ABC-like Diffuse Large B-cell Lymphoma Research Articles

Clinical pathology of primary central nervous system lymphoma in HIV-positive patients-a 41 Chinese patients retrospective study

ObjectivesThe purpose of this study was to investigate the clinicopathological characteristics of primary central nervous system lymphoma (PCNSL). MethodsWe collected 41 PCNSL formalin-fixed, paraffin-embedded (FFPE) samples from human immunodeficiency virus (HIV)-positive patients and performed HE (haematoxylin-eosin) staining, immunohistochemistry (IHC) staining, in situ hybridization, fluorescence in situ hybridization (FISH). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in 9 cases of FFPE samples. Meanwhile, we analysed the clinical pathological significance of the results. ResultsSeven patients had diffuse large B-cell lymphoma (DLBCL) with germinal centre B-cell (GCB)-like DLBCL, 32 had activated B-cell (ABC)-like DLBCL, and 2 had Burkitt lymphoma (BL). GCB-like DLBCL patients were older at onset (P = 0.040).A lower CD4+ T-cell count and a decrease in cerebrospinal fluid (CSF) glucose content were more frequent in ABC-like DLBCL (P = 0.012, P = 0.006). Overexpression of P53 was more in ABC-like DLBCL (P = 0.041). 73.2 % cases were Epstein–Barr encoding region (EBER) positive, which was more likely in ABC-like DLBCL patients (P = 0.037). EBV DNA were detected in 5/7 EBER-negative DLBCL cases and none (0/2) of the BL cases. All the cases were negative for HHV8 staining. None of the 7 Double expressor lymphoma (DEL) cases had BCL2, BCL6, or c-MYC genetic rearrangements. ConclusionsHIV-related PCNSL showed unique clinical pathological significance. None of EBV detected in HIV-related BL and without HHV8 infectious are new sights in our single-center study of Chinese HIV-related PCNSL patients.

Comprehensive Metabolome Analysis Identified Novel Biomarkers for the Diagnosis and Prognosis of Diffuse Large B-Cell Lymphoma

Background Metabolic reprogramming is a major hallmark of cancer, providing tumor cells with vital energy and metabolites. Diffuse large B-cell lymphoma (DLBCL) is metabolically heterogeneous, and there was also evidence that altered metabolism is associated with tumor progression and prognosis. This study was aimed to assess the characteristics of metabolic reprogramming and novel biomarkers in DLBCL, thereby proposing a novel therapeutic strategy for DLBCL. Methods Serum sample were obtained from100 patients with newly diagnosed DLBCL and 70 age-gender matched healthy controls in the study. Non-targeted metabolomics studies were performed based on liquid mass spectrometry (LC-MS) technology. The normalized peak areas (normalized by IS) metabolites were introduced into SIMCA software for multivariate statistical analysis. Metabolomic pathway analysis of metabolic biomarkers was performed using MetbraAnalyst5.0. Results We first analyzed differential metabolite profiles in the serum of DLBCL patients. The metabolites detected in positive and negative ion modes were analyzed separately. Compared to the healthy control, DLBCL patients were featured with a specific metabolic profile, indicating the differential metabolism in disease state. We then established a model containing metabolic information by orthogonal partial least squares discriminant analysis (OPLS-DA) and tested its ability to identify new samples in seven cross-validations. The variable importance in the projection (VIP) was also calculated for each metabolite. We further screened for differentially expressed metabolites by plotting collapsed changes (fold change>1.5 or <0.667, p<0.05). In DLBCL patients, 33 up-regulated metabolomes and 48 down-regulated metabolites were detected in positive ion mode, while 19 up-regulated metabolites and 29 down-regulated metabolites were in negative ion mode. Enrichment analyses showed that differential metabolites were mainly enriched in D-Glutamine and D-glutamate metabolism, caffeine metabolism, and ascorbate and aldarate metabolism. We then attempted to verify whether the differential metabolites had diagnostic value in DLBCL patients. By using random forest analysis, 2-Phenylpropionicacid, 4-oxododecanedioicacid, cis-4-Hydroxy-D-proline, Hypoxanthine Octanedioicacid under positive ion mode, and Xanthosine, Subericacid, Diethyloxalpropionate, 4-Oxoproline Azelaicacid under negative ion mode, were screened out as potential biomarkers. Based on the above biomarkers, diagnostic models were developed and revealed the mean accuracies of 0.9460 in positive mode and 0.943 in negative mode. Receiver operating characteristic (ROC) curves then verified the significant sensitivity and specificity of the diagnostic models. Notably, the Area Under Curve（AUC）of the combined diagnostic model was superior to that of each metabolite, indicating that combined model values could significantly improve the diagnostic performance in DLBCL. Further investigations were aimed to explore the correlation of metabolite profiles with clinical features in DLBCL. Venn diagram profiled the differential metabolites between patients with GCB-like DLBCL and ABC-like DLBCL. Correlation analysis then suggested that the differential metabolites were correlated with clinical prognostic indicators in DLBCL patients. In particular, D-Glucuronic acid and Lignoceric acid were significantly associated with Ki-67 expression (tumor proliferation index), cis-4-Hydroxy-D-proline was significantly associated with LDH and β2-MG, and 2-Phenylpropionic acid significantly correlated with LDH, PLT and ALB. Conclusions Our study revealed the metabolic profile of DLBCL and found a relatively high diagnostic value of metabolite-based diagnostic models constructed. Furthermore, the combination with clinical data suggests that D-glucuronide, lignoceric acid, cis-4-hydroxy-D-proline and 2-phenylpropionic acid may be potential prognosis-related biomarkers in DLBCL. Targeting metabolic reprogramming is expected to provide novel strategies for the treatment of DLBCL. Keywords DLBCL, metabolomics, biomarkers, D-glucuronide, tumor Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lenalidomide As Maintenance Therapy after R-CHOP Has No Protecting Effect for Central Nervous System Relapse in Frontline Treatment of Diffuse Large B-Cells Lymphoma. an Ancillary Studies of the Remarc Study

Lenalidomide As Maintenance Therapy after R-CHOP Has No Protecting Effect for Central Nervous System Relapse in Frontline Treatment of Diffuse Large B-Cells Lymphoma. an Ancillary Studies of the Remarc Study

MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11.

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

Acquisition of Mannoses on the Surface Immunoglobulin Binding Site Reveals Functional Status and Cell of Origin in Diffuse Large B Cell Lymphomas

Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown.While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010).In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars).We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into “DC-SIGN binders” (DB-DLBCL) vs “DC-SIGN non-binders” DLBCL (NB-DLBCL).Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites.We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC.Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by ‘antigen’. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. DisclosuresPackham:Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

A New Stromal Signature Applicable to Formalin-Fixed Paraffin-Embedded Tissues Identifies Patients at Risk in Prospective Clinical Trials of the German High-Grade Non-Hodgkin Lymphoma Study Group

Diffuse large B-cell-lymphoma (DLBCL) is a heterogeneous disease in its sites of origin, genetic alterations, and clinical behavior. Gene expression profiling (GEP) has led to the identification of two molecular subtypes, GCB-like and ABC-like DLBCL, that follow different molecular circuits and hence, likely represent different diseases. Next to the ABC-like GEP, the rearrangement and/or expression of MYC and TP53 mutations in tumors characterize patient subsets with inferior prognosis. However, the clinical impact of cell of origin (COO) subtyping and the identification of prognostic biomarkers differ between studies. In many clinical trials, patients identified “at risk” experience cure and long-time survival. Important factors modulating the impact of tumor cell-specific factors may be conferred by the host microenvironment, and stromal signatures have been shown to be correlated to survival in DLBCL. The robust analysis of stromal signatures is hampered by the lack of assays applicable to routinely used formalin-fixed paraffin-embedded (FFPE) materials. We have constructed a molecular signature applicable to FFPE, interrogating the quantitative and qualitative composition of the microenvironment in DLBCL. The signature was trained using an algorithm that extracts prognostic information out of the ratios of pairs of genes. The genes that drive prognosis are expressed in T-cells and macrophages and have function in the communication between both cell types. The model was validated using the NanoString assay in a cohort of 466 DLBCL patients enrolled in 7 prospective clinical trials (MInT, MegaCHOEP phase III and observation, RICOVER-60, RICOVER-noRTh, DENSER, SMARTER) of the German High grade non-Hodgkin's lymphoma study group (DSHNHL). Grouping of the patients into quartiles according to the expression of the continuous stromal signature score (ranging from -1.880to 4.441) resulted in three quartiles (Q1-3) with comparable clinical behavior (stromal signature low). Patients from quartile 4 (Q4), however, characterized by high expression of the signature (stromal signature high), showed a clearly inferior outcome in EFS, PFS and OS (Figure 1 a-c). This result could be independently reproduced in the seven clinical trials named above, thus clearly depicting the robustness of the signature. Multivariate analysis revealed that high expression of the stromal signature is a prognostic risk factor independent of the IPI factors in EFS (HR 1.7, 95 % CI 1.2-2.4, p-value =0.001), PFS (HR 1.8, 95 % CI 1.2-2.5, p-value =0.001) and OS (HR1.8, 95 % CI 1.3-2.7, p-value =0.001). Combining stromal signature count with IPI-score led to the identification of a high-risk cohort (Fig. 1 d-f). Of importance, additional multivariate analyses adjusted for the IPI factors performed within selected trials showed that the stromal signature provides prognostic information independent of the COO status, MYC and dual MYC/BCL2 rearrangements, TP53 mutations and the MYC/BCL2 double expresser status.In summary, our data from prospectively randomized trials of the DSHNHL underline the importance of the microenvironment in the prognostic stratification of DLBCL patients and suggest that the composition and quality of the tumor stroma is an independent risk factor in DLBCL. Analysis of stromal features, therefore, may provide a rationale for targeted treatment approaches, e.g. with immunomodulatory substances (IMIDs), in patients at risk. [Display omitted] DisclosuresKlapper:Regeneron: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Richter:HTG Molecular Diagnostics, Inc.: Research Funding. Poeschel:Roche: Other: Travel grants; Amgen: Other: Travel grants. Held:BMS: Consultancy, Other: Travel grants, Research Funding; Spectrum: Research Funding; Roche: Consultancy, Other: Travel grants, Research Funding; Amgen: Research Funding; MSD: Consultancy.

VEGF121, is predictor for survival in activated B-cell-like diffuse large B-cell lymphoma and is related to an immune response gene signature conserved in cancers.

Tumor microenvironment including endothelial and immune cells plays a crucial role in tumor progression and has been shown to dramatically influence cancer survival. In this study, we investigated the clinical relevance of the gene expression of key mediators of angiogenesis, VEGF isoforms 121, 165, and 189, and their receptors (VEGFR-1 and R-2) in a cohort of patients (n = 37) with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) from the Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL). In patients with ABC-like DLBCL, but not in patients with GCB-like DLBCL, low VEGF121 expression was associated with a significantly better survival than in those with high VEGF121 level: 4-year overall survival at 100% vs 36% (p = .011), respectively. A specific gene signature including 57 genes was correlated to VEGF121 expression level and was analyzed using a discovery process in 1,842 GSE datasets of public microarray studies. This gene signature was significantly expressed in other cancer datasets and was associated with immune response. In conclusion, low VEGF121 expression level was significantly associated with a good prognosis in relapsed/refractory ABC-like DLBCL, and with a well-conserved gene-expression profiling signature related to immune response. These findings pave the way for rationalization of drugs targeting immune response in refractory/relapsed ABC-like DLBCL.

Clinical Impact of the Cell-of-Origin Classification and the MYC/ BCL2 Dual Expresser Status in Diffuse Large B-Cell Lymphoma Treated Within Prospective Clinical Trials of the German High-Grade Non-Hodgkin's Lymphoma Study Group.

Purpose To explore the prognostic impact and interdependence of the cell-of-origin (COO) classification, dual expression (DE) of MYC and BCL2 proteins, and MYC, BCL2, and BCL6 translocations in two prospectively randomized clinical trials of patients with diffuse large B-cell lymphoma (DLBCL). Patients and Methods Overall, 452 formalin-fixed paraffin-embedded samples from two prospective, randomized DLBCL trials (RICOVER-60, prospective, randomized study for patients > 60 years, all IPI groups; and R-MegaCHOEP, prospective, randomized study for patients ≤ 60 years with age-adjusted IPI 2,3) of the German High-Grade Non-Hodgkin Lymphoma Study Group were analyzed with the Lymph2Cx assay for COO classification, with immunohistochemistry for MYC and BCL2, and with fluorescent in situ hybridization for MYC, BCL2, and BCL6 rearrangements. Results COO classification was successful in 414 of 452 samples. No significant differences with respect to COO (activated B-cell [ABC]-like DLBCL v germinal center B-cell [GCB]-like DLBCL) were observed in event-free survival, progression-free survival, and overall survival in patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in the RICOVER-60 trial. Also, no differences with respect to COO were observed in multivariable analyses adjusted for International Prognostic Index factors in event-free survival (hazard ratio [HR] of ABC-like disease v GCB-like disease, 1.0; 95% CI, 0.6 to 1.6; P = .93), progression-free survival (HR, 1.1; 95% CI, 0.6 to 1.8; P = .82), and overall survival (HR, 1.0; 95% CI, 0.6 to 1.8; P = .96). Similar results were observed in the R-MegaCHOEP trial. In patients treated with R-CHOP, DE status was associated with significantly inferior survival compared with nonDE within the GCB, but not within the ABC subgroup. DE status was associated with significantly inferior outcome compared with patients with ABC-like DLBCL without DE (5-year PFS rate, 39% [95% CI,19% to 59%] v 68% [95% CI, 52% to 85%]; P = .03) and compared with patients with GCB-like DLBCL without DE. When data from patients with nonDE were analyzed separately, the outcome of patients in the ABC subgroup was inferior to that of patients in the GCB subgroup (5-year PFS rate, 68% [95% CI, 52% to 85%] v 85% [95% CI, 74% to 96%]; P = .04). Conclusion COO profiling in two prospective randomized DLBCL trials failed to identify prognostic subgroups, whereas dual expression of MYC and BCL2 was predictive of poor survival. Evaluation of prognostic or predictive biomarkers in the management of DLBCL, such as the COO, within prospective clinical trials will be important in the future.

PD-L1 Expression Identifies High Risk Diffuse Large B-Cell Lymphoma and Is Associated with Several Genomic Markers

Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors and the most common form of non-Hodgkin lymphoma (NHL). DLBCLs arise from germinal center (GC) or post-GC B cells and display marked heterogeneity in genetic features and clinical outcomes DLBCLs, similar to other NHL and to solid tumors, are known to evade host immune response by usurping immune checkpoint pathways such as CTLA4 and PD-1. The initial Phase I and Phase II trials of checkpoint inhibitors in NHL revealed promising results, but rational and reliable biomarkers for selecting patients that benefit from such therapies is missing. Even in the case of the better-studied PD1-PD-L1 pathway, the characterization of expression of those proteins in DLBCLs is not standardized, and their relationship with molecular and clinical features in DLBCLs are still largely unknown. We set out to study PD-L1 expression using three commonly used anti-PD-L1 antibodies in a well-characterized single-institutional cohort of 52 primary DLBCLs from Keck School of Medicine of USC treated with R-CHOP or R-CHOP-like therapy with clinical follow up. All cases were characterized by aCGH; Focus::Lymphoma NGS test (Cancer Genetics Inc), which includes 220 most commonly mutated genes in B NHL; FISH including bcl2 and c-myc; and immunohistochemical staining to define cell of origin (Hans classification) and with three anti-PD-L1 antibodies (Abcam EPR1161, Abcam 28-8 and Ventana SP263 clones).We observed variable sensitivity, specificity and dynamic range of three anti-PD-L1 antibodies. EPR1161 and SP263 clones had highest correlation (Pearson correlation=0.69) and similar staining characteristics demonstrating robust linear membranous staining on neoplastic cells and on reactive infiltrating Immune Cells (IC). PD-1 expression on IC had inverse correlation with PD-L1 expression on neoplastic cells, suggesting immunosuppressive effect of PD-L1 positive neoplastic cells on tumor microenvironment. Non-GCB DLBCLs had higher PD-L1 positivity than GCB DLBCLs (p=0.0001, 0.01 and 0.06 respectively for EPR1161, DAKO 28-8 and Ventana SP263 Abs, t-test).Using molecular characterization, we observed that PD-L1 expression marked high risk aggressive DLBCLs, which also had higher TP53 expression, TP53 mutation and/or del 17p13 (Pearson correlation= 0.176). Moreover, higher expression of PD-L1 had negative correlation with mutations associated with GCB-like DLBCLs including BCL2, FOXO1, KMT2D, EZH2 (p=0.003, 0.01, 0.06, 0.08; t-test) and positive correlation with mutations associated with ABC-like DLBCLs including FAT2 (p=0.04, t-test). Interestingly, while PD-L1 expression correlated with high risk molecular and cytogenetic groups of DLBCL, it did not show correlation with BCL2 or C-MYC expression nor with BCL2/C-MYC double expressors.Most importantly, Kaplan Meyer analysis showed that even in the settings of chemotherapy, higher PD-L1 expression correlated with more aggressive disease, using all three antibodies ( p=0.03, 0.01, 0.04 respectively for Abcam EPR1161, Abcam 28-8 and Ventana SP263 clones).We conclude that PD-L1 expression reflects the biology of aggressive subsets of DLBCLs, which are also characterized by high risk cytogenetic and molecular biomarkers. PD-L1 expression can be measured by several antibody clones, which have different performance characteristics but show positive correlation. DisclosuresNo relevant conflicts of interest to declare.

Clinicopathological features of 49 primary gastrointestinal diffuse large B-cell lymphoma cases; comparison with location, cell-of-origin, and frequency of MYD88 L265P.

The gastrointestinal (GI) tract is the most common primary site of extranodal diffuse large B-cell lymphoma (DLBCL), with approximately one-third of extranodal DLBCL occurring in the GI tract. We investigated the clinicopathological features and immunohistochemically-assessed cell-of-origin of 49 GI DLBCL cases (stomach, 24; small intestine, 10; colon, 15) and also examined the presence of MYD88 L265P as recently this mutation has been frequently identified in ABC-like DLBCL, particularly in extranodal sites. Small intestinal DLBCL was characterized by the preponderance of women (P = 0.041) and elevated LDH (P = 0.002) and soluble interleukin-2 receptor (P = 0.033). Small intestinal DLBCL more frequently showed anemia (P = 0.031) and elevated CRP (P = 0.029) than gastric DLBCL. ABC-like phenotype was seen in 71.4% cases (stomach, 79%; small intestine, 70%; colon, 60%). MYD88 L265P was detected in 6.1% cases; all were primary gastric DLBCL with ABC-like phenotype but had no distinct clinicopathological features. In conclusion, GI DLBCL had different clinicopathological features according to the primary site especially in the small intestine. Also, MYD88 L265P had little involvement in GI DLBCL compared with other extranodal DLBCLs, suggesting that its pathogenesis might be different from that of organs with a high frequency of MYD88 L265P.

MiR-181a negatively regulates NF-κB signaling and affects activated B-cell–like diffuse large B-cell lymphoma pathogenesis

AbstractDistinct subgroups of diffuse large B-cell lymphoma (DLBCL) genetically resemble specific mature B-cell populations that are blocked at different stages of the immune response in germinal centers (GCs). The activated B-cell (ABC)–like subgroup resembles post-GC plasmablasts undergoing constitutive survival signaling, yet knowledge of the mechanisms that negatively regulate this oncogenic signaling remains incomplete. In this study, we report that microRNA (miR)-181a is a negative regulator of nuclear factor κ–light-chain enhancer of activated B-cells (NF-κB) signaling. miR-181a overexpression significantly decreases the expression and activity of key NF-κB signaling components. Moreover, miR-181a decreases DLBCL tumor cell proliferation and survival, and anti–miR-181a abrogates these effects. Remarkably, these effects are augmented in the NF-κB dependent ABC-like subgroup compared with the GC B-cell (GCB)–like DLBCL subgroup. Concordantly, in vivo analyses of miR-181a induction in xenografts results in slower tumor growth rate and prolonged survival in the ABC-like DLBCL xenografts compared with the GCB-like DLBCL. We link these outcomes to relatively lower endogenous miR-181a expression and to NF-κB signaling dependency in the ABC-like DLBCL subgroup. Our findings indicate that miR-181a inhibits NF-κB activity, and that manipulation of miR-181a expression in the ABC-like DLBCL genetic background may result in a significant change in the proliferation and survival phenotype of this malignancy.

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

MiR-181a Is a Master Regulator of the Nuclear Factor-κB Signaling Pathway in Diffuse Large B Cell Lymphoma

AbstractAbstract 417MicroRNAs (miRNAs) exhibit differential expression in cancer and can be used as prognostic biomarkers. MiR-181a expression is reported to be associated with survival and outcome in acute myeloid and chronic lymphocytic leukemia patients. We demonstrated that miR-181a levels are independently associated with improved survival of diffuse large B cell lymphoma (DLBCL) patients treated with R-CHOP (Rituximab, Cyclophosphamide, Adriamycin, Oncovin, Prednisolone). However, the mechanism underlying this observation and the function of miR-181a in DLBCL pathogenesis are unknown.MiR-181a was expressed at higher levels in centroblasts compared to naïve and memory B cells, and at significantly higher levels in GCB-like compared to ABC-like DLBCL cell lines (p=0.017). These observations suggested that miR-181a may differentially target critical signaling pathways in GCB and ABC DLBCL. NF-kB serves a critical role in ABC DLBCL survival. Utilizing 3 miRNA target prediction algorithms, multiple NF-κB signaling pathway transcripts harbored putative miR-181a binding sites. Consequently, we tested the effect of miR-181a on CARD11, IBKα, p105/p50, and C-Rel expression in DLBCL cell lines (HBL1, VAL). Compared with a scrambled miRNA control, miR-181a expression decreased protein and mRNA levels of these targets. To confirm the effect was direct, we fused the 3′-UTR sequences of CARD11, IBKα, p105 and C-Rel, each containing miR-181a putative binding sites, to a luciferase reporter gene. Co-transfecting miR-181a with the corresponding constructs, we demonstrated that all the constructs had significantly repressed luciferase activity compared with a non-targeting control. The effect was specific, since miR-181a did not affect luciferase activity of CARD11, IBKα, p105 and C-Rel reporter constructs with mutated binding sites. Using an NF-κB luciferase reporter assay, we next demonstrated that compared to a scrambled control, miR-181a significantly decreased NF-κB reporter activity in DLBCL cell lines (VAL, SUDHL6, OCILY7, OCILY19, HBL1, RCK8). MiR-181a also decreased NF-κB reporter activity induced by anti-IgM and TNFα stimulation. Concordantly, anti-miR-181a increased endogenous p105/p50 and C-Rel protein levels. Because ubiquitinated-IKKγ drives NF-κB signaling, we tested the effect of miR-181a in TNFα-stimulated 293T cells on ubiquitinated-IKKγ. MiR-181a decreased levels of ubiquitinated-IKKγ, corroborating the observed inhibitory effects on NF-κB signaling. We reasoned that NF-κB signaling repression should coincide with a decrease in endogenous transcription activity from NF-κB promoters. Indeed, miR-181a decreased mRNA expression levels of NF-κB target genes (BCL2, IRF4, IL-6, IKBa, FN1, PIM1, BLR1, CCL3, CFLAR, FCER2, TP53) as measured by qRT-PCR in miR-181a-transfected HBL1 cells. Because miR-181a directly targets p105/p50 and REL proteins, we postulated that this may be one of the main mechanisms of NF-κB signaling repression. Indeed, an electrophoresis mobility shift assay along with super-shifts analyses showed a decrease in the p105/p50 protein in HeLa nuclear extracts. To examine the biological significance of differential miR-181a expression between GCB- and ABC-like DLBCL and elucidate its potential role in DLBCL pathogenesis, we next assessed cell death (Annexin V, 7AAD) and cell proliferation (BrdU, 7AAD) in GCB (SUDHL4, OCILY7, OCILY19, VAL) and ABC (HBL1, OCILY10, RCK8, U2932) DLBCL cell lines transfected with GFP labeled precursor miR-181a. MiR-181a expression significantly increased cell death and apoptosis of ABC versus GCB DLBCL (p=0.006). This was associated with a more pronounced G1 phase growth arrest in the ABC DLBCL cells.Our studies demonstrate that miR-181a is a master regulator of canonical NF-kB signaling by regulating the expression of multiple components of this pathway, an effect that may underlie the distinct prognosis of DLBCL with different miR-181a expression levels. Furthermore, miR-181a down regulation may contribute to the pathogenesis of ABC DLBCL. Disclosures:No relevant conflicts of interest to declare.

Association of low expression of VEGF121 soluble isoform with prognostic impact and survival in patients with activated B-cell-like (ABC-like) diffuse large B-cell lymphoma (DLBCL) from the CORAL trial.

8048 Background: Gene expression profiling (GEP) has demonstrated that survival of DLBCL is influenced by the origin of the tumor cells (COO), germinal center-like (GC-like) vs ABC-like (Alizadeh et al. 2000), but also by the tumor microenvironment, particularly the angiogenesis (Lenz et al., 2008). To further understand the prognostic impact of these tumoral characteristics, we analysed the prognostic impact of COO and the expression of biomarkers involved in angiogenesis, in a selected population of 36 patients with relapsed/refractory DLBCL included in CORAL (Gisselbrecht, ASCO 2011). Methods: Expression of 5 biomarkers including VEGF (isoforms 121, 165, and 189), and their receptors (VEGFR-1 and R-2) was assessed by quantitative qRT-PCR after total RNA extraction and cDNA synthesis of tumor samples. COO was determined by GEP, and progression free- (PFS) and overall- (OS) survivals were analysed. Results: In the study population, VEGF121 expression below the median was associated to a better outcome, with 4 year-PFS at 63% vs 33% (p=.053), and a 4-year-OS at 79% vs 37% (p = 0.032), respectively. VEGF-165 -189, VEGF-R1, -R2 did not have any significant impact. Eighteen patients were predicted as ABC-like DLBCL and 18 as GCB-like DLBCL. In patients with ABC-like DLBCL, low VEGF121 level was associated to a significantly better survival than in those with high VEGF121 level: 4-year OS at 100% vs 36% (p=.011). The type of induction treatment, R-ICE or R-DHAP, did not influence the outcome. The differences in outcome according to VEGF isoforms were not significant among CGB patients. Conclusions: Our data suggest that angiogenesis plays a central role in ABC-like DLBCL. VEGF121 seemed to be a key player in this context and should be proposed as a target in the treatment of patients with ABC-like DLBCL.

Targeting of TNFAIP3 (A20) Links MiR-125a and MiR-125b to Constitutive Activation of the NF-KB Pathway and Lymphomagenesis

Abstract 774The most aggressive subtype of diffuse large B-cell lymphoma (DLBCL), ABC-DLBCL, is characterized by constitutive activation of NF-κB signals, in association with somatic mutations in regulators of this pathway. However, in a large fraction of cases the molecular basis for the elevated NF-κB activity is unclear. MicroRNAs (miRNA) play a central role in immune function, in part by modulating the NF-κB system; yet, their role in the NF-κB deregulation found in DLBCL is unknown.To address this issue, we created a copy number/expression map of miRNAs in ∼100 DLBCLs, and integrated these data with miRNA target prediction models. This supervised strategy showed that miR-125a and miR-125b, which we found to be amplified/overexpressed in ∼ 30% of DLBCLs, are predicted to target TNFAIP3 (A20) via two fully conserved binding sites. A20 is an ubiquitin editing enzyme that negatively regulates NF-κB complexes and acts as a lymphoma suppressor gene, suggesting that its abnormal downregulation by miRs-125a/b may impinge on DLBCL pathogenesis.To validate these initial findings, we stably expressed miR-125a and miR-125b in multiple DLBCL cell lines, and used western blots to show that both miRNAs downregulate A20. In parallel assays, we used anti-miR oligos and miRNA sponge constructs in cell lines expressing high levels of miRs-125a/b, and found that their inhibition upregulates A20 levels. To study the interaction between miRs-125a/b and A20, we created reporter constructs with A20 sequences wild-type (WT) or mutated for the miR-125 binding sites; luciferase activity of the WT (but not the mutant) constructs was inhibited by miRs-125a/b confirming that they directly target A20.We hypothesized that miRs-125a/b-mediated inhibition of A20 activates NF-κB in DLBCL. Indeed, phosphorylation and degradation of the NF-κB inhibitory protein IκBα was markedly enhanced in DLBCL cell lines ectopically expressing miRs-125a/b, in comparison to their isogenic counterparts lacking these miRNAs. Further, nuclear accumulation of RelA was also elevated in miRs-125a/b expressing cells. Finally, we used a NF-κB reporter construct, to show that DLBCL cells stably expressing miRs-125a/b had significantly higher NF-κB activity (p<.005) than those lacking these miRNAs. In key additional assays, we found that stable expression of miRs-125a/b-specific sponges blocked the phosphorylation/degradation of IκBα in association with A20 upregulation.Each miRNA targets multiple genes. Thus, to confirm that the effects of miRs-125a/b on NF-κB activity result mainly from A20 inhibition, we stably expressed an A20 construct lacking miRs-125a/b binding sites in DLBCL cell lines genetically modified to overexpress these miRNAs. The ratios of phosphorylation/degradation of IκBα and the nuclear localization of RelA showed that A20 re-expression rescued the miRs-125a/b-induced activation of NF-κB in DLBCL, asserting the key role of A20 targeting on miRs-125a/b function.MiR-125a and −125b are thought to act as oncomiRs in the hematopoietic lineage. In probing this concept, we found that DLBCLs stably expressing miRs-125a/b grow at significantly faster rate than their empty-vector counterparts (p<0.05), and have enhanced clonogenicity potential (p<0.05), tested by colony forming assays. The poor response to chemotherapy found in ABC-like DLBCL may be related to high NF-κB signals, and resistance to apoptosis. To investigate if miRs-125a/b render DLBCLs resistant to apoptosis in a therapeutic setting, we exposed cells lines expressing miRs-125a/b to H2O2, a reactive oxygen species generated by anticancer agents; we found that miRs-125a/b expressing DLBCLs were significantly more resistant to H202-induced apoptosis than isogenic cells lacking these miRNAs (20±2.8% vs. 45±3.8% apoptosis rate, p< 0.05). Finally, we examined the interplay between miRs-125a/b and A20 in primary DLBCLs, and found that tumors with the highest miRNA levels have significantly lower A20 protein expression (p<0.05, densitometric calculation of A20 levels).Together, we defined miRNA deregulation as a novel mechanism for aberrant activation of NF-κB signals in DLBCL, and showed that A20 targeting by the lymphomagenic miRs-125a/b enhances lymphoma aggressiveness. These data bring mechanistic insight into the oncogenic activities of miRs-125a/b, and may guide the development of miRNA-based therapeutic strategies in hematological cancers. Disclosures:No relevant conflicts of interest to declare.

STAT3 Inhibition Is a Therapeutic Strategy for ABC-like Diffuse Large B-Cell Lymphoma

Persistent STAT3 signaling contributes to malignant progression in many diverse types of human cancer. STAT3 is constitutively active in activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCL), a class of nongerminal center derived DLBCL cells for which existing therapy is weakly effective. In this report, we provide a preclinical proof of concept that STAT3 is an effective molecular target for ABC-like DLBCL therapy. Direct inhibition of STAT3 with short hairpin RNA suppressed the growth of human ABC-like DLBCL in mouse models in a manner associated with apoptosis, repression of STAT3 target genes, and inhibition of a tumor-promoting microenvironment. Together, these results suggest that STAT3 is essential to maintain the pathophysiology of ABC-like DLBCL and therefore that STAT3 inhibition may offer a promising approach in its therapy.

Abstract 1637: STAT3 as a new molecular target for treating ABC-like DLBCL

Abstract Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. STAT3 has been found to be constitutively active in activated B cell (ABC)-like diffuse large B cell lymphomas (DLBCL). We investigated the effect of STAT3 silencing in human DLBCL Ly3 cells both in vitro and in vivo. For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells and control lentivirus Ly3 cells. The stable expression of STAT3 shRNA resulted in 40-50% reduction of STAT3 protein levels compared to the control cells. In vitro experiments revealed that STAT3 downregulation was associated with a higher percentage of dead cells in STAT3 shRNA cells compared with the control cells, and this percentage was even higher in serum starvation conditions. STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc associated with downregulation of IL-10-dependent STAT3 activation and inhibition of IL-10-inducible cell growth. STAT3 inhibition also reduced adhesion to the bone marrow stroma layer and migration toward SDF-1 alpha. Moreover, analysis of inflammatory cytokines and receptors indicated a substantial downregulation of CXCL6 mRNA levels in STAT3 shRNA cells compared to the control cells. Tumors in control Ly3-bearing mice grew progressively, whereas tumors in STAT3 shRNA Ly3-bearing mice regressed 4-5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis and significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin. Moreover, immunofluorescence analysis of tumor specimens showed that while granulocytes and endothelial cells infiltrated into the control cells-bearing tumors, tumor bearing STAT3 shRNA cells showed less or no infiltration of these cells. We also observed reduction of IL-10 production and the secretion of other cytokines was altered as well. Thus, our results show that even partial down-regulation of STAT3 could achieve complete suppression of the tumorigenesis of ABC-like DLBCL Ly3 cells by affecting also the tumor microenvironment. Furthermore, we tested two STAT3 inhibitors, STATTIC and S31-201, in this system. The viability of Ly3 cells grown in vitro was reduced by both compounds tested in a dose-dependent manner and the antiproliferative effect was associated with inhibition of STAT3 phosphorylation. The antilymphoma activity of STATTIC was also observed in vivo. This is the first demonstration in vivo that direct STAT3 inhibition in ABC-like DLBCL suppresses tumor growth. While developing a small-molecule STAT3 inhibitor as well as siRNA delivery for clinical trials is still a challenge, our studies validate STAT3 as a good target for therapy in DLBCL and also establish that STAT3 siRNA-based gene therapy is a feasible approach for DLBCL. In Sum, our data support continued development of new STAT3 inhibitors as well as STAT3 siRNA delivery strategies for treatment of DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1637. doi:10.1158/1538-7445.AM2011-1637

BLIMP1 against Lymphoma: The Verdict Is Reached

BLIMP1, a master regulator of plasma-cell differentiation, is implicated in the pathogenesis of Activated B cell (ABC)-like Diffuse Large B cell Lymphoma (DLBCL). In this issue of Cancer Cell, Mandelbaum and colleagues and Calado and colleagues unequivocally demonstrate that BLIMP1 functions as a tumor suppressor and guardian of ABC-like DLBCL lymphomagenesis.

STAT3, Constitutively Activated In ABC-Like DLBCL, Regulates Expression of the Prognostic Factor Cyclin D2

AbstractAbstract 705As the most common subtype of non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL) remains clinically heterogeneous. Genome-scale expression profiles have defined two distinct molecular subtypes of DLBCL: Germinal Center B-Cell like (GCB-like) and Activated B-Cell like (ABC-like), with separate postulated tumor cells of origin and different pathogenesis, e.g. activation of NF-kB and Signal Transducer and Activator of Transcription 3 (STAT3) in the ABC-like DLBCLs. These subtypes are also characterized by distinct clinical outcomes. Since global gene expression is not practical for routine prognosis determination, we established an IPI independent 6-gene model based on the expression of BCL6, LMO2, FN1, BCL2, SCYA3 and cyclin D2 (CCND2) genes, which was capable of predicting patients' survival. While some of these genes may merely reflect different postulated tumor cells of origin, others may represent distinct pathogenetic mechanisms. Herein we demonstrate that CCND2, whose expression correlates with shorter survival, is positively regulated by the STAT3 signaling pathway, activated in the ABC-like DLBCL. Immunohistochemical staining of phosphorylated STAT3 (pSTAT3) demonstrated strong positive pSTAT3 staining (greater than 30% of nuclei) in 85 of 204 (41.7%) primary DLBCLs. Hierarchical clustering analysis of 143 DLBCL specimens stained for GCB and ABC markers demonstrated that expression of the pSTAT3 protein correlated with the expression patterns of non-GCB markers MUM1/IRF4 and BCL2 but not with GCB-specific markers HGAL, BCL6, and CD10. However, pSTAT3 expression was not different between the GCB-like and non-GCB-like DLBCLs defined by Han's model (Hans et al, Blood 2004). Examination of pSTAT3 levels in GCB-like DLBCL cell lines (OCI-LY7, OCI-LY19, and Sud-HL6) and the ABC-like cell lines (OCI-LY3 and OCI-LY10) by Western blotting demonstrated its expression in OCI-LY3 and OCI-LY10 but not in other cell lines. pSTAT3 expression in these cells was confirmed by phospho-flow cytometry. Interestingly, pSTAT3 expressing cell lines also expressed high levels of Cyclin D2, while it was not detected by Western blotting in the DLBCL cell lines not expressing pSTAT3. Analysis of the CCND2 promoter showed a single potential STAT3 binding site. Chromatin immunoprecipitation (ChIP) assay confirmed specific binding of STAT3 to this site in the CCND2 promoter. BCL6 is reported to negatively regulate expression of CCND2 and to competitively bind to STAT3 binding sites. Consequently, using quantitative ChIP assay we measured BCL6 and STAT3 binding to the CCND2 promoter in Sud-HL6, Oci-LY7 and Oci-LY19 GCB-like and in Oci-LY3 and Oci-LY10 ABC-like DLBCL cell lines. Levels of the bound STAT3 and BCL6 proteins were significantly higher in the Oci-LY3 and Oci-LY10 cell lines compared to their GCB counterparts. Treatment with BCL6 peptide inhibitor (BPI) slightly increased CCND2 mRNA expression in both GCB-like and ABC-like DLBCL, confirming the inhibitory effect of BCL6 on the CCND2 expression. To examine the effect of pSTAT3 on Cyclin D2 in the physiological context, we have knocked-down expression of STAT3 and pSTAT3 by specific siRNA in the OCI-LY3 cell line. A 37% decrease in the total levels of STAT3 led to a 52% decrease in pSTAT3 and a 55% reduction in the Cyclin D2 protein levels and was associated with a statistically significant decrease in cell proliferation as measured by the MTT assay. Overall our results demonstrate that a constitutively active STAT3 signaling pathway contributes to the Cyclin D2 expression and proliferation of the ABC-like DLBCLs. These findings elucidate the mechanism regulating the expression of prognostic factor Cyclin D2 and link it to the STAT3 signaling pathway constitutively activated in ABC-like DLBCL. Disclosures:Patricelli:ActivX Biosciences: Employment. Nomanbhoy:ActivX Biosciences: Employment.

MiR155 DOWNREGULATES EXPRESION of HGAL and INCREASES LYMPHOMA CELL MOTILITY

AbstractAbstract 464HGAL is a germinal center (GC)-specific gene that inhibits lymphocyte and lymphoma cell motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Its specific expression only in the GC B-cells and GC-derived lymphomas suggest the existence of a tight regulatory process. We have previously reported that IL-4 and IL-13 can induce HGAL RNA and protein expression (Lossos et al, Blood. 2003; Natkunam et al, Blood. 2007) and at this meeting we also report that HGAL transcription is regulated by BLIMP1 protein (Cubedo et al). However, recent observations suggest that the fine-tuned control of gene expression, necessary for the immune cell differentiation-specific gene expression, is frequently accomplished by miRNAs. Therefore we have searched three prediction algorithms, PicTar (http://pictar.mdc-berlin.de/), miRanda (http://cbio.mskcc.org/ mirnaviewer/), and TargetScan (http://www.targetscan.org/) to identify miRNAs potentially regulating HGAL expression. We have focused on miR155, predicted to target HGAL in the TargetScan algorithm, since miR155 is overexpressed in aggressive activated B-cell like (ABC) type of DLBCL, characterized by low expression of HGAL. Furthermore, miR155 plays an important role in the regulation of GC reaction (Thai et al, Science. 2007) and its overexpression in mice B-lymphocytes leads to preleukemic B cell proliferation followed by full blown B-cell malignancy (Costinean et al, Proc Natl Acad Sci USA. 2006). To test HGAL regulation by the miR155, precursor of hsa-miR-155 was transfected into VAL and Raji lymphoma cells that express endogenous HGAL protein. Western blotting of whole-cell lysates showed a decrease of native HGAL in both cell lines compared with control miRNA transfection. Transfection of hsa-miR155 precursor into 3 lymphoma cell lines did not result in uniform decreases in HGAL mRNA expression levels, suggesting main regulation at the protein translation level. Transfection efficacy in each experiment was confirmed by measurements of miR155 by TaqMan MicroRNA Assay. To confirm direct effect on HGAL expression, we fused 2614 bp 3'-UTR sequences of HGAL containing two putative binding sites (M1 positions 2285–2291 and M2 positions 1844–1849) to a luciferase reporter gene. Cotransfection of the hsa-miR155 precursor with the luciferase reporter significantly repressed luciferase activity compared to a nontargeting control. To identify the binding site and to further demonstrate the specificity of the interaction, we generated luciferase reporter constructs with mutations in the M1 and M2 binding sites. Mutagenesis of M2 but not M1 site reversed the inhibitory effect of miR155, thus establishing M2 as the binding site. Since HGAL inhibits motility and chemotaxis of B lymphocytes and B cell lymphoma cells, we next examined effect of mir155 on SDF1 and IL-6-induced chemotaxis and spontaneous motility of VAL and Raji cells. Decrease in HGAL protein levels by hsa-miR155 precursor led to increased cell chemotaxis, cell motility, and cell velocity and was associated with decreased actin polymerization (F-actin content). The-miR155 effect on chemotaxis was rescued by transfection of HGAL-encoding vector. Since HGAL exerts its motility inhibitory effects by activation of RhoA, we next evaluated effect of miR155 on levels of activated GTP-bound RhoA. Overexpression of miR155 resulted in decreased levels of GTP-bound RhoA in both Raji and Val cell lines. Our findings demonstrate that miR155 may not only lead to oncogenic transformation of B lymphocytes, but also increases lymphoma cell motility by down regulation of HGAL expression. This effect may contribute to lymphoma cell dissemination and aggressiveness, characteristic of ABC-like DLBCL typically expressing high levels of miR155 and lacking HGAL expression. Disclosures:No relevant conflicts of interest to declare.