ALBERT

Description: Abstract 1589 Introduction: Acquired potentially N-glycosylation sites are produced by somatic hypermutation (SHM) in the immunoglobulin (Ig) variable region. This phenomenon is produced in ∼9% of normal B-cells and seems to be related to certain B-cell lymphoproliferative disorders (B-LPDs) such as follicular lymphoma (FL, 79%), endemic Burkitt lymphoma (BL, 82%) and diffuse large B-cell lymphoma (DLBCL, 41%). These data suggest that new potential N-glycosylation sites could be related to germinal center B (GCB)-LPDs. By contrast, in other B-LPDs, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), MALT lymphoma, Waldenström macroglobulinemia (WM) or multiple myeloma (MM), these modifications have not been analyzed in deep. Aims: To evaluate the acquisition of potential N-glycosylation sites in B-LPDs, including immunohystochemical DLBCL subtypes (GCB and non-GCB) and specific non-GCB-LPDs, such as hairy cell leukemia (HCL), splenic marginal-zone lymphoma (SMZL), CLL, MCL, ocular extranodal marginal zone lymphoma (OAEMZL), MM and WM. Patients: A total of 953 sequences (203 from our group and 750 previously published sequences) of B-LPDs were included. Diagnosis distribution was as follows: DLBCL (n=235), MCL (n=235), CLL (n=166), MM (n=96), OAEMZL (n=82), SMZL (n=68), WM (n=38) and HCL (n=33). Methods: Acquired N-glycosylation sites were counted according to the sequence Asn-X-Ser/Thr, where X could be any amino acid except Pro. Natural motifs in germline sequences of IGHV1–08, IGHV4–34 e IGHV-5a were not considered. Fisher test was used to perform comparisons between groups. To distinguish DLBCL biological subtypes (GCB and non-GCB DLBCL), Hans' algorithm was used. Results: A total of 83 out of the 235 DLBCL cases acquired at least a new N-glycosylation site, a higher value than in normal B-cells (35% vs. 9%, p

Description: 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.

Description: Abstract 3660 Poster Board III-596 Introduction Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease. Current predictive systems, based on clinical and analytical parameters, fail to identify accurately this significant fraction of patients with short failure-free survival (FFS). Transcriptional analysis has identified genes and pathways associated with clinical failure, but the biological relevance and clinical applicability of these data await further development. Robust molecular techniques for the identification of biological processes associated with treatment response are necessary for developing new predictive tools. Patients and Methods We used a multistep approach to design a quantitative RT-PCR-based assay to be applied to routine formalin-fixed, paraffin-embedded samples (FFPEs), integrating genes known to be expressed either by the tumor cells and their reactive microenvironment, and related with clinical response to adriamycin-based chemotherapy. First, analysis of 29 patient samples allowed the identification of gene expression signatures related to treatment response and outcome and the design of an initial RT-PCR assay tested in 52 patient samples. This initial model included 60 genes from pathways related to cHL outcome that had been previously identified using Gene Set Enrichment Analysis (GSEA). Second, we selected the best candidate genes from the initial assay based on amplification efficiency, biological significance and treatment response correlation to set up a novel assay of 30 genes that was applied to a large series of 282 samples that were randomly split and assigned to either estimation (194) or validation series (88). The results of this assay were used to design an algorithm, based on the expression levels of the best predictive genes grouped in pathways, and a molecular risk score was calculated for each tumor sample. Results Adequate RT-PCR profiles were obtained in 264 of 282 (93,6%) cases. Normalized expression levels (DCt) of individual genes vary considerably among samples. The strongest predictor genes were selected and included in a multivariate 10-gene model integrating four gene expression pathway signatures, termed CellCycle, Apoptosis, NF-KB and Monocyte, which are able to predict treatment response with an overall accuracy of 68.5% and 73.4% in the estimation and validation sets, respectively. Patients were stratified by their molecular risk score and predicted probabilities identified two distinct risk groups associated with clinical outcome in the estimation (5-year FFS probabilities 75.6% vs. 45.9%, log rank statistic p≈0.000) and validation sets (5-year FFS probabilities 71.4% vs. 43.5%, log rank statistic p

Description: Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: The stem cell factor c-kit signaling pathway (SCF/c-kit) has been previously implicated in normal hematopoiesis, melanogenesis, and gametogenesis through the formation and migration of c-kit+ cells. These biologic functions are also determinants in epithelial–mesenchymal transitions during embryonic development governed by the Snail family of transcription factors. Here we show that the activation of c-kit by SCF specifically induces the expression of Slug, a Snail family member. Slug mutant mice have a cell-intrinsic defect with pigment deficiency, gonadal defect, and impairment of hematopoiesis. Kit+ cells derived from Slug mutant mice exhibit migratory defects similar to those of c-kit+ cells derived from SCF and c-kit mutant mice. Endogenous Slug is expressed in migratory c-kit+ cells purified from control mice but is not present in c-kit+cells derived from SCF mutant mice or in bone marrow cells from W/Wv mice, though Slug is present in spleen c-kit+ cells of W/Wv (mutants expressing c-kit with reduced surface expression and activity). SCF-induced migration was affected in primary c-kit+ cells purified from Slug−/− mice, providing evidence for a role of Slug in the acquisition of c-kit+ cells with ability to migrate. Slug may thus be considered a molecular target that contributes to the biologic specificity to the SCF/c-kit signaling pathway, opening up new avenues for stem cell mobilization.