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Memory B cells (MBCs) have a long lifespan compared with naive B cells (NBCs), remaining viable for years. This could predispose them to suffer misbalances in the gene expression pattern in the long term, which might be involved in the development of age-related B-cell disorders. In order to identify genes whose expression might change during life, we analysed the gene expression patterns of CD27− NBCs versus CD27+ MBCs in young and old subjects. Using microarray assays we observed that the expression pattern of CD27− NBCs versus CD27+ MBCs is significantly different. Furthermore, to evaluate the age effect, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the CD27− NBC population between young and aged individuals, whereas we found 925 genes differentially expressed in CD27+ MBCs. Among these genes, 193 were also differentially expressed in CD27+ MBCs compared with CD27− NBCs, most of them involved in cell survival, cell growth and proliferation, cellular development and gene expression. We conclude that gene expression profiles of CD27− NBCs and CD27+ MBCs are different. Moreover, whereas the gene expression pattern of CD27+ MBCs varies with age, the same does not happen in CD27− NBCs. This suggests that MBCs undergo time-dependent changes, which could underlie a higher susceptibility to dysfunction with age.

Solitary plasmacytoma represents a heterogeneous group of patients; approximately half develop multiple myeloma (MM) in 2 or 3 years,whereas others remain disease-free at 10 years. By definition, these patients do not have morphologic bone marrow (BM) plasma cell (PC) infiltration. Here, we investigated whether sensitive BM evaluation of patients withsolitaryboneplasmacytoma(SBP; n = 35) andextramedullary plasmacytoma (EMP; n = 29) through multiparameter flow cytometry (MFC) would unravel the presence of clonal PCs in otherwise disease-free BM, and whether BMclonality predicted higher risk of progression. BMclonal PCs were detected in 17 of 35 SBP (49%) and 11 of 29 EMP (38%) patients. Seventy-one percent of flow-positive vs only 8% of flow-negative SBP patients evolved to MM (median time to progression of 26 months vs not reached; hazard ratio, 17.4; P < .001). No significant differences were observed among EMP cases. Our results highlight the importance of MFC for sensitive BM evaluation of SBP patients, to predict risk of developing treatment-requiring MM and to plan disease monitoring., This work was supported by the Cooperative Research Thematic Network grants RD12/0036/0058, RD12/0036/0069 and RD12/0036/0052 of the Red de Cancer (Cancer Network of Excellence), Instituto de Salud Carlos III, Spain, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339, 06/1354, 02/0905, 01/0089/01-02, PS09/01897/01370, G03/136, Sara Borrell: CD13/00340), Consejería de Educacion, Junta de Castilla y León Valladolid, Spain (HUS396A12-1), and Asociación Española Contra el Cáncer (AECC, GCB120981SAN), Spain.

Introduction: Although survival rates have improved over the years, chronic graft-versus-host disease (cGvHD) remains the most frequent and severe complication in the long term after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All the strategies developed to reduce its incidence are based on procedures aimed to decrease the risk of acute GvHD that, consequently, can also reduce the risk of cGvHD, mainly using immunosuppression in the early post-transplant period. These strategies induce apoptosis of donor T lymphocytes, responsible for a/cGvHD but also for graft-versus-tumor response. In the present project, we have evaluated a new strategy aimed to reduce the risk of cGvHD by manipulating the immune response not in the early post-transplant period, but in later phases. cGvHD develops via a complex cellular and molecular network involving thymus damage and unusual antigen presentation leading to aberrant T- and B-cell reactions characterized by Th17/Tc17 differentiation, macrophage sequestration in tissue, alloantibody formation, and fibrosis. Most of these cell populations are dependent on NF-kB for their activation. Ixazomib is a second-generation proteasome inhibitor for oral administration, representing an ideal candidate for prophylaxis in GvHD. Objective: We propose to develop a murine model of cGVHD and progressive onset cGvHD to test the efficacy of delayed administration of ixazomib as a novel strategy to decrease the risk of cGvHD. Methods: For in vitro studies, peripheral blood mononuclear cells from healthy donors were stimulated in the presence of different concentrations of ixazomib. After 48h and 120h, apoptosis was analyzed, and activation markers were evaluated. For in vivo studies, a murine model of progressive cGvHD (pcGvHD - allowing the recipient to survive to mild aGvHD, thus favoring autoreactive T cells to expand and cause cGvHD) and a scleroderma model of cGvHD were used. Ixazomib at 0.75mg/Kg/twice weekly, 2X from day +21 in the pcGvHD, with or without cyclosporine A (CyA) at 5mg/Kg/day from day 0 until the end of the study. For scleroderma cGvHD group, 3mg/Kg/2X from day +30 was used up to 120 days post-transplant. Flow cytometry was used to evaluate the different lymphocyte populations in the different target organs of the GvHD. Results: In vitro results showed that activated T lymphocytes are sensitive to the proapoptotic effect of ixazomib (>100nM), while high concentrations of the drug are necessary to cause apoptosis in the non-activated cells (5000nM). Through CD27 and CD45 expression, we verified that ixazomib has a proapoptotic effect mainly on naïve and effector cells. We also verified a decrease in the activation markers of CD3+CD25+INF-γ+ cells (p˂0.01, 1000nM, n = 4). In the animal trial, the survival of the pcGvHD model mice treated with ixazomib was significantly higher as compared to untreated mice, with a significant decrease in the signs of pcGvHD (Figure 1A). In addition, the combination of CyA and ixazomib improved survival as compared to those mice receiving CyA or ixazomib alone as well as untreated controls (Figure 1B). In the scleroderma cGvHD model, we observed that the animals treated with ixazomib showed significantly less signs of GvHD (p˂0.0001) (Figure 2). Multiparametric flow cytometer analyses showed that in the pcGvHD model, mice treated with ixazomib had a decrease of effector CD4 T-cells in bone marrow (p=0.06) and spleen (p=0.015) when compared to untreated mice. Also an increase of CD19+ cells in the colon (p=0.04), liver (p=0.035), lymph nodes (LN) (p=0.037) and lungs (p=0.03) was observed. Regarding the regulatory T cells (Foxp3+), the treated mice had a significant increase in LN (p=0.02), Peyer patches (p=0.015) and thymus (p=0.028) as compared to untreated mice. Conclusion: In vitro studies show that ixazomib induces apoptosis on activated T lymphocytes and decreases the expression of activation markers. Our in vivo model indicates that the combination of CyA and ixazomib for the prevention of pcGvHD and cGVHD after allogeneic HSCT is promising and merits further investigation in clinical trials. Figure 1. Survival of pcGvHD. Figure 2. Score graphic of scleroderma cGvHD. BM - Bone marrow, mice that were transplanted with BM from BALBc mice (Syngeneic). Disclosures Ramos: Takeda Oncology: Research Funding.

Introduction Several studies have shown that the minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has a prognostic value after induction and consolidation therapy. Nevertheless the relapse is the most important cause of treatment failure in these patients, although they achieved a negative MRD, and even after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays, the value of the MRD before allogeneic BMT is still controversial. Method Multicentric study where we have studied correlative AML patients who went under an allo-HSCT in a situation of complete response, between 2012 and April'18. The MRD was analyzed by 8-coloured multiparametric flow cytometry, at least with 2 tubes per patient and 1,000,000 events per tube. We evaluated the prognostic value of the MRD before allo-HSCT. Results Between January'12 and April'18 we have gathered 90 allogeneic BMT in AML patients who were in CR, with a median age of 45 years old (17 - 66). The pre-HSCT situation was 1st complete remission (CR) in 75 patients and 2nd CR in 15. In 45 patients the conditioning regimen was myeoablative. In the group of patients (67) where we could know the risk group at diagnosis, the distribution was: low risk 18%, intermediate risk 59.7% and high risk 22.4%. The 46.7% of the donors were not related. In the last follow-up after allo-HSCT 24 patients have suffered a relapse (26.7%) and 41 (45.5%) have died (17 cases of mortality related to the transplant and 24 not related). In the global analysis the median follow-up of the overall survival (OS) was 37.5 months. Among the 90 patients, MRD was valuable in 86. Ten of 59 patients (16.9%) with negative MRD relapsed vs 12/27 (44.4%) with positive MRD, p= 0.016. If we consider only patients in 1st CR, 9/50 (18%) patients with negative MRD relapsed vs 10/22 (45.5%) with positive MRD, p= 0.02. This statistically significant difference does not exist if we consider only patients in 2nd CR. The median follow-up of OS and event free survival (EFS) was not reached in the negative MRD group and 571 days and 299 days in the positive MRD group. OS and EFS at 2 years after transplantation were 65% and 64% in the negative MRD group and 42% and 37% in the positive MRD group, p= 0.03 and p= 0.008 respectively (figure 1). Conclusions The detected MRD by 8-colour multiparametric flow cytometry previous an allo-HSCT in patients with AML in 1st CR is a prognostic factor in terms of relapse. Patients with a positive MRD before the allo-HSCT have a poorer OS and EFS than the patients with a negative MRD. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.

Purpose: We describe the results of a prospective multicenter phase I/II trial evaluating the impact of the use of vitamin D (VitD) from day −5 to +100 on the outcome of patients undergoing allogeneic transplantation (EudraCT: 2010-023279-25; ClinicalTrials.gov: NCT02600988). Experimental Design: A total of 150 patients were included in three consecutive cohorts of 50 patients each group: control group (CG, not receive VitD); low-dose group (LdD, received 1,000 IU VitD daily); and high-dose group (HdD, 5,000 IU VitD daily). We measured levels of VitD, cytokines, and immune subpopulations after transplantation. Results: No significant differences were observed in terms of cumulative incidence of overall and grades 2–4 acute GVHD in terms of relapse, nonrelapse mortality, and overall survival. However, a significantly lower cumulative incidence of both overall and moderate plus severe chronic GVHD (cGVHD) at 1 year was observed in LdD (37.5% and 19.5%, respectively) and HdD (42.4% and 27%, respectively) as compared with CG (67.5% and 44.7%, respectively; P < 0.05). In multivariable analysis, treatment with VitD significantly decreased the risk of both overall (for LdD: HR = 0.31, P = 0.002; for HdD: HR = 0.36, P = 0.006) and moderate plus severe cGVHD (for LdD: HR = 0.22, P = 0.001; for HdD: HR = 0.33, P = 0.01). VitD modified the immune response, decreasing the number of B cells and naïve CD8 T cells, with a lower expression of CD40L. Conclusions: This is the first prospective trial that analyzes the effect of VitD postransplant. We observed a significantly lower incidence of cGVHD among patients receiving VitD. Interestingly, VitD modified the immune response after allo-SCT. Clin Cancer Res; 22(23); 5673–81. ©2016 AACR.

In septic patients, the onset of septic shock occurs due to the over-activation of monocytes. We tested the therapeutic potential of directly targeting innate immune cell activation to limit the cytokine storm and downstream phases. We initially investigated whether caspase-8 could be an appropriate target given it has recently been shown to be involved in microglial activation. We found that LPS caused a mild increase in caspase-8 activity and that the caspase-8 inhibitor IETD-fmk partially decreased monocyte activation. Furthermore, caspase-8 inhibition induced necroptotic cell death of activated monocytes. Despite inducing necroptosis, caspase-8 inhibition reduced LPS-induced expression and release of IL-1β and IL-10. Thus, blocking monocyte activation has positive effects on both the pro and anti-inflammatory phases of septic shock. We also found that in primary mouse monocytes, caspase-8 inhibition did not reduce LPS-induced activation or induce necroptosis. On the other hand, broad caspase inhibitors, which have already been shown to improve survival in mouse models of sepsis, achieved both. Thus, given that monocyte activation can be regulated in humans via the inhibition of a single caspase, we propose that the therapeutic use of caspase-8 inhibitors could represent a more selective alternative that blocks both phases of septic shock at the source., This work was supported by grants SAF2012-39029 and SAF2015-64171R (MINECO/FEDER, UE), the Junta of Andalucía (P10-CTS-6494), the Swedish Research Council, the Swedish Childhood Cancer Foundation, the Swedish Cancer Foundation, Swedish Parkinson Foundation and the Swedish Brain Foundation.

It is established that hematopoietic stem cells (HSC) in the hypoxic bone marrow have adapted their metabolism to oxygen-limiting conditions. This adaptation includes suppression of mitochondrial activity, induction of anerobic glycolysis, and activation of hypoxia-inducible transcription factor 1α (Hif1α)-dependent gene expression. During progression of hematopoiesis, a metabolic switch towards mitochondrial oxidative phosphorylation is observed, making this organelle essential for determining cell fate choice in bone marrow. However, given that HSC metabolism is essentially oxygen-independent, it is still unclear whether functional mitochondria are absolutely required for their survival. To assess the actual dependency of these undifferentiated cells on mitochondrial function, we have performed an analysis of the hematopoiesis in a mouse mutant, named SDHD-ESR, with inducible deletion of the mitochondrial protein-encoding SdhD gene. This gene encodes one of the subunits of the mitochondrial complex II (MCII). In this study, we demonstrate that, in contrast to what has been previously established, survival of HSC, and also myeloid and B-lymphoid progenitors, depends on proper mitochondrial activity. In addition, gene expression analysis of these hematopoietic lineages in SDHD-ESR mutants calls into question the proposed activation of Hif1α in response to MCII dysfunction. Ministerio de Ciencia e Innovación SAF2009-06970 Junta de Andalucía CTS-4589 Instituto de Salud Carlos III PI-0355-2013

PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies.

Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP+-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP- cells, which confirms their differentiation into corneal tissues. GFP+-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.

Current graft-versus-host disease (GVHD) inhibition approaches lead to abrogation of pathogen-specific T-cell responses. We propose an approach to inhibit GVHD without hampering immunity against pathogens: in vitro depletion of alloreactive T cells with the preoteasome inhibitor bortezomib. We show that PBMCs stimulated with allogeneic cells and treated with bortezomib greatly reduce their ability to produce IFN-γ when re-stimulated with the same allogeneic cells, but mainly preserve their ability to respond to citomegalovirus stimulation. Unlike in vivo administration of immunosuppressive drugs or other strategies of allodepletion, in vitro allodepletion with bortezomib maintains pathogen-specific T cells, representing a promising alternative for GVHD prophylaxis. © 2011 Elsevier Ltd., Belén Blanco was supported by a fellowship from the Fondo de Investigación Sanitaria and by a grant of Gerencia Regional de Salud de Castilla y León.

The gastrointestinal (GI) tract is the main target site of graft-versus-host disease (GVHD). Diagnosis is based on endoscopic and histological findings. Helicobacter pylori (HP) is a Gram-negative spiral bacterium linked to gastritis, peptic ulcer disease, gastric mucosa–associated lymphoid tissue lymphoma, and adenocarcinoma and is frequently observed on endoscopy in patients who have undergone transplantation. The role, if any, played by HP infection in the development of acute GVHD is unknown. We conducted a retrospective study between January 1, 1990, and December 31, 2008, of 338 upper GI endoscopies (gastroscopies) performed on patients who underwent allogeneic stem cell transplantation with clinical suspicion of GVHD (198 patients). Acute and chronic GVHD were confirmed from histological features in 97 patients (51.3%) and 68 patients (36%), respectively. HP infection was detected in 69 patients (35%) and had a negative modulating effect on the development of acute GVHD (relative risk [RR], 0.60; 95% confidence interval, 0.46-0.79; P = .001) and chronic GVHD (RR, 0.75; 95% confidence interval, 0.61-0.92; P = .016). Furthermore, the presence of HP was inversely correlated with the histological severity of GVHD ( P = .003). Our findings suggest that infection with HP may have a negative modulating effect on GVHD.

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality among patients undergoing allogeneic hematopoietic stem cell transplantation. Although animal models have been clearly established for the study of skin, liver, and gut, currently there is no equivalent experiemental model for analyzing ocular involvement, which is rather common, especially among patients diagnosed with chronic GVHD. In the current study we have developed a murine model of ocular GVHD and, for the first time, we describe the histopathologic features involving cornea and limbus, which could play a role in the physiopathology of the disease at the ocular level. Our results represent a major finding that allows us to define a model for evaluating new therapeutic strategies for treating ocular GVHD prior to their use in clinical setting.

Large studies, mostly based on series of patients receiving CSA/tacrolimus (TKR) plus MTX as immunoprophylaxis, have demonstrated a deleterious effect on survival of the presence of a single mismatch out of eight loci after allogeneic hematopoietic SCT (alloHSCT). We retrospectively analyzed a series of 159 adult patients who received sirolimus(SRL)/TKR prophylaxis after alloHSCT. We compared overall outcomes according to HLA compatibility in A, B, C and DRB1 loci at the allele level: 7/8 (n = 20) vs 8/8 (n = 139). Donor type was unrelated in 95% vs 70% among 7/8 vs 8/8 pairs, respectively (P = 0.01). No significant differences were observed in 3-year OS (68 vs 62%), 3-year EFS (53 vs 49%) and 1-year non-relapse mortality (9 vs 13%). Cumulative incidence of grades II-IV acute GVHD (aGVHD) was significantly higher in 7/8 alloHSCT (68% vs 42%, P < 0.001) but no significant differences were found for III-IV aGVHD (4.5% vs 11%), overall (35% vs 53%) and extensive (20% vs 35%) chronic GHVD in 7/8 vs 8/8 subgroups, respectively. In summary, the present study indicates favorable outcomes after alloHSCT using the combination of SRL/TKR combination as GVHD prophylaxis with OS in the range of 55-70%, and non-significant differences in overall outcomes, irrespective of the presence of any mismatches at obligatory loci.

Memory B cells (MBCs) have a long lifespan compared with naive B cells (NBCs), remaining viable for years. This could predispose them to suffer misbalances in the gene expression pattern in the long term, which might be involved in the development of age-related B-cell disorders. In order to identify genes whose expression might change during life, we analysed the gene expression patterns of CD27− NBCs versus CD27+ MBCs in young and old subjects. Using microarray assays we observed that the expression pattern of CD27− NBCs versus CD27+ MBCs is significantly different. Furthermore, to evaluate the age effect, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the CD27− NBC population between young and aged individuals, whereas we found 925 genes differentially expressed in CD27+ MBCs. Among these genes, 193 were also differentially expressed in CD27+ MBCs compared with CD27− NBCs, most of them involved in cell survival, cell growth and proliferation, cellular development and gene expression. We conclude that gene expression profiles of CD27− NBCs and CD27+ MBCs are different. Moreover, whereas the gene expression pattern of CD27+ MBCs varies with age, the same does not happen in CD27− NBCs. This suggests that MBCs undergo time-dependent changes, which could underlie a higher susceptibility to dysfunction with age.

We evaluated the feasibility, safety, and efficacy of the administration of 4 sequential doses (intravenously administered on days 1, 4, 11, and 18) of cryopreserved bone marrow–derived mesenchymal stromal cells (MSC) expanded with platelet lysate and obtained from third-party donors as a second-line treatment for steroid-refractory acute graft-versus-host (aGVHD) disease in a series of 25 patients. All patients received at least 2 doses of MSC, whereas 21 received 3 doses and 18 received the initially planned 4 doses. Because of the achievement of partial response, 4 patients received additional doses of MSC. Median single cell dose administered was 1.1 × 106 MSC/kg of recipient body weight. There were no adverse events related to the MSC infusion in the 99 procedures performed, with the exception of a cardiac ischemic event that occurred twice in a patient with prior history of cardiac ischemia. Response to MSC at 60 days after the first dose was evaluable in 24 patients. Seventeen patients (71%) responded (11 complete and 6 partial responses), with a median time to response of 28 days after the first MSC dose, whereas 7 patients did not respond. In summary, we can conclude that sequential cryopreserved third-party MSC therapy administered on days 1, 4, 11, and 18 is a safe procedure for patients with steroid-refractory aGVHD. This strategy may provide a high rate of overall responses of aGVHD with a low toxicity profile.

Introduction: Allogeneic haematopoietic stem cell transplantation (alloHCT) is a potentially curative approach for patients (pts) with multiple myeloma (MM). The high transplant related mortality (TRM) rate with myeloablative conditioning has resulted in a shift to reduced intensity conditioning regimens (RIC). However, most MM pts who receive an alloRIC ultimately relapse and their treatment remains a challenge. Since alloHCT can modify the biology of the disease, including the immune environment, responses after alloHCT to rescue therapies previously used before alloHCT could be improved. Our main objective was to evaluate the efficacy of regimens including new drugs in MM pts relapsing after alloHCT comparing the efficacy achieved before and after alloHCT. Material and Methods: We report a retrospective multicenter analysis of 126 consecutive pts that underwent alloHCT for MM from 2010 to 2013 at 8 Spanish centers. Results: Baseline pts and transplant characteristics are shown in Table I. The median of prior therapies prior to alloHCT was 3 (1-9), 83% had received previous autologous HCT and 26% had high risk cytogenetic. 71 (56%) and 48 pts (38%) had been treated with regimens containing proteasome inhibitors (PI) and immunomodulatory drugs (IMIDs) before transplantation, respectively. Disease status at transplant was complete remission (CR) in 16 (13%) pts, partial response (PR) or very good PR in 86 (68%) and 24 (18.5%) had relapsed/progressive disease. 35 pts (28%) had active extramedullary disease at transplant. The majority of pts received peripheral blood HCT (90%), RIC (90%) with fludarabine plus melphalan based conditioning regimen (61%) and calcineurin inhibitor plus MTX as GVHD prophylaxis (68%). 19% receiving allo-HCT from an unrelated donor (91% 10/10 HLA matched). All pts engrafted. Grade II-IV acute GVHD occurred in 54% pts (grade IV 8%) and chronic GVHD in 45% (moderate 15%, severe 12%). TRM within the first 100 days after transplant was 6% (overall TRM 28%). 60% pts improved their pre-transplant response, with an overall response rate of 74% (56% CR). After a median follow-up of 92 months for pts alive (22-197), the OS was 51 and 43% at 2 and 5 years respectively. 75 pts (59.5%) relapsed after alloHCT, 57 of them with extramedullary involvement. Median time to relapse was 8 months post-transplant (1-141). The cumulative incidence of relapse was 79% at 3 years. Median OS after relapse was 22 months (8-33). Seventeen out of 75 pts who relapsed received IP both in the pre-transplant and in the post-transplant period. Sixteen pts out of 17 who received IP achieved at least PR pre-transplant while 10 out of these 16 pts responded again to PI post-HCT. Moreover, 1 patient reached a deeper response (CR) post as compared to pre alloHCT (PR) and 1 patient who was refractory pre-alloHCT did respond post-alloHCT. In addition, 6 out of 7 pts who did not respond to IP post-transplant reached stable disease with time to progression (TTP) lasting 4 to 12 months. Twelve out of 75 pts who relapsed received IMIDs both pre and post-alloHCT. Ten pts out of 12 who received IMID pre-alloHCT achieved at least PR, and 8 out of these 10 pts responded again to IMIDs post-alloHCT. Moreover, 1 patient who had been refractory to IMIDs in the pre-transplant period reached CR after alloHCT. In addition, 2 out of 4 pts who were refractory to IMIDs in the post-transplant period reached stable disease with TTP of 8 to 13 months. Remarkably, among pts who respond both in the pre and the post-transplant period to IP or IMIDs, the time to response (TTR) and time to progression (TTP) was similar despite the regimens used in the pre-transplant setting were more aggressive (TTR 3 vs 3.5 and TTP 9 vs 7 months before and after alloHCT for IP, and TTR 4 both before and after alloHCT and TTP 10 vs 9.5 months before and after alloHCT for IMIDs). All but 2 pts received first generation IP pre and post-alloHCT (two pts received carfilzomib) and all but 5 received first generation IMIDs (5 pts were treated with pomalidomide). Conclusions: MM pts relapsing after alloHCT should be considered candidates to receive new drugs, as they can achieve response rates at least in a similar proportion and durability to those observed in the pre-transplant setting. This finding is in contrast to the usual course of the disease outside the alloHCT setting, where response rates and TTP decreases with consecutive lines of treatment. Disclosures Mateos: Janssen: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells). Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro, ex vivo and in vivo and studied the molecular signaling pathways involved in this effect. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor. For their design and synthesis, computational techniques of docking, analytical and spectroscopic techniques such as mass spectrometry (MS) were used. To assess the anti-leukemia effect of the different cannabinoids, we analyzed cell viability by MTT and flow cytometry using six human AML cell lines, primary cells from healthy donors (hematopoietic progenitor cells (HPC) and lymphocytes) and blasts from AML patients. Cannabinoids induced a potent proapoptotic effect on AML cell lines and on primary leukemic cells, which was not observed in normal HPC and lymphocytes from healthy donors. Fragmentation of PARP and activation of caspases 2, 3, 8 and 9 were confirmed by western-blot. Other proteins involved in the effect of cannabinoids were p-AKT, p-ERK 1/2, p-38 and p- JNK. Also studies on p-PERK, p-IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Mitochondrial damage was analyzed by flow cytometry using TMRE and by MitoSOXTMRed. These assays confirmed a very early mitochondrial damage in leukemic cells which was not observed in normal hematopoietic progenitor cells. Moreover, we analyzed the ceramide levels, a membrane lipid associated with death/survival cell processes by HPLC and immunohistochemistry. Remarkably, we observed significant differences in the amounts of certain subtypes of ceramides in untreated versus treated leukemic cells. The proapoptotic effect of cannabinoids on AML cells was abolished upon co-culture with either CB2 receptor antagonists or with pancaspase inhibitors. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60 cell line. We confirmed disease infiltration in bone marrow (BM) by BM aspirates and flow cytometry assays. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. We observed a significantly increased survival among mice treated with WIN-55 cannabinoid as compared to both the control group and the group treated with ARA-C. In addition, we tested in vivo the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. We confirmed that cannabinoids did not affect the viability of the different populations of hematopoietic progenitors (LK, GMP, CMP) and, moreover, an increased platelet count was observed in treated mice. Our findings indicate that cannabinoids display a highly selective proapoptotic effect against leukemic cells. Several pathways are involved in this effect, the modification in the ceramide pattern playing a main role. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

The current study was designed to assess the safety and efficacy of bortezomib in combination with fludarabine and melphalan as reduced intensity conditioning before allogeneic stem cell transplantation in patients with high risk multiple myeloma. Sixteen patients were evaluable. The median number of previous line of treatment was 3; all patients had relapsed following a prior autograft and 13 had previously received bortezomib. Fifteen of them either remained stable or improved disease status at day +100 post-transplant, including 11 patients with active disease. More specifically, nine patients (56%) and five patients (31%) reached complete remission and partial response, respectively. 25% developed grade III acute graft-versus-host disease. The cumulative incidence of non-relapse mortality, relapse and overall survival were 25%, 54% and 41%, respectively, at 3 years. Regarding the non-haematological toxicity (grade>2), two patients developed peripheral neuropathy, two patients liver toxicity and 1 pulmonary toxicity early post-transplant. The haematological toxicity was only observed during the first three cycles mostly related to low haemoglobin and platelet levels. The current trial is the first one evaluating the safety and efficacy of bortezomib as part of a reduced intensity conditioning regimen among patients with high risk multiple myeloma., This study was partially supported by a grant from the Ministery of Health (EC10-289); Teresa Caballero was supported by a grant Rio Hortega (code: CM10/00161) from the Instituto de Salud Carlos III. The study was supported by Jassen-Cilaj providing bortezomib free of charge as well as financial support.

Background aims Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive properties. Nevertheless, it has been previously reported that MSCs might also trigger the immune response. We studied whether MSCs may act as carriers, capturing antigens that can be endocytosed by antigen-presenting cells later on. Methods We measured the cellular uptake of mannose receptor-mediated fluid phase macropinocytosis, assessed as cellular uptake of fluorescein isothiocyanate-dextran, and PKH-67-labeled cell lysates as a surrogate marker for antigen capture among dendritic cells (DCs, positive control), T lymphocytes (negative control) and MSCs. Results All experiments confirmed that MCSs displayed pinocytic and endocytic capacities, which were lower than those observed for DCs but significantly higher than those observed for T cells. We also demonstrated that MSCs release previously endocytosed antigens, which subsequently can be captured by DCs. Conclusions MSCs have the ability to capture and release antigens.

This is an open-access paper., [Background]: We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reducegraft- versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. [Design and Methods]: We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. [Results]: The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. [Conclusions]: The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect., Supported by a grant Rio Hortega (code: CM10/00161) from the Instituto de Salud Carlos III and grant PI080047 from FIS.

Chronic graft-versus-host disease (cGVHD) is the major late complication after allogeneic hematopoietic stem cell transplant [1,2]. In this article, we have analyzed the value of noninvasive day 1100 tests as predictors of severe cGVHD development in 165 patients undergoing allogeneic peripheral blood stem cell transplant (allo-PBSCT) from a matched related donor. The cumulative incidence of overall, extensive, and severe cGVHD was 67, 56, and 23%, respectively, among patients surviving >100 days after transplant. In univariate analysis, patients displaying an abnormal liver function tests (LFTs) (total bilirubin, alkaline phosphatase, and GGT > 2 times above the upper normal limit) and a low absolute lymphocyte count (ALC) (

[ES] El trasplante de progenitores hematopoyéticos (TPH) representa la única posibilidad de curación para numerosos pacientes con hemopatías malignas, lo que justifica el número creciente de trasplantes realizados a nivel mundial. Se basa en la sustitución de un sistema hematopoyético neoplásico, defectuoso o insuficiente por otro normal obtenido de un donante sano histocompatible. Para ello los pacientes reciben un régimen de acondicionamiento basado en la administración de quimioterapia a menudo combinada con radioterapia o depleción de linfocitos T. Esta “preparación al trasplante” está diseñada con dos finalidades: la primera, conseguir una inmunosupresión del receptor para evitar el rechazo del injerto y la segunda, erradicar el clon maligno o población celular anormal. Clásicamente se ha considerado que el acondicionamiento tenía un tercer objetivo: crear espacio en la médula ósea para que los progenitores pudieran anidar mediante un tratamiento mieloablativo (9). Sin embargo, se ha podido comprobar que un tratamiento inmunosupresor adecuado permite evitar el rechazo y favorecer el injerto de los progenitores hematopoyéticos del donante sin necesidad de administrar dosis mieloablativas de quimio o radioterapia., [EN] The hematopoietic stem cell transplantation (HSCT) represents the only possibility of cure for many patients with hematologic malignancies, which justifies the increasing number of transplants performed worldwide. It is based on the replacement of neoplastic hematopoietic system, defective or insufficient for other normal obtained from a healthy donor histocompatible. For this, patients receive a conditioning regimen based on chemotherapy often combined with radiotherapy or T cell depletion This "preparation for the transplant, " is designed with two purposes: first, get a receiver immunosuppression to prevent graft rejection and second, to eradicate the malignant clone or abnormal cell population. Traditionally been considered that the upgrading had a third goal: to create space in the bone marrow to the nest by parents could myeloablative therapy (9). However, it has been shown that an appropriate immunosuppressive treatment can prevent rejection and promote graft donor hematopoietic progenitors without myeloablative doses of chemotherapy or radiotherapy.

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G0 to G1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.

Producción Científica, Background In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells.Design and Methods Conventional or regulatory CD4+ T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4+ T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN-γ and CD40L expression of stimulated responder T cells by flow cytometry.Results We observed that naturally occurring CD4+CD25+ regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4+ T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN-γ production and CD40L expression among stimulated effector T cells.Conclusions These results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases.

The most common approach for the treatment of chronic graft-versus-host disease (cGVHD) has been the long-term use of systemic steroids. Beclomethasone dipropionate (BDP) is a topically active corticosteroid with relatively low absorption from the gastrointestinal mucosa. It has been successfully used to treat acute GVHD (aGVHD), but its use in the cGVHD setting is far more limited. In the current study, BDP was administered to 33 patients who underwent allogeneic transplantation and had biopsy-proven gastrointestinal cGVHD (GI cGVHD). Twenty-six patients with GI cGVHD received BDP as first-line and 7 as either second- or third-line treatment. All patients received BDP together with a calcineurin inhibitor, except for 1 patient who was also receiving mycophenolate mofetil (MMF). BDP was administered for a minimum of 16 weeks and was tapered during 4 additional weeks. Of those patients receiving BDP as the first line of treatment, 22 (84.6%) achieved complete remission (CR) of GI cGVHD, 2 (7.7%) achieved a partial response (PR) and 2 (7.7%) did not respond or progressed. Median time to response was 28 days. Nevertheless, only 7 (27%) patients had maintained the response at last follow-up, whereas 19 (73%) finally relapsed or progressed. Median time to relapse was 147 days after the end of BDP. In the case of the patients who received BDP as a second- or third-line treatment, 3 (42.9%) achieved CR and 2 (28.6%) PR. For the whole series of patients, 13 patients (39.4%) were not receiving immunosuppressive treatment at final follow-up. Only 4 patients developed cytomegalovirus (CMV) reactivation, which was successfully treated with antiviral drugs. No fungal infection was observed during the treatment period. In conclusion, the current study shows that BDP, in the absence of systemic steroids, is a highly effective initial therapeutic approach for GI cGVHD, which helps to avoid complications related to systemic steroids.

Introduction Memory B cells (MBCs) have a very long half-life, remaining viable in a quiescent state for years. However, elderly individuals have less amount of MBCs and they produce lower amounts of antibodies. Thus, in aged individuals immunization become less efficient in terms of quantity and quality. Furthermore, several hematologic malignancies involving B-cell lineage, such as non-Hodgkin lymphomas, chronic lymphocytic leukemia or multiple myeloma, are increasingly common in aging population. With this background, we analyzed the gene expression patterns of naïve B cells (NBCs) versus MBCs in both younger and older subjects, in order to identify genes related to longevity that could be altered in the elderly population and related to an increased risk of developing certain lymphoid malignancies. Methods NBCs and MBCs were obtained by immunomagnetic separation from buffy coats of 10 healthy donors: 5 young (20-25 years) and 5 elderly (65-70 years). By microarray techniques we analyzed the expression of 45000 genes in all samples. Unsupervised hierarchical clusters of gene expression data were performed using the average linkage and the Euclidean distance. To identify differentially expressed genes between experimental groups we applied non-parametric Mann-Whitney test. The differences in expression with a p value < 0.05 were considered significant. All analyses were performed using the Multi-experiment Viewer 4.7.1 software. Results Firstly, we confirmed that the expression pattern of NBCs versus MBCs is significanly different in 3548 genes in young and 2145 genes in elderly individuals. Next, in order to evaluate the effect of age in NBCs and MBCs, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the naïve population between young and aged individuals, whereas we found 1593 genes differentially expressed in young versus elderly subjects within MBCs; therefore, age affects the gene expression pattern of MBCs but not NBCs. Furthermore, we identified 467 genes which displayed a higher or lower expression in MBCs from aged as compared to younger individuals and NBCs, most of them involved in proliferation and cell cycle, apoptosis, cell survival and hematological diseases (Table 1). Conclusions Gene expression profiles of NBCs and MBCs are different. While there are no significant differences in NBCs of young versus elderly individuals, we detected significant differences in expression patterns of MBCs between both age groups, which indicates that MBCs could be more susceptible to age related alterations. Disclosures: No relevant conflicts of interest to declare.

Introduction Memory B cells (MBCs) remain viable in a non-proliferative state for years. These cells express genes involved in cell survival and anti-apoptotic factors, while repress the expression of cell cycle regulatory genes. During their differentiation into plasma cells (PCs), these cells develop an opposite gene expression pattern, with a higher expression of genes implicated in cell proliferation and activation, and a lower expression of survival genes. In multiple myeloma (MM) the PCs accumulate into the bone marrow due, at least in part, to failure of pro-apoptotic mechanisms normally expressed in PCs. In the present study we analyzed the gene expression patterns of MBCs and PCs from healthy donors and patients with MM, in order to determine whether or not myelomatous PCs share characteristics of MBCs and/or normal PCs, and to identify possible genes related to the pathophysiology of the disease. Methods MBCs were obtained by immunomagnetic separation from buffy coats of 5 aged healthy donors. Likewise, PCs were isolated from bone marrow of 6 healthy donors and of 5 patients with MM. Using microarray techniques we analyzed the expression of 45000 genes in all samples. We performed unsupervised hierarchical cluster of gene expression data using the average linkage and the Euclidean distance. To identify differentially expressed genes among experimental groups we applied non-parametric Kruskal Wallis test. The differences in expression with a p value < 0.05 were considered significant. All analyses were performed using the Multi-experiment Viewer 4.7.1 software. Results From the hierarchical cluster obtained we clearly identified two groups, one which included normal PCs samples and the other one containing myelomatous PCs and MBCs. Interestingly, myelomatous PCs displayed intermediate features between MBCs and normal PCs (Figure 1). We found 5159 genes differentially expressed between normal and myelomatous PCs. Among these, we identified 3455 genes which displayed a similar expression pattern between MBCs and myelomatous PCs, including caspases inhibitors, MAP kinases, ubiquitins, transcription and translation factors. Conclusion Myelomatous PCs display an intermediate gene expression pattern between normal PCs and MBCs. These cells display high expression of genes involved in cell survival that should be normally inactivated in the transit of MBC to a normal PC, so that its expression pattern is closer to a MBC than a normal PC. Disclosures: No relevant conflicts of interest to declare.

Introduction Chronic Graft-versus-Host disease (cGVHD) remains a major cause of morbidity and mortality in long-term survivors after allogeneic hematopoietic stem cell transplantation (ASCT). Among the major risk factors previously noted is sex-mismatch, acute GVHD and peripheral hematopoietic blood stem cell grafts (PBSC). Our aim in this study was to determine risk factors for cGVHD and evaluate the impact of ATG on cGVHD in a multi-centre setting. Methods Patients from three centers (Stockholm, Sant Pau, Barcelona and Salamanca) were included. Retrospective data analysis was conducted for all patients (n=820) transplanted between 2000 and 2006. In our cohort 91% had malignant disease, 57% received HLA-identical sibling donor grafts, 13% received grafts with one HLA-A, -B or –DR antigen mismatch and 30% received grafts from HLA-A, -B and –DR matched unrelated donors. Reduced intensity conditioning was given to 65% of the patients. Chronic GVHD was classified according the National Institute of Health consensus criteria. Results Overall incidence of cGVHD was 46% for patients surviving more than three months after ASCT (n=747). Older patient age HR 1.15 (95% CI 1.07-1.24), p Conclusion RIC increases the risk for both overall and moderate to severe cGVHD. Acute GVHD and older recipient age are significant risk-factors for overall cGVHD and female donor to male recipient and PBSC for moderate to severe cGVHD. ATG significantly reduces the risk of all grades of cGVHD without having a negative outcome on RFS. Therefore, in order to prevent overall and moderate-extensive cGVHD it should be added to the RIC regimen in older patients and in male patients with female donors after PBSC grafts. Disclosures: Ringden: Gilead : Invited to Gilead on July 28, 2011, to participate in an Advisory Board Meeting on Treatment of invasive fungal infection. Other. San Miguel:Jansen, Celgene, Onyx, Novartis, Millenium: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Abstract 3025 Introduction: Although allogeneic stem cell transplantation (allo-SCT) is the only curative treatment for MM, it is associated to a high morbility and mortality. Moreover, relapses are common after allo-RIC. Accordingly, new strategies are required to reduce both the risk of relapse and the toxicity of the procedure. As we have previously demonstrated, Bz induces a selective depletion of alloreactive T-cells and has immunomodulatory properties which might be of potential benefit for GVHD control. The primary end point of this study was to evaluate the efficacy of allo-RIC in terms of response when Bz was added as part of a reduced intensity conditioning prior to allo-SCT. Secondary end points included incidence of GVHD and analysis of the toxicity of the procedure when Bz is also administered post-infussion as part of the GVHD prophylaxis. Method: Prior to allo-RIC, patients received two cycles of Bz plus dexamethasone. Conditioning consisted of fludarabine (30 mg/m2 intravenously on days -9 to -5) and melphalan (70 mg/m2 intravenously on days -4, -3) plus Bz 1, 3mg/m2 on day - 11 and -2. GVHD prophylaxis included cyclosporine (CsA) and methotrexate for the first 9 patients and CsA plus MTX and Bz on days +3 and +7 for the remaining 7 patients. From day +50 post allo-RIC 7 cycles of Bz (+1, +8, +15) were administered, the first two cycles every 28 days and the rest every 56. Results: 16 patients from the Twenty-one initially enrolled, were evaluable. All 16 patients had received at least 2 lines of therapy including autologous-SCT. Disease status was CR or nCR in 4 patients, 9 had PR and the remaining 3 patients had relapsed / progressive disease. 15 patients maintained or improved status at transplant including all × patients with active disease at transplant. Eight patients (50%) relapsed, four with extramedullary involvement. No patient developed grade 4 aGVHD.Grades 2–3 aGVHD occurred in 6 patients (37%). Interestingly, two out of the nine (29%) patients who received Bz on days +3 and 7 developed grades 2–3 acute GVHD as compared to four of the nine (44%) who did not receive it. In terms of toxicity, one patient did not achieve platelet engraftment and 2 patients developed peripheral neuropathy requiring treatment withdrawal. 8 patients died, four of them due to relapse (MRT: 25%). With a median follow-up of 457 days overall survival was 46%. Conclusions: The current trial is the first evaluating the efficacy and safety of Bz as part of a reduced intensity conditioning regimen among patients with high risk MM undergoing allogeneic transplantation. Regarding the efficacy of the procedure all but one patient improved disease status post-alloRIC although relapse rate was still high in this heavily pretreated population. In addition, Bz post-alloSCT is well tolerated and may decrease the incidence of GVHD. Disclosures: Perez-Simón: Janssen-Cilag: Patents & Royalties. Off Label Use: This study evaluates the efficacy of Bortezomib as part of a reduced intensity conditioning regimen among patients with high risk MM undergoing allogeneic transplantation. Rosiñol:Celgene: Honoraria; Janssen: Honoraria. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

Abstract 2771 Introduction: We have previously shown that 5-azaC inhibits T-cells proliferation and favours the development of Tregs which decreases the risk of GVHD after allogeneic transplantation. This is at least in part due to the effect of the drug on the expression of genes such as FOXP3. Nevertheless, considering the unspecific effect of 5-azaC, it could also favour the overexpression of other genes related to the regulation of the immune response such as TBET, GATA3, IFNg or IL-2, which in turn would favour the development of a Th1, Th2 or Th17 polarization instead of a Treg expansion. In the current study we have evaluated the effect of 5-azaC on these genes in order to know the mechanisms involved in the effect of the drug on the immune response. Methods: We analyzed TBET, GATA3, FOXP3, IFNg and IL-2 mRNA expression of T lymphocytes by RT-PCR after exposure to 1mM 5-azaC during eleven days of culture. These T cells were cultured in medium alone or stimulated with plate-bound anti-CD3 (5 mg/ml) plus soluble anti-CD28 (2.5 mg/ml). Furthermore, we analyzed the methylation status of the promoters of these genes before and after 5-azaC treatment. Results: The expression of TBET, GATA3 and RORγ is not significantly affected by the exposure to the drug whereas the expression of FOXP3 significantly increases along the culture. Regarding IFNg and IL-2 expression no increased expression was observed after exposure to the drug at different time-points along the 11 days of culture. Upon analyzing the mathylation status of the promoter of these genes, we observed that in steady state the promoter of TBET and GATA is demethylated, which is in contrast to FOXP3 promoter. For this reason, the exposure to the drug decreases the methylation status of the promoter of FOXP3 while there is no effect on the promoters of TBET or RORg, thus justifying the absence of effect on the expression of these genes. By contrat, the promoter of both IFNg and IL-2 is methylated prior to the exposure to 5-azaC and it is demethylated after exposure to the drug, which is in contrast to the absence of increased expression of these genes. Accordingly, other mechanisms in addition to the epigenetic regulation of the promoter of IFNg and IL-2 are responsible for their expression in this model. Conclusions: In the current study we show by the first time the effect of 5-azaC on the promoters of genes which regulate the immune response. While no effect was observed for TBET, and GATA3 5-azaC induces a strong demethylation in the promoter of IFN or IL-2. In spite of this effect there is no increase in their expression which could be due to the overexpression of FOXP3 or to additional mechanisms involved in their regulation which are currently being evaluated. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: 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: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is the inhibition of graft versus host disease.

Abstract 3615 Introduction: Preferentially expressed antigen of melanoma (PRAME) was first isolated as a human melanoma antigen by cDNA expression cloning using melanoma-reactive cytotoxic Tcells (CTL). PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies and solid tumors. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). In addition, several authors have suggested that PRAME could be used as a target for anticancer T-cell therapy. PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies. Methods: We analyzed PRAME mRNA expression of blast cells from AML patients at diagnosis versus CD34+ stem cells from healthy donors by RT-PCR without treatment or after exposure to 1mM 5-azaC during the four days of culture and correlated the expression of PRAME with the methylation status of the promoter. Results: PRAME is significantly over-expressed in blasts from AML patients (n=11) compared with normal CD34+ cells (n=8) ((700±1102 vs. 1.8±2.5 p=0.002). Interestingly, we found an inverse correlation between PRAME expression and the degree of methylation in the promoter among both AML samples and healthy donors (r=-0.77 p=0.010). In order to evaluate the effect of 5-azaC on PRAME gene expression, we treated blast cells and CD34+ cells from healthy donors with the drug and we observed that the exposure to the drug induced a decrease in the percentage of methylation in the promoter and subsequently increased the expression of PRAME but, interestingly, the higher the basal methylation of the promoter the more intense the effect of the drug among AML cells. By contrast, CD34+ cells from healthy donors were resistant to the effect of the drug so that no significant changes were observed neither in terms of methylation status of the promoter nor in the expression of PRAME prior to or after exposure to the drug among healthy donors. Conclusions: The promoter region is highly methylated in normal CD34+ cells compared to AML cells and this pattern correlates with a higher expression of PRAME in blasts. Furthermore, the level of PRAME methylation was reduced in AML patients after exposure to 5-azaC which correlated with an increase in the expression of PRAME. By contrast, the effect of 5-azaC on the methylation pattern of the promoter was significantly lower in CD34+ cells from healthy donors. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: 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: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is to increase PRAME expression in blasts from AML patients and generate CTL CD8+ specific response against tumor cells.

Frequency of additional chromosomal abnormalities in chronic myeloid leukemia (CML) is estimated to be 7% in chronic phase and increases to 40–70% in advanced disease. Progression of CML from chronic phase to accelerated phase or blast crisis is often associated with secondary chromosomal aberrations. We report an exceptional case of CML as debut in lymphoblastic blast crisis and a subsequent progression in myeloblastic blast crisis with rare cytogenetic abnormalities.