ALBERT

Description: Introduction Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in in the promoter region of cytokine genes have shown to alter their expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 17 (IL-17) is secreted by CD4+ T-cells and has been implicated in the pathogenesis of various autoimmune diseases but its importance in SCT is not well-known. Objective To analyse the influence of IL-17A SNP genotypes on the risk and severity of GvHD and other complications after HLA-identical allo-SCT. Patients and Methods Genomic DNA obtained from peripheral blood samples belonging to 546 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). Genotyping of the polymorphisms of interest, rs8193036 (-737C〉T), rs2275913 (-197G〉A), rs3819024 (-444A〉G), rs4711998 (-877A〉G), were performed by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Results Genotype frequencies are shown in Table 2 and the association between IL-17A genotypes and complications after allo-SCT are shown in Table 3. Patients transplanted from donors harboring genotype CC for the SNP rs8193036 show increased risk of grade III-IV acute GvHD (7/26 vs 47/397, p=0.035) and of grade II-IV acute GvHD (13/26 vs 133/409, p=0.048). Patients transplanted from donors harboring allele A in the SNP rs4711998 show increased risk of extensive chronic GvHD (53/161 vs 43/177, p=0.045). Relapse rate was not related with IL-17A SNP genotypes. Finally a higher risk of toxicity-related mortality (TRM) was observed in patients transplanted from donors harboring allele A for SNP rs2275913 (78/293 vs 46/227, p=0.048), donors harboring allele G for SNP rs3819024 (78/279 vs 46/242, p=0.011) and donors harboring allele A for SNP rs4711998 (68/250 vs 55/229, p=0.044). Conclusions IL-17A SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore, to improve the clinical management of transplanted patients. This results further support the idea of a genetic predisposition to certain complications after allo-SCT. Paper presented on behalf of the GvHD/Immunotherapy committee of the Spanish Group for Hematopoietic Transplantation (GETH). Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 132 Background: There is increasing evidence on the importance of epigenetic mechanisms such as DNA methylation and acetylation in the pathogenesis of multiple myeloma (MM). A global DNA hypomethylation pattern with selective genes hypermethylated has been described in myeloma plasma cells when compared with normal plasma cells. This fact could constitute a potential target for the use of demethylating agents. The response to bortezomib, a widely used agent against myeloma cells through proteasome inhibition, is particularly variable in patients with relapsed or refractory disease. We examined both, the global DNA methylation pattern and methylation state in 30 genes, in DNA from bone marrow cells and correlated our findings with response, progression (PFS) and overall-survival (OS) to bortezomib in patients with relapsed myeloma. Methods: Seventy-five patients (37M/38F; median age 65 years, range 29 to 80) with relapsed MM were treated from December 2002 to March 2010 with bortezomib-based regimens at our institution. Median follow-up for patients alive was 31 months (range 6 to 45). Genomic DNA was isolated from bone marrow slides with plasma cell infiltration at the time of relapse using a commercial kit (Qiagen). Global methylation was determined in all patients by ELISA (Epigentek), obtaining the percentage of 5-methylcytosine (5-mC) present in total DNA. CpG island DNA methylation profile of 30 genes was determined in 42 patients by a DNA methylation PCR system based on methylation sensitive and/or dependent restriction enzymes digestion (Qiagen). These genes were selected based on either their potential impact on prognosis in previous reports, or on the pathogenesis of MM, involving several cellular pathways such as innate immune response (CD40, EP300, MIF, CBP, TGFB1, TGFBR2), cytokine receptors (CXCR4, CXCL12, IL6R, IL17RA), transcription factors (NFKB1, NFKBIB, IRF4), cytokine stimulus response (SOCS3), apoptosis (TNFRSF13C, TNFRSF21, TNFRSF25, BCL2L11), tumor suppression (TP53, BRCA1, DAPK1, CDH1, RASD1), cellular cycle control (CCNB1, CCND1, CCNA2, CCNE1, CDKN2A, CDKN1A) and efflux transporter (ABCG2). Results: Overall response (OR) was achieved in 62% of the patients (complete remission 6.7%, partial response 44% and minor response 10.7%), while 9 (12%) and 20 (26.7%) showed no response (NR) or progressive disease (PD), respectively. The median PFS and OS after bortezomib therapy were 6 and 19.6 months, respectively. A low global methylation status was observed (median 4.68% of 5-mC, range 0.02 to 13.6) and patients with more than 3.95% of total DNA methylated achieved better OS than patients with more unmethylated DNA (median 30 versus 15 months) (p=0.004; Figure 1). Concerning methylation on specific-genes, a methylation status lower than 3.97% in CXCR4 was correlated with a longer PFS after bortezomib treatment (p=0.009; Figure 2). Clustering analysis with methylation status for these genes, showed that NFkB presented a differential profile according to response to bortezomib (p=0.037). A relative low methylation percentage (lower than 6.7%) in this gene was also associated with longer OS after bortezomib treatment (p=0.015; Figure 3). A positive correlation was observed with high methylation status in NFkB and other genes involved in the same cellular pathway (NFKBIB, EP300, CBP, CCNA2, CCNB1) (p

Description: Abstract 468 AML is worldwide the most frequent indication for allo-SCT. This is most likely due to the curative potential of the graft versus leukemia (GVL) effect associated with the procedure. Unfortunately, GVL and GVHD are intimately linked. Thus, it is important to identify markers predictive of severe GVHD, to balance the risk of this complication and the risk of relapse in a particular AML patient. The innate immune system, as the initial regulator of the inflammatory response, mediates an important role in these reactions. After conditioning regimen, toll-like receptors initiate the innate inflammatory response by activating intracellular signaling cascades that converge on the activation of NF-κB and interferon regulatory factor-3 (IRF3). IRF3 activation in bone marrow derived dendritic cells (DCs) results in natural killer (NK) cell activation inducing an anti-tumor NK response. We hypothesized that genetic variability in IRF3 in either the donor or the recipient could impact on the degree of the inflammatory response and on GVHD and GVL effect allo-SCT. We analysed the effect of two frequent single nucleotide polymorphisms (SNPs) in IRF3 (rs7251 and rs2304205), which are inherited in a haplotype, on the GVHD and GVL in 249 patients diagnosed with AML and submitted to HLA-identical sibling allo-SCT. Those patients with a donor carrying dominant GG gene variant in rs7251 (45% of the donors) had, as compared to GC (44%) and CC (11%) variants, lower aGVHD III-IV incidence (4% vs 11% vs 27%; p=0.0078) (Figure 1A), higher relapse incidence (49% vs 35% vs 26%; p=0.018) (Figure 1B), and lower TRM (7% vs 24% vs 18%; p=0.0065). This clinical impact on severe aGVHD, relapse, and TRM was retained at multivariate analysis. Further, GG gene variant in rs7251 when present in the patient was also associated with lower aGVHD III-IV incidence (4% vs 13% vs 24%; p=0.009), higher relapse incidence (50% vs 34% vs 31%; p=0.014), and with a trend for a lower TRM (9% vs 19% vs 23%; p=0.064). Patients carrying AA dominant gene variant in rs2304205 in IRF3 presented a higher relapse incidence than the rest of genotypes (50% vs 39% vs 18%, p=0.0068). However, this impact was not retained at multivariate analysis. Functional studies in 180 healthy individuals showed that after stimulation of peripheral blood with cytomegalovirus (CMV) peptides, GG gene variant in rs7251 presented lower IFN-γ serum production than the rest of individuals, and GG gene variant was associated with lower number of IFN-γ producing mature NK cells, lower number of cytotoxic NK cells against K562 cell line and lower proliferation of T cells after antigen presentation by DCs. In conclusion, we show that a particular gene variant in IRF3 in the donors is associated with a low incidence of severe aGVHD and high incidence of relapse in AML patients submitted to allo-SCT. This finding could be explained by its effect in the inflammatory and adaptive immune response, with lower IFN-g production, lower lymphocyte proliferation after antigen presentation by DCs and lower mature NK cell response. Thus, these results suggest that, if possible, when transplanting an AML patient with a low risk of relapse it might be preferable to select a donor harbouring GG in rs7251 in IRF3, and when transplanting an AML patient with a high risk of relapse after the transplant it might be preferable to consider select a donor GC or CC in rs7251 in IRF3. Disclosures: No relevant conflicts of interest to declare.

Description: Cellular senescence was first described as a physiological tumor cell suppressor mechanism that leads to cell growth arrest with production of the senescence-associated secretory phenotype known as SASP. The main role of SASP in physiological conditions is to attract immune cells to clear senescent cells avoiding tumor development. However, senescence can be damage-associated and, depending on the nature of these stimuli, additional types of senescence have been described. In the context of cancer, damage-associated senescence has been described as a consequence of chemotherapy treatments that were initially thought of as a tumor suppressor mechanism. However, in certain contexts, senescence after chemotherapy can promote cancer progression, especially when immune cells become senescent and cannot clear senescent tumor cells. Moreover, aging itself leads to continuous inflammaging and immunosenescence which are responsible for rewiring immune cells to become defective in their functionality. Here, we define different types of senescence, pathways that activate them, and functions of SASP in these events. Additionally, we describe the role of senescence in cancer and its treatments, including how aging and chemotherapy contribute to senescence in tumor cells, before focusing on immune cell senescence and its role in cancer. Finally, we discuss potential therapeutic interventions to reverse cell senescence.

Description: Multiple myeloma (MM) remains an incurable hematological malignancy characterized by clonal proliferation of malignant plasma cells in bone marrow. In the last 20 years, the introduction of autologous stem cell transplantation, followed by proteasome inhibitors and immunomodulatory agents, increased the survival of MM patients by 50%. However, still a high proportion of patients relapse and become refractory, especially, high-risk patients with adverse cytogenetics where these treatment combinations have shown limited benefit. Therefore, novel strategies, such as immunotherapy, have been developed in the last few years to help improve the survival of these patients. Immunotherapy treatments include a high number of different strategies used to attack the tumor cells by using the immune system. Here, we will review the most successful immunotherapy strategies published up to date in patients with relapsed or refractory (R/R) MM, including monoclonal antibodies targeting specific antigens on the tumor cells, antibodies combined with cytotoxic drugs or Antibodies Drug Conjugates, immune checkpoint inhibitors which eliminate the barriers that damper immune cells and prevent them from attacking tumor cells, bi-specific T-cell engagers antibodies (BiTEs), bi-specific antibodies and the infusion of chimeric antigen receptor-modified T cells. We overview the results of clinical studies that have been presented up to date and also review pre-clinical studies describing potential novel treatments for MM.

Description: In previous work we have demonstrated that CB-NK exert a cytotoxicity towards MM cells which involves lipid-protein vesicle transfer, which it is secondarily transferred from recipient MM cells to neighboring MM cells. This transmissible cytotoxicity causes a 5-7% of neighboring MM cell death (CDD 2015;22(1):96-107). However, this transmission between MM cells could have a diluent effect of the CB-NK cytotoxicity which could be also a tumor cell survival mechanism. To further analyze this mechanism, we determined the proteins transferred between cells and the role of lipids in this transfer. We performed TRANS-SILAC proteomics using two different approaches: 1) Labeling CB-NK cells with heavy amino-acids (hAA) to identify both CB-NK proteins transferred to MM (1ºMM) and secondary CB-NK proteins transferred from 1ºMM to neighboring MM cells (2ºMM). 2) Labeling MM cells, before CB-NK contact, with hAA to identify transferred proteins from 1ºMM to 2ºMM cells, and from 1ºMM to CB-NK. We found that 1ºMM cells acquired 9.5% of CB-NK proteins, and that these proteins were transferred to 2ºMM cells. As a consequence, 1ºMM cells diluted the CB-NK proteins from 9.5% to 3.8%; which represented 7.2% of proteins in 2ºMM cells. In the second approach, we observed that, MM cells transferred to neighboring MM cells only 1.9% of MM proteins in resting conditions. However, in the presence of CB-NK, this transfer was increased up to 7.7%. Furthermore, CB-NK cells acquired 7.3% of 1ºMM proteins. These findings demonstrate a secondary CB-NK protein transfer between MM cells, which represents a CB-NK protein dilution content from 1ºMM to 2ºMM, and an increased transfer of 1ºMM proteins to 2ºMM (Fig.1A). Proteomic analysis showed that transferred proteins were involved in FAS signaling, apoptosis, inflammation, chromatin organization, glycolysis, spliceosome, and rRNA metabolic process. Among these proteins we focused on histones and the 14-3-3 family which are associated to both cell death and cell survival. Confocal microscopy confirmed transfer of histones and 14-3-3 proteins. Protein transfer occurred within neutral lipid vesicles-structures. We also observed that MM cells were inter-connected with a much higher number of neutral lipid structures (nanotubes and bigger structures similar to bags) than in K562 cells (Fig. 1B). Thus, we next analyzed the role of these neutral lipids in MM cell-cell communication. The cholesterol synthesis and lipid transport inhibitor U18666A significantly decreased the number of these lipid connective structures between MM cells, and it was toxic for MM cells. Furthermore, breaking the lipid connection between MM cells with U18666A increased CB-NK cytotoxicity (p

Description: Abstract 3020 Introduction: Hematopoietic progenitors cells (HPCs) used in allogenic transplantation (allo-HSCT) may have different biological properties depending on their source of origin: mobilized peripheral blood (PB), bone marrow (BM) or umbilical cord (UC), which may be reflected in miRNAs or gene expression. The identification of different patterns of expression could have clinical implications. The aim of this study was to determine differences in miRNAs and gene expression patterns in the different sources of HPCs used in allo-HSCT. Materials and Method: CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors per type of source: UC, BM and PB mobilized with G-CSF. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and gene expression using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed between the different sources of HPCs statistical Kruskal Wallis test was applied. All analysis were performed using the Multiexperiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology and TargetScan Human). Results: Forty-two miRNAs differentially expressed between the different sources were identified. As compared to BM or UC, in mobilized PB most miRNAs were overexpressed, including the miRNA family of miR515, which is characteristic of embryonic stem cells. On the other hand, 47 genes differentially expressed between the different sources were identified. Interestingly, a similar pattern of expression was observed between movilized PB and UC as compared to BM. Interestingly, 13 of these genes are targets of the miRNAs also identified in this study, which suggests that their expression might be regulated by these miRNAs. Conclusion: There are significant differences in miRNAs and gene expression levels between the different sources of HPCs Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) plasma cells are equipped with an extensive endoplasmic reticulum (ER) for antibody production. Misfolding of newly synthesized immunoglobulins leads to ER stress and cell death. Plasma cells evade this by enhancing protein degradation through the proteasome pathway and the autophagy machinery. Because of this, MM cells are sensitive to proteasome inhibitors (PIs). However, autophagy induced by PIs and by reactive oxygen species (ROS) can increase drug resistance and lead to eventual relapse. We have previously demonstrated in vitro and in vivo sensitivity of MM cells to cord blood-derived natural killer (NK) cells, and have observed that this effect may involve NKG2D and NKP30. In this study we used confocal microscopy, chromium cytotoxicity assays and flow cytometry and we aimed to determine if there is a unique mechanism of NK cell-mediated cytotoxicity on MM cells involving ER stress, ROS and its dependence on NK cell receptors NKG2D and NKp30. We first demonstrated a difference in NK-mediated killing of MM cells vs K562 cells. As expected inhibition of GranzymeB (GrB) and Caspase3 pathway yielded a 13% reduction in NK-cytotoxicity against K562 cells; however, a 12% increase in killing was seen against ARP1 cells (p

Description: Introduction. Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in cytokine genes have shown to influence gene expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 1 (IL1) is a proinflammatory cytokine that induces the production of cytokines and chemokines and plays an important role in the inflammatory processes. IL1 is involved in various cellular functions, including cell proliferation, differentiation, and apoptosis. The IL1 family consists of two biologically actives forms (IL1A and IL1B). Several polymorphisms of these genes have been implicated in the pathogenesis of autoimmune diseases, however, their importance in SCT is not well-known. Objective To investigate the relationship between 4 SNPs in IL1A and IL1B genes and the susceptibility to the development of GvHD and other complications after HLA-identical allo-SCT. Patients and methods Genomic DNA obtained from peripheral blood samples belonging to 509 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). One SNP, rs1800587 (-889 C〉T), in the promoter region of the IL1A gene and three SNPs in the IL1B gene (two in the promoter region rs16944 (-511C〉T), rs1143627 (-31 T〉C) and one in exon 5 rs1143634 (+3954 C〉T) were genotyped by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Univariate and multivariate regression analysis were performed using Cox regression in the presence of competing risks except for chronic GvHD for which we used logistic regression model. All variables with p≤0.10 according to univariate analysis were included in the multivariate analysis. For multivariate analyses, p values were two sided and the outcomes were considered to be significant for p