CT with intravenous contrast enhancement at initial staging and response assessment to chemotherapy in these patients. Methods and Materials A retrospective single-center study that included 34 patients, diagnosed of MZL between 1998 and 2012, with at least one PET/CT available. A total of 55 PET/ CT were performed: 25 at initial diagnosis, 19 for first- line response assessment, 6 in relapse and 5 after relapse- treatment. Locations of involved areas were registered comparing staging CT and PET/CT and were classified as discrepancy or not. Results Patients´ baseline characteristics are shown in table 1. At diagnosis, all patients presented with at least one abnormal focal FDG uptake except for one, which reflected a sensitivity of 96%. Median SUVmax was higher in nodal marginal zone lymphoma (NMZL) and extranodal marginal zone lymphoma (ENMZL): 6,1 (4- 8,4) and 6,9 (2- 13,8) respectively, in comparison to splenic marginal zone lymphoma (SMZL) 3,4 (3,2- 3,6) p=0,3. SUVmax was much higher in a patient with histological transformation to a DLBCL (SUVmax 37). Among 17 patients with both radiological imaging at the time of diagnosis, there were 8 patients (47%) with more involved areas demonstrated by PET/ CT than by CT alone, 75% of them were extranodal lesions. PET/CT upstaged 5 patients but in only 2 of them entailed a change in therapeutic management. Four patients did not show FDG avidity by PET/CT in some areas suspected to be involved by CT, what generated a CT sensitivity of 76%. Overall, CR was attained in 24 patients (66%) with 5-y OS 78%. Among 19 patients with a PET/CT available for first-line response assessment, responses were: 12 CR, 2 PR, 1PD and isolated residual lesions in 4 patients. Progression was documented in 2 of the 4 patients with residual lesions which were considered positive, and in 2 patients who maintained remission, the image was interpreted as a false positive (FP). The response assessment was performed by both radiological imaging in 13 patients. Discrepancies were found in 4 cases: CT showed CR in 3 patients while PET/CT detected localized residual disease and in another patient, CT showed stable disease whereas PET/CT demonstrated CR. Overall, none of the patients in CR by PET/CT relapsed. Five-year OS was 100% in contrast to 64% for those patients with a positive PET/CT after completing treatment (p=0,2), with a mean follow-up of 50 months (10-152). The NPV was 100% and PPV was 71% (5/7). Relapse was detected in 9 patients (37.5%). Six patients had PET/CT for re-staging and 5 for response assessment. All re-staging PET/CT had FDG-avidity with a median SUVmax of 9.9 (4.6-17.2). PET/CT for response after salvage treatment demonstrated 3 CR and 2 localized residual lesions. The NPV and PPV was 100% and 50%, respectively. Conclusions MZL shows higher 18F- FDG avidity in NMZL y ENMZL subtypes. PET/CT is more sensitive than CT at initial staging, chiefly in identifying extranodal involvement. Response assessment PET/CT had a NPV of 100% and PPV of 71 and 50% after first and second-line treatment, respectively. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION: Cannabinoids are the active components of Cannabis sativa. The interest in cannabinoid research has triggered only two decades ago following the discovery of the endocannabinoid system, mainly from the molecular characterization of endogenous receptors: CB1 (mostly expressed in the central nervous system) and CB2 (in immune cells). In the last few years, several groups have described their use as potential therapeutic agents in the treatment of pain, multiple sclerosis or Alzheimer. Moreover, increasing evidences have suggested their potential role as antitumor drugs. Despite the abundant expression of CB2 in immune cells, very few studies have examined its use in hematological malignancies. Considering the high expression levels of CB2 in B-cells, we hypothesized that plasma cells (PCs) could also express high levels of CB2 and therefore might be an excellent target for cannabinoids. DEVELOPMENT: Our objective was to evaluate the anti-tumor effect of cannabinoids in MM and identify the mechanisms involved. We use the synthetic cannabinoids WIN-55 (CB1 and CB2 mixed agonist) and JWH-133 (CB2 selective agonist). We used MM cell lines U266, MM1R, MM1S and RPMI8226 and primary PCs from patients and CD34+ cells from patients and healthy donors. Viability studies were carried out by MTT and cytometric analyses and the expression of receptors and the study of signaling pathways by Western blot (WB). Further, we tested the cannabinoid effects in vivo in murine models (NSG xenograft mice). We observed a high expression of CBs in CPs and a remarkable proapoptotic effect of cannabinoids on myelomatous cells. By contrast, the viability of the CD34 + hematopoietic progenitor cells remained unaffected irrespective of the dose used. In this regard, in MM cells lines and primary cells from patients we observed cleavage of PARP as well as activation of caspases 8, 9, 3 and 2, the latter related to endoplasmic reticulum stress induced by the cannabinoids. This cannabinoid-induced apoptotic effect was also mediated by AKT and MAPKs signaling pathways, as assessed by WB. In addition, Fluorometric analyses confirmed that cannabinoids induce an early mitochondrial damage. Next we confirmed that cannabinoids increase the expression of SPT, the limiting enzyme for the synthesis of ceramides (membrane sphingolipids). The upregulation of SPT following cannabinoid incubation induced accumulation of ceramide, as assessed by immunohistochemistry. Furthermore, the incubation with myoricine, an SPT inhibitor, partially inhibited caspase 3 activation. Finally, we checked the antimyeloma effect of the cannabinoids in vivo, using a model of human MM xenografted in immunodeficient mice NOD/SCID. Our results demonstrate a significant reduction in tumor growth, even tumor regression, as well as a significant increase of survival of cannabinoid-treated as compared to mice receiving vehicle. CONCLUSIONS: Cannabinoids have a very selective antitumor effect against MM cells. This effect involves activation of apoptosis processes and alterations in the composition of membrane sphingolipids (ceramides). In vivo studies confirmed the efficacy of these agents in the treatment of MM. This study lays the groundwork for the design of new anti-myeloma therapies. Disclosures No relevant conflicts of interest to declare.