Expression In Diffuse Large B-cell Lymphoma Research Articles

CXCL-8/IL8 Produced by Diffuse Large B-cell Lymphomas Recruits Neutrophils Expressing a Proliferation-Inducing Ligand APRIL.

Tumor-infiltrating neutrophils have been implicated in malignant development and progression, but mechanisms are ill defined. Neutrophils produce a proliferation-inducing ligand APRIL/TNFSF13, a factor that promotes development of tumors from diverse origins, including diffuse large B-cell lymphoma (DLBCL). High APRIL expression in DLBCL correlates with reduced patient survival, but the pathway(s) dictating APRIL expression are not known. Here, we show that all blood neutrophils constitutively secrete APRIL, and inflammation-associated stimuli, such as TNF, further upregulate APRIL. In a significant fraction of DLBCL patients, tumor cells constitutively produced the ELC-CXC chemokine CXCL-8 (IL8), enabling them to recruit APRIL-producing blood neutrophils. CXCL-8 production in DLBCL was unrelated to the cell of origin, as APRIL-producing neutrophils infiltrated CXCL-8+ DLBCL from both germinal center (GC) and non-GC subtypes. Rather, CXCL-8 production implied events affecting DNA methylation and acetylation. Overall, our results showed that chemokine-mediated recruitment of neutrophils secreting the tumor-promoting factor APRIL mediates DLBCL progression. Cancer Res; 77(5); 1097-107. ©2016 AACR.

AICDA Introduces Epigenetic Plasticity in Germinal Center-Derived Lymphomas and Accelerates Lymphomagenesis

Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center B cells (GC) B cells. Previous work from our group established that inferior outcome in DLBCL is associated with higher degrees of intra-tumor and inter-tumor cytosine methylation heterogeneity, although the molecules driving this epigenetic perturbation are unknown. We investigated the contribution of activation-induced cytidine deaminase (AICDA) to cytosine methylation heterogeneity in DLBCLs. AICDA is highly expressed in GC B cells where it drives somatic hypermutation (SHM) of immunoglobulin (Ig) and non-Ig genes. AICDA is also expressed in DLBCLs and high level of AICDA in CHOP-treated DLBCL patients is associated with unfavorable prognosis. In addition to mutagenesis, AICDA can cause DNA demethylation through the removal of modified cytosine residues. Indeed, we previously reported that GC B cells manifest a marked AICDA-mediated DNA hypomethylation signature as well as a high degree of epigenetic heterogeneity as compared to other B-cell subsets. Thus, we hypothesized that AICDA could contribute to the progression of GC-derived lymphomas by facilitating epigenetic plasticity through the redistribution of cytosine methylation. We overexpressed AICDA in VavP-Bcl2 transgenic mice -which develop B cell lymphomas of GC origin- using an AICDA-expressing retroviral vector (VavP-Bcl2+Aicda) vs empty vector (VavP-Bcl2) as control and transplanted them into lethally irradiated recipient mice. We observed more aggressive lymphomas based on greater disruption of the splenic architecture and higher degree of B cell infiltration in organs such as lung, liver and kidney in VavP-Bcl2+Aicda mice (n=7) compared to VavP-Bcl2 mice (n=6). Notably, the overexpression of AICDA reduced significantly the lifespan of the mice (Log-rank test p=0.0289) with a median survival of 214 days for VavP-Bcl2+Aicda mice (n=10) and 293 days for VavP-Bcl2 animals (n=9). Neoplastic B cells from VavP-Bcl2+Aicda and VavP-Bcl2 micedisplayed similar SHM rate in Ig genes (JH4 and Sm) and AICDA off-targets (Bcl6, Cd83 and Pax5), suggesting that the more aggressive phenotype is not likely due to increased mutagenesis. We then profiled the DNA methylation landscape of these lymphomas using enhanced reduced representation bisulfite sequencing. We analyzed the DNA methylation level and heterogeneity of all represented CpGs by calculating the mean methylation difference and interquartile range (IQR) between VavP-Bcl2+Aicda and VavP-Bcl2 tumors. A principal component analysis of all CpGs, each one represented by its mean methylation and IQR differences across replicates, revealed methylation loss and increased intertumor heterogeneity within VavP-Bcl2+Aicda methylomes compared to VavP-Bcl2. AICDA-perturbed CpGs (n=49,750) in particular showed significant intratumor heterogeneity (p<1e-300) in AICDA overexpressing tumors. These altered CpGs were depleted in promoters and enriched in introns and intergenic regions. We observed a remarkably similar pattern of hypomethylation and increased methylation heterogeneity in Aicda+/+ GC B cells compared to Aicda-/- GC B cells (AICDA-perturbed CpGs n=64,323) and also in primary DLBCLs with high AID compared to low AID expression (AICDA-perturbed CpGs n=37,557), suggesting a conserved epigenetic function of AICDA in GC B cells and human and mouse GC-derived lymphomas. We performed RNA sequencing on BCL2-driven lymphomas and found that expressed genes containing AICDA-perturbed CpGs displayed significantly lower expression in VavP-Bcl2+Aicda compared to VavP-Bcl2 tumors (gene set enrichment analysis (GSEA) NES=-1.68, FDR<0.001). Pathway analysis revealed that these AICDA-epigenetic target genes were associated with MAPK- and KMT2D-signaling pathways, which are related to B cell activation and lymphomagenesis. Similarly to mouse tumors, genes enriched for AICDA-perturbed CpG in human lymphomas exhibited lower expression in AID high DLBCLs compared to AICDA low DLBCLs (GSEA NES=-1.65, FDR<0.001) and were associated with MAPK and KMT2D pathways. These results demonstrate that AICDA acts as a methylome modifier in GC-derived lymphomas, introducing epigenetic plasticity and leading to gene expression changes, thus contributing to clonal evolution and higher adaptability to an evolving environment. DisclosuresMelnick:Janssen: Research Funding.

CD30 Expression in Diffuse Large B-Cell Lymphoma (DLBCL) Correlates with Non-GCB Subtype but Does Not Have Prognostic Impact in Patients Treated with First Line R-CHOP/R-CHOP-like

Background: DLBCL is a heterogeneous disease with 30% of patients presenting as refractory/relapsing disease following R-CHOP treatment. Therefore, new prognostic factors to identify the poor-risk population and alternative therapies for them are needed. CD30 is a member of the tumour necrosis factor receptor (TNFR) super family and is expressed by several types of T- and B-cell non-Hodgkin lymphomas. Brentuximab vedotin, an anti CD30 antibody, is used in clinical practice for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. The significance of CD30 expression in DLBCL remains unknown. We aimed to study the expression of CD30 in a large cohort of the novo DLBCL patients homogeneously treated with R-CHOP or R-CHOP-like protocols to evaluate its correlation with clinical and biological characteristics at diagnosis and its impact in clinical outcome.Methods: we prospectively registered 155 patients diagnosed of DLBCL between January 2012 to December 2015 in our center. Twenty-one patients with primary central nervous system DLBCL, transformed lymphoma or post-transplant DLBCL were excluded from the study. Anatomopathology review was performed in all cases, and 14 cases with not enough histological tissue to perform additional staining were also excluded. Immunohistochemistry was performed in 120 cases on 4 microns' paraffin sections using routine protocols,CD30 expression was analyzed using the antibodyCD30 clone BerH2, dilution 1:30; Dako; staining in more than 10% of the malignant cells was considered positive. Samples were classified as germinal center B-cell-like (GCB) vs. non-GCB subtypes by immunohistochemistry according to Hans algorithm. Univariate analyses were assessed by Chi square test. Survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Multivariate analyses for progression-free survival (PFS) were assessed Cox proportional hazard regression model.Results: among the 120 cases analyzed, CD30 was expressed in 40 (33.3%). Clinical and biological characteristics at disease presentation are shown in Table 1. The expression of CD30 was significantly higher in the non-GCB subtype cases (p=0.0001). One hundred and six patients were homogeneously treated with R-CHOP (100 patients) or R-CHOP-like (1 R-COMP, 2 Burkitt scheme, 1 GA101-CHOP and 2 R-Bortezomib-CAP), and were included in the outcome analyses. Response rates and survival of this subgroup of patients are shown in Table 2. With a median follow-up of 16.4 months, no differences were found neither in PFS or OS between CD30 positive and CD30 negative patients (Table 2). In the multivariate analyses including the following independent variables: gender, B-symptoms, Bulky mass (>5cm), cell of origin (GCB vs non-GCB), International Prognostic Index (IPI) and CD30 expression, IPI 3-5 was de only factor significantly related with poor PFS (HR 2.92, 95% CI:1.18-7.24, p=0.02).Conclusion: CD30 is expressed in one third of DLBCL patients, in whom an anti-CD30 therapy could be used. CD30 expression is significantly higher in patients with non-CGB DLBCL, however and in our experience, its expression does not influence either response to R-CHOP or R-CHOP- like therapy or survival. [Display omitted] [Display omitted] DisclosuresGonzalez Barca:Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau.

The Adhesion Molecule ICAM-1 in Diffuse Large B-Cell Lymphoma Post-Rituximab Era: Relationship with Prognostic Importance and Rituximab Resistance

Background: Intercellular adhesion molecule-1 (ICAM-1) is a cell-surface receptor involving in cell-to-cell adhesion and as well as co-stimulatory molecule involved in T-cell lymphocyte infiltration and activation. In the pre-rituximab era, high level of ICAM-1 was reported to be a prognostic indicator of better clinical outcomes in diffuse large B-cell lymphoma (DLBCL). The value of ICAM-1 expression in the post-rituximab era remains unclear. To investigate the impact of ICAM-1 expression in DLBCL, we conducted a retrospective study in R+CHOP treated patients.Methods: Patients (N=102) with histologically proven DLBCL treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. Forty-three patients were treated with CHOP and 59 patients with R+CHOP. ICAM-1 expression was determined by immunohistochemistry (IHC). ICAM-1 expression was quantified by staining intensity or percentage of positive cells. Tumors were considered positive when at least 75% of tumor cells expressed ICAM-1. Progression free survival (PFS) and overall survival (OS) was plotted by Kaplan-Meier method and curves were compared with the long-rank test in both groups. Correlation between the ICAM-1 expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of <0.05 was considered to be statistically significant. Finally, ICAM-1 expression was determined in a panel of rituximab-sensitive (RSCL) and rituximab-resistant lymphoma cell lines (RRCL) by western blot. Correlation of ICAM-1 expression and rituximab anti-tumor activity was determined by standard complement-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC) assays.Result: ICAM-1 expressionwas detected in 28 (27.5%) DLBCL patients. The rest of the patients tested negative for ICAM-1 (N=74; 72.5%). Differences in ICAM-1 expression were observed according to cell of origin DLBCL subtype. ICAM-1 expression was higher among germinal center B-cell (GCB) DLBCL (41.4%) vs. non-GCB subtype patients (19.6%, P=0.019). The response rates for R-CHOP and CHOP were, respectively, 89.4% and 77.7% (P=0.409) in ICAM-1 positive patients and 92.5% and 73.5% (p=0.027) in ICAM-1 negative patients. As previously described, ICAM-1 expression was associated with an improved PFS/OS (P=0.03/ P=0.05) in CHOP-treated DLBCL. On the other hand, no differences in PFS/OS were observed between ICAM-1 positive or negative DLBCL patients treated with R+CHOP. The addition of rituximab to CHOP chemotherapy resulted in an improved PFS in ICAM-1 negative DLBCL (P=0.018). On the other hand, the clinical outcome of ICAM-1 positive DLBCL patients treated with CHOP chemotherapy was similar than R+CHOP treated patients. Finally, loss of ICAM-1 expression level was found in our rituximab resistant cell lines, along with reduction of rituximab activity.Conclusion: Before therituximab era, ICAM-1 low expression correlated with worse PFS and OS. In our retrospective clinical analysis, we found that rituximab significantly improved the overall response rate and PFS in ICAM-1 negative subset patients. Our data also indicated that ICAM-1 expression level was higher in GCB than non-GCB subtypes, and this may contribute to better rituximab response in GCB cell types. Gradually exposed lymphoma cell to rituximab lost ICAM-1 expression and may contribute to the resistance to rituximab and chemotherapy agents. Further investigation of ICAM-1 expression needed to be study to enhance the rituximab therapy in lymphoma. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of OCT4 and SALL4 in Diffuse Large B-cell Lymphoma: An Analysis of 145 Consecutive Cases and Testicular Lymphomas.

OCT4 and SALL4 are transcription factors within a complex network that functions to maintain pluripotency in primitive stem cells and germ cells. Nuclear expression of OCT4 is widely cited as sensitive and specific for primary and metastatic germ cell tumors and is commonly used in the diagnosis of central nervous system (CNS) germinomas. Studies have failed to systematically examine the expression of OCT4 or SALL4 in diffuse large B-cell lymphoma (DLBCL), although this entity enters the morphologic differential diagnosis of some germ cell tumors. A retrospective review was conducted on 145 consecutive cases of DLBCL and testicular lymphoma to evaluate the prevalence of OCT4 and SALL4 expression. Nuclear OCT4 expression was present in 2/11 (18%) testicular DLBCLs and 6/134 (4.5%) nontesticular DLBCLs. Most OCT4 cases demonstrated moderate to strong expression in >50% of neoplastic cells. Rare, weak nuclear SALL4 expression was detected in only 3 nontesticular DLBCLs. Within the extratesticular DLBCL group, 2/6 (33%) primary CNS DLBCLs expressed nuclear OCT4. In addition, OCT4 DLBCL showed an overall predilection toward non-germinal center B-cell phenotype (7/8; 88%) and had a higher than expected rate of CD5 coexpression (4/8, 50%). These results are cautionary against using OCT4 as a sole marker of germ cell differentiation in testicular and extratesticular sites, especially in the CNS. The apparent associations of OCT4 expression with primary CNS DLBCL, non-germinal center B-cell phenotype, and CD5 coexpression raise the question of whether OCT4 expression in DLBCL may reflect more aggressive biology.

Prognostic Implication of Semi-quantitative Immunohistochemical Assessment of CD20 Expression in Diffuse Large B-Cell Lymphoma.

Background:Immunohistochemical demonstration of CD20 in diffuse large B-cell lymphoma (DLBCL) is prerequisite not only for the diagnosis but also for assigning patients to rituximab-containing chemotherapy. However, little is known about the impact of abundance of CD20 expression assessed by immunohistochemistry on the clinical outcome of DLBCL. We performed a semi-quantitative immunohistochemical analysis of CD20 expression in DLBCL to examine the prognostic implication of the level of CD20 expression.Methods:Pre-treatment diagnostic tissue samples from 48 DLBCL patients who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen were represented in a tissue microarray and immunostained for CD20. The relative abundance of CD20 expression was semi-quantitatively scored using a web-based ImmunoMembrane plug-in. Receiver operating characteristic curve analysis was used to determine a prognostically relevant cut-off score in order to dichotomize the patients into CD20-high versus CD20-low groups.Results:The levels of CD20 expression were heterogeneous among the patients, with a wide and linear distribution of scores. Patients in CD20-low group showed significantly poor clinical outcome.Conclusions:The levels of CD20 expression in DLBCL are heterogeneous among the patients with DLBCL. A subgroup of the patients with CD20 expression levels below the cut-off score showed poor clinical outcome.

CD4-positive diffuse large B-cell lymphoma: A variant with aggressive clinical potential.

CD4 expression is rare in diffuse large B-cell lymphoma (DLBCL), with 4 previously reported cases. Its significance is uncertain. We report five patients with CD4+ DLBCL and one CD4+ primary mediastinal large B-cell lymphoma. Cases were identified by searching the electronic database of the department; each was reviewed. Average age was 56 years. Neoplastic cells expressed CD20 (5/6 tested cases). BCL2/BCL6 expression were seen in 3/3 tested cases, suggesting a germinal center origin. Additionally, expression of T-cell antigens CD2 and CD5 was noted in 2/2 and CD7 in 1/1 tested case. CD3 was negative in all. Lymph nodes were commonly involved (67%). Patients received chemotherapy +/- radiation (6/6) and bone marrow transplant (2/6). Average survival was 44.2 mo. CD4 expression in DLBCL raises questions of lineage commitment. CD4+ DLBCL is rare; care should be exercised not to diagnose these as T-cell lymphomas. A subset behaves aggressively.

Expression and prognostic value of CARD11 in diffuse large B cell lymphoma

目的了解CARD11在弥漫大B细胞淋巴瘤（DLBCL）中的表达情况及其与预后的关系。方法收集经福尔马林固定、石蜡包埋的DLBCL患者治疗前淋巴结活检病理标本及临床资料，对标本进行组织芯片制备，并采用EnVision两步法对CARD11进行免疫组化染色，同时利用Hans法（CD10、BCL6、MUM1）对DLBCL标本进行分型。分析CARD11表达与患者特征及预后的关系。结果共纳入79例初治DLBCL患者，采用2例反应性增生患者的淋巴结标本作为对照。对77例患者进行生存分析，75例患者可判定CARD11表达结果。在DLBCL组织的肿瘤细胞中，CARD11表达阳性率为65.33%（49/75）。CARD11与患者特征均无显著相关性。CARD11阳性组2年无事件生存（EFS）率低于阴性组（52.03％对86.12%，P=0.036）。在国际预后指数3~5分的患者中CARD11阴性组与阳性组的中位EFS率差异有统计学意义（未达到对18.7个月，P=0.033）。CARD11不是影响预后的独立危险因素。结论在DLBCL中，CARD11阳性患者EFS时间显著短于CARD11阴性患者，在国际预后指数高中危和高危的患者中，这种差异依然存在，提示CARD11有助于对高危DLBCL患者进一步进行预后分层。

FOXO1-p300-Txn Circuit Regulates Oxidative Stress Responses in Diffuse Large B-Cell Lymphomas Characterized By Enhanced Oxidative Phosphorylation

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. The comparison of DLBCLs transcriptional profiles using multiple clustering algorithms led to the identification of distinct DLBCL subtypes reflecting tumor-intrinsic features. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation, a major source of potentially toxic reactive oxygen species (ROS). Therefore, we investigated the role of potential mechanisms attenuating ROS toxicity in this DLBCL subtype.We found significantly increased thioredoxin (TXN) mRNA abundance in DLBCLs classified as OxPhos subtype compared to other subtypes. The overall survival (OS) of patients with high TXN mRNA expression was significantly shorter than of those with low TXN mRNA expression, regardless of treatment regimen (R-CHOP and CHOP). TXN overexpression in OxPhos-DLBCLs was also confirmed in a cell line panel at protein level using immunoblotting. To explain transcriptional mechanisms responsible for differential TXN expression in different DLBCLs, we analyzed the TXN promoter and identified two BCL6 binding sites within 1kb upstream of TXN transcription start site. Unlike in other molecular subtypes, BCL6 does not exhibit a repressor activity in OxPhos-DLBCLs (PNAS, 2007; 104: 3207-12). Using luciferase reporter assays and shRNA-mediated gene expression knock-down, we demonstrated that relative differences in TXN abundance between DLBCL subtypes are at least in part caused by the lack of BCL6 transcription repressor activity.We next tested the consequences of TXN depletion in DLBCLs. We found that OxPhos cells with silenced TXN expression were uniformly more sensitive to apoptosis induced by ROS production than control cells. TXN inhibition sensitized all tested cell lines to doxorubicin, the fundamental drug used in DLBCL chemotherapy acting in part as a ROS inducer.In addition to its role in maintaining redox homeostasis, TXN also regulates transcriptional responses to ROS. For example, TXN reduces disulfide bonds between FOXO4 and acetyltransferase p300, which results in reduced FOXO4 acetylation and impaired proapoptotic signaling. For this reason, we assessed whether p300 and TXN are involved in acetylation of FOXO1, a major FOXO member expressed in DLBCLs. In cells with blocked TXN activity, FOXO1 acetylation was significantly higher compared to cells overexpressing wild-type TXN. TXN decreased p300-mediated FOXO1 acetylation, reduced its proapoptotic activity and expression of FOXO1-dependent genes (TRAIL, p27, BIM). We found that FOXO1 and p300 interact in redox and TXN-dependent manner and identified a conserved FOXO1's Cys612 to be responsible for FOXO1-p300 binding. We mutated FOXO1 C612 to Ala and found that when cotransfected with p300, C612A FOXO1 exhibited dramatically suppressed ROS-induced acetylation. Blockade of FOXO1 acetylation resulted in markedly lower expression of FOXO1 target genes, higher cellular proliferation and lower apoptosis. Furthermore, TXN inhibited FOXO1 nuclear translocation in response to oxidative stress in OxPhos-DLBCLs. Finally, silencing FOXO1 in OxPhos cells with knocked-down TXN expression markedly inhibited DLBCL cell line apoptosis in response to oxidative stress, suggesting that FOXO1 is an essential TXN-regulated sensor and effector of ROS toxicity in OxPhos-DLBCL cells.Taken together, these results demonstrate that TXN is overexpressed in a subset of DLBCLs, and high TXN mRNA abundance is related to shorter OS of DLBCL patients. TXN knock-down enhances oxidative stress toxicity in OxPhos cell lines at least in part by facilitating FOXO1 nuclear retention and increasing acetylation of this transcription factor, thus augmenting its proapoptotic activity. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome Sequencing Reveals Thousands of Novel Long Non-Coding RNAs in B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of lymphoma. Roughly 40% of patients do not respond or relapse after current therapy. Gene expression profiling of DLBCL has revealed broad gene expression deregulation compared to normal B Cells and categorized DLBCLs into two main subtypes: germinal center B-cell like and activated B-cell like (GCB-DLBCL and ABC-DLBCL), both associated with distinct clinical outcomes. Recent RNAseq studies have demonstrated that large portion of the genome consists of long non-coding transcripts (lncRNAs) that exhibit cell type specific expression patterns and are shown to play critical roles in disease specific epigenetic gene regulation. We sought to discover and characterize novel lncRNAs expressed in DLBCL that are responsible for the disease phenotypes. We performed a systematic de novo lncRNA discovery analysis on RNAseq data obtained from 116 primary DLBCL tumors (dbGaP, phs000235.v6.p1), and 8 normal B-cell counterparts (4 Naïve B-cell and 4 Germinal Center B-cell samples). We identified 2,632 novel expressed lncRNAs in DLBCL. These novel lncRNAs exhibit characteristics similar to known lncRNAs, such as low coding potential, > 200 nt in length, and exon counts (>=2 exons). Further comparison between tumors and normal B-cells revealed that 942 lncRNAs were expressed in DLBCL only. We noticed that many of these lncRNAs were located nearby coding-genes that are important in B-cell biology, such as BCL6, IKZF1, IL10, IRF4, PRDM1, TNFAIP3, etc. Indeed, using a computational tool GREAT, we found that the coding-genes located nearby DLBCL-specific lncRNAs were significantly enriched in immune cell activation and differentiation pathways (FDR < 0.05), suggesting that novel DLBCL lncRNAs are preferentially located near key B-cell and lymphoma genes perhaps contributing to the expression regulation of these genes. Furthermore, through a systematic approach, we found that 88% of these lncRNAs correlated significantly with at least one coding gene expression (Spearman correlation, FDR < 0.2), and collectively these co-expressed coding genes were enriched in B-cell specific functional pathways, including CD40-regulated pathways. Interestingly, we observed that 81 lncRNA were located within 17 DLBCL locus control regions (LCRs or super enhancers), representing a significant enrichment of lncRNAs at LCRs (p < 0.001). This suggests that super-enhancers may coordinate the co-expression of lncRNAs and their neighborhood coding-genes. Moreover, through supervised analysis, we derived a lncRNA expression signature that can differentiate ABC and GCB subtypes of DLBCL. There were 465 lncRNAs significantly differentially expressed between ABC and GCB DLBCLs (FDR < 0.05). When we performed pathway analysis on the protein coding genes significantly co-expressed with these 465 subtype-specific lncRNAs, we found they were enriched in signatures that can define the two subtypes, including Germinal_center_B-cell_DLBCL and ABC_gt_GCB_Affy signatures.To understand the regulation of lncRNA expression in DLBCL, we examined the histone mark profiles at lncRNA loci. First, we found in any given DLBCL cell line, there was a significant overlap of lncRNA promoters and H3K4me3 peaks (p < 2.2e-16), suggesting like coding genes, lncRNA promoters are marked with H3K4me3. Second, we identified that expression of 323 lncRNAs were negatively correlated with BCL6 expression (FDR < 0.2) in this cohort of DLBCL patients, among which 104 exhibited increased expression upon BCL6 knocking down. BCL6 is a transcription repressor and a master regulator of germinal center transcription programming. Taken together, our data suggest that expression of novel DLBCL lncRNAs is potentially under the control of B-cell specific transcription factors.Finally, we performed single nucleotide variant analysis on these novel lncRNAs by using VarScan. We identified 9,713 SNVs within the exonic regions of these lncRNAs. We calculated the Minimum Free Energy Scores (MFEs) by using RNAfold to examine whether these SNVs would have an impact on the stability of the lncRNA secondary structure. We observed a significantly higher than background shift towards the stabilization of the secondary structure with the SNVs (p < 2.2e-16, Wilcoxon test), indicating a potential genetic mechanism of aberrant expression of lncRNA in DLBCL. DisclosuresMelnick:ROCHE: Other: Research; Genentech: Speakers Bureau; Janssen: Other: Research; Celgene: Consultancy; Eli Lilly: Consultancy; Epizyme: Consultancy.

Identification of Blut, a Novel Long-Noncoding RNA Differentially Expressed in Burkitt Lymphoma

The class of the so-called long non-coding RNAs (lncRNAs) is defined by their lacking coding potential and their length of more than 200 nucleotides. LncRNAs are readily detected in transcriptome analysis but have so far not been characterized or catalogued exhaustively. Although lncRNAs make up a major portion of the mammalian non-coding transcriptome, it is challenging to predict their functions due to their diversity in terms of size, sequence and biogenesis. However, several lncRNAs have already been shown to be involved in crucial cellular processes such as imprinting, regulation of cell proliferation and cellular differentiation. Their mode of action generally involves epigenetic modifications as well as transcriptional and post-transcriptional regulation.Within the ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium, which aims at fully characterizing a total of 250 germinal center derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), we conducted a search for yet unidentified genes.We here report the identification of a novel transcript, which we have named BLUT (Burkitt Lymphoma Unknown Transcript). BLUT is highly expressed in BL, with only very low expression levels in FL and no detectable expression in DLBCL. It is around 1050 nucleotides long with splice evidence for three exons, whereby exons 1 and 3 are shared between all isoforms.BLUT represents a putative lncRNA since it lacks conserved protein domains and has a splice site conservation pattern typical of non-coding transcripts. Furthermore, it is predicted as non-coding by the software Coding Potential Calculator and there was not a single peptide match between a published peptide database (Human Proteome Map) and any potential BLUT peptide (all isoforms). Moreover, BLUT shares a high sequence identity with two (so far) uncharacterized non-coding RNAs (Pan Troglodyte, Chlorocebus sabeus).We tested a cell line panel (consisting of 16 different cell lines including non-lymphoma cell lines) for BLUT expression but could only detect it in the two BL cell lines Namalwa and Raji.Cellular fractionation experiments showed that BLUT is predominantly localized in the nucleus in both Namalwa and Raji.Overexpression of BLUT in SU-DHL-4 resulted in the identification of 222 differentially regulated genes (FDR < 0.05) with a logFC of ± 0.25, among which 49 genes had a logFC of ± 0.50. Several lymphoma relevant genes were part of this list including BIN1, CCL22, CCR7, EBI3, ID2, JAG1, NQO1, and RGS1.Given its nuclear localization, we currently perform chromatin isolation by RNA purification experiments to identify the genomic regions which BLUT binds to. In combination with our overexpression data this will allow further insights into the functions of BLUT.In summary, we have identified a novel long-noncoding RNA termed BLUT, which is differentially expressed between lymphoma subtypes with a high expression in Burkitt lymphoma. Overexpression of BLUT resulted in the deregulation of several genes with known roles in lymphomagenesis. Given its predominantly nuclear localization, we are currently investigating its functions on chromatin regulation.(Supported by BMBF through 01KU1002A-J and by the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Center Duesseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf)) DisclosuresTruemper:Amgen, roche, Mundipharma: Research Funding; Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board.

Prognostic Significance of EBV Association in Diffuse Large B-Cell Lymphoma in the Rituximab Era

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases in terms of morphology, immunohistochemistry and molecular features. The prognostic factors of DLBCL in the rituximab era are different from previously reported prognostic factors. Hans, et al. reported that patients with non-germinal center B-cell like (GCB)-type DLBCL had a significantly poorer prognosis than those with GCB-type DLBCL in the pre-rituximab era. In patients who received rituximab combination chemotherapy, there is a report that did not find a difference in survival ratio between those with GCB DLBCL or non-GCB DLBCL. On the other hand, there was a report that found a difference in survival ratio between patients with GCB DLBCL and those with non-GCB DLBCL. In addition, the previously reported that patients with Epstein-Barr virus (EBV)-positive DLBCL had a significantly poorer prognosis than those with EBV-negative DLBCL in the pre-rituximab era. In the present study, we considered the prognostic factors of DLBCL patients who received rituximab combined chemotherapy.Patients and Methods: The subjects were 209 DLBCL patients in whom immunohistochemical markers (CD20, CD5, CD10, BCL-2, BCL-6, MUM-1) could be analyzed. In addition, 86 patients were examined EBV-encoded RNA (EBER) in situ hybridization. Pathologic evaluation of the materials from each patient was performedat several central review meetings by six hematopathologists in the ALTSG pathology review board. Patients were treated with cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, and prednisolone (CyclOBEAP) regimen or CHOP regimen. Rituximab was administered to all patients. One hundred forty-six DLBCL patients who underwent CHOP-like therapy were assumed as historical controls. The median follow-up period was 69 months (range, 46-88months).Results: In the R-chemotherapy and CHOP-like groups, patients with stage III or IV disease comprised 57% and 67%, respectively, patients with performance status≧2 comprised 32% and 23%, respectively, and patients with serum LDH >normal comprised 61% and 69%, respectively. There were no significant differences in clinical characteristics between the R-chemotherapy group and CHOP-like group. BCL2 was positive in 72 (55%) of the 131 patients who received the R-chemotherapy and in 71 (50%) of the 142 patients who received the CHOP-like regimen. When the 209 patients who received the R- chemotherapy group were divided into the GCB group and the non-GCB group, the GCB group consisted of 74 patients (35%) and the non-GCB group consisted of 135 patients. The relationships between immunohistological markers and outcome among the patients with DLBCL who received CHOP-like therapy were studied. CD5, CD10, and BCL6 had no prognostic impact on 5-year overall survival (OS) and progression-free survival (PFS) rates. When the patients were divided into the GCB DLBCL and non-GCB DLBCL groups, the 5-year PFS of the GCB group was 78% and that of the non-GCB group was 48% (p=0.0008). Next, the effect of rituximab with chemotherapy was examined. CD5, CD10, BCL2, BCL6, and MUM-1 had no prognostic impact on 5-year OS and PFS rates. When the patients were divided into the GCB DLBCL (n=74) and non-GCB DLBCL (n=135) groups, the 4-year PFS of the GCB group was 79% and that of the non-GCB group was 73%, showing no significant difference.We also evaluated the significance of EBER expression among patients by incorporating the EBV-positive DLBCL (n=14) and EBV-negative DLBCL (n=72).There were no significant differences in immunophenotyping analysis between the EBV-positive DLBCL and EBV-negative DLBCL. As for EBER expression in DLBCL, the 4-year PFS of the EBER-positive group was 37% and that of the EBER-negative group was 75% (P=0.007), indicating that the EBER-positive group showed significantly poorer prognosis. The 4-year OS of the EBER-positive group was 50% and the EBER-negative group was 86% (P=0.0005).Conclusions: The EBER may be an important prognostic factor in patients with DLBCL who underwent R-chemotherapy therapy. DisclosuresNo relevant conflicts of interest to declare.

MiR-31 and miR-17-5p levels change during transformation of follicular lymphoma

The 30% of patients whose indolent follicular lymphoma transforms to aggressive diffuse large B-cell lymphoma (DLBCL) have poor survival. Reliable predictors of follicular B-cell lymphoma transformation to DLBCL are lacking, and diagnosis of those that will progress is challenging. MicroRNA, which regulates gene expression, has critical functions in the growth and progression of many cancers and contributes to the pathogenesis of lymphoma. Using 5 paired samples from patients who presented with follicular lymphoma and progressed to DLBCL, we identified specific microRNA differentially expressed between the two. Specifically, miR-17-5p levels were low in follicular lymphoma and increased as the disease transformed. In contrast, miR-31 expression was high in follicular lymphoma and decreased as the lymphoma progressed. These results were confirmed in additional unpaired cases of low-grade follicular lymphoma (n = 13) and high-grade follicular lymphoma grade 3 or DLBCL (n = 17). Loss of miR-31 expression in DLBCL was not due to deletion of the locus. Changes in miR-17-5p and miR-31 were not correlated with immunophenotype, genetics, or status of the MYC oncogene. However, increased miR-17-5p expression did significantly correlate with increased expression of p53 protein, which is indicative of mutant TP53. Two pro-proliferative genes, E2F2 and PI3KC2A, were identified as direct messenger RNA targets of miR-31, suggesting that these may contribute to follicular lymphoma transformation. Our results indicate that changes in miR-31 and miR-17-5p reflect the transformation of follicular lymphoma to an aggressive large B-cell lymphoma and may, along with their targets, be viable markers for this process.

LincRNA-p21 predicts favorable clinical outcome and impairs tumorigenesis in diffuse large B cell lymphoma patients treated with R-CHOP chemotherapy.

Despite improved survival for the patients with diffuse large B cell lymphoma (DLBCL), the prognosis after relapse is poor. LincRNA-p21 is a long intergenic noncoding RNA, which is located on chromosome 17, approximately 15kb upstream from the Cdkn1a (p21) gene. However, its clinical importance and biological role in DLBCL prognosis are unknown. In this study, we conducted quantitative reverse-transcription polymerase chain reaction to investigate the lincRNA-p21 expression in DLBCL. We found that lincRNA-p21 levels were markedly decreased in DLBCL tissues compared with normal. Its expression level was significantly correlated with Ann Arbor stages, B symptoms, performance status, IPI score and serum LDH. Moreover, patients with high levels of LincRNA-p21 expression had a favorable overall survival and progression-free survival. Furthermore, ectopic expression of lincRNA-p21 inhibited cell proliferation, arrested cycle progression and modulated cyclin D1, CDK4 and p21 expression in DLBCL cell lines. These results demonstrated lincRNA-p21 can be identified as a potential novel prognostic biomarker for prognosis in DLBCL and regulate cell proliferation and cycle in vitro. Our findings highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL.

Prognostic and Clinicopathological Value of Survivin in Diffuse Large B-cell Lymphoma

Up to date, survivin, a well-known inhibitor of apoptosis, has attracted considerable attention as a potential biomarker and therapeutic target in diffuse large B-cell lymphoma (DLBCL). Nevertheless, there still remains no consensus on heterogeneous results. Herein, a meta-analysis was performed to clarify a convincing significance of survivin status on prognosis and clinicopathology of DLBCL patients.Eligible studies were identified by searching Medline, Embase, Scopus, CNKI, and Wanfang databases (last updated on November 30, 2014). Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Heterogeneity and sensitivity were also analyzed. Moreover, Begg, Egger test, and funnel plots were applied to evaluate the publication bias.We finally included 17 eligible studies with the total number of 1352 patients in the meta-analysis. The pooled results showed that positive survivin expression in DLBCL was associated with inferior overall survival (OS) (HR: 1.880, 95% CI: 1.550-2.270) in patients. Moreover, a significant association was revealed between survivin expression and advanced clinical stage (III + IV) (OR: 0.611, 95% CI: 0.452-0.827), higher International Prognosis Index (IPI) score (Score 3-5) (OR: 0.559; 95% CI: 0.410-0.761), elevated serum lactic dehydrogenase (LDH) (OR: 0.607, 95% CI: 0.444-0.831), presence of bone marrow involvement (OR: 2.127, 95% CI: 1.154-3.921) together with reduced complete remission (CR) rate (OR: 0.478, 95% CI: 0.345-0.662).The results suggest that survivin could be a useful prognostic biomarker, and a promising target for DLBCL therapeutic intervention. Considering limited HR data adjusted for standard prognostic variables could be retrieved, future high-quality studies will be needed in evaluating the independent prognostic value of survivin expression in DLBCL.

Loss of HLA-DR expression is related to tumor microenvironment and predicts adverse outcome in diffuse large B-cell lymphoma

The interaction between tumor cells and the tumor microenvironment is essential in the development and progression of diffuse large B-cell lymphoma (DLBCL). Loss of human leukocyte antigen DR (HLA-DR) in DLBCL is a robust adverse prognostic marker. We evaluated the immunohitochemical expression of HLA-DR in lymphoma and the biologic implications of the loss of HLA-DR. The loss of HLA-DR correlated with clinical stage (p < 0.05), International Prognosis Index (p < 0.05), soluble interleukin-2 receptor (p < 0.05) and poor outcome in patients with DLBCL, especially among elderly patients. Flow cytometry analysis of the infiltrating T-cells showed that the mean CD4 + CD25 +/CD8 ratio of the infiltrating T-cells was higher in the HLA-DR positive group than in the HLA-DR negative group (p < 0.05). These data suggest that loss of HLA-DR expression in DLBCL decreases the ratio of helper T-cell within the T-cell population in the tumor microenvironment and might contribute to escape from immunosurveillance.

The expression of CD30 based on immunohistochemistry predicts inferior outcome in patients with diffuse large B-cell lymphoma.

The prognostic value of CD30 expression indiffuse large B-cell lymphoma (DLBCL)remains controversial. Herein, we performed this retrospective study to investigate the clinical and prognostic significance of CD30 expression in patients with DLBCL.Among all the 146 patients, the expression of CD30 was observed in 23 cases (15.7%).The DLBCL patients with CD30 expression showed more likely to present B symptoms, bone marrow involvement, non-germinal centre B-cell-like (Non-GCB) DLBCL, BCL-2 and Ki-67overexpression(p<0.05). Patients with CD30 expression showed significantly poor overall and event-free survivalcompared with CD30 negative patients(p = 0.031 and 0.041, respectively), especially those with the high intermediate/high-risk international prognostic index (IPI)(p = 0.001 and 0.007, respectively). The prognostic value of CD30expression retained in DLBCL patients treated with eitherCHOP (cyclophosphamide, doxorubicin, vincristine,prednisone) or R-CHOP(rituximab+CHOP). The multivariate analysisrevealed that the expression of CD30 remained an unfavorable factor for both overall and event-free survival (p = 0.001 and 0.002, respectively).In conclusion, these data suggest that CD30 is expressed predominantly in Non-GCBDLBCL. The expression of CD30 implied poor outcomein DLBCL patientstreated with either CHOP or R-CHOP, especially those with the high intermediate/high-risk IPI, possibly indicating that anti-CD30 monoclonal antibody could be of clinical interest.

CD43 expression in diffuse large B-cell lymphoma, not otherwise specified: CD43 is a marker of adverse prognosis.

CD43 (leukosialin) is a transmembrane glycoprotein expressed in a variety of hematopoietic cells, including B lymphocytes, and a variety of malignancies including lymphoma, leukemia, and solid tumors. CD43 plays an important role in the development of many diseases, and coexpression of CD43 and CD20 on peripheral B cells is a predictive factor of hematopoietic malignancy. Although CD43 is expressed in approximately 25% of diffuse large B-cell lymphomas (DLBCLs), its prognostic significance remains unclear. To analyze CD43 expression in DLBCL, not otherwise specified (DLBCL, NOS), and assess its prognostic value, we analyzed clinical data from 160 patients with DLBCL, NOS. We observed that CD43 expression was detected in 47 (29.4%) of 160 cases. CD43 expression was positively correlated with old age (>60 years), high serum lactate dehydrogenase level, B symptoms, non-germinal center type, and DLBCL, NOS, mortality. Patients with CD43-positive DLBCL, NOS, had poorer overall survival (P < .001, log-rank test) and event-free survival (P < .001, log-rank test) than CD43-negative patients. Univariate analysis showed that CD43 expression, age, sex, Ann Arbor stage, International Prognostic Index category, and germinal center phenotype were prognostic factors for DLBCL, NOS, patient survival. Multivariate analysis showed that CD43 expression was an independent significant prognostic factor for event-free survival (P < .001) and overall survival (P < .001). Based on these data, we conclude that CD43 expression is a novel adverse prognostic factor for patients with DLBCL, NOS.

Expression of LMO3 and SNAP25 in Diffuse Large B-Cell Lymphoma Cells and Its Relation to Clinical Features

Diffuse large B-cell lymphoma (DLBCL) is a clinicopathologically heterogeneous group of lymphomas. Gene expression profiling (GEP) has identified two distinct forms of DLBCL: activated B-cell-like (ABC) type and germinal center B-cell-like (GCB) type. Approximately 10% of DLBCL are positive for CD5, and most of CD5-positive (CD5+) DLBCLs are classified as ABC DLBCL. CD5+ DLBCL is also characterized by aggressive clinical course, poor prognosis, and frequent central nervous system relapse (Yamaguchi M, et al. Blood 2002, Haematologica 2008; Miyazaki K, et al. Ann Oncol 2011).We previously analyzed 90 patients with DLBCL by GEP and identified a signature gene set that enabled categorization of DLBCLs into two groups: CD5+ group and CD5-negative (CD5-) group. Our analysis of samples from 78 patients with ABC DLBCL showed that the CD5+ ABC DLBCL signature gene set comprised many genes which are related to the neuronal system (Miyazaki K, et al. ASH2010, #4164). To clarify the clinical significance of protein expression of these genes not only in CD5+ DLBCL but also in whole DLBCL, we examined the protein expression of the genes in DLBCL cells and analyzed the relationship with clinical features.The study comprised 167 patients with DLBCL who were diagnosed between 1993 and 2010 at Mie University Hospital. Of the 90 patients who were previously analyzed by GEP, 60 patients were included in the present study. The histologic diagnosis of DLBCL was made according to the 2008 WHO classification. Thirty (18%) of 167 patients were diagnosed as having CD5+ DLBCL. We selected six genes which were included in the CD5+ ABC DLBCL signature gene set: LMO3, SNAP25, SYT5, SV2C, NEUROD1, and SYN2. Protein expression of these genes was analyzed by means of immunohistochemistry using frozen sections.We identified LMO3 and SNAP25 expressions in DLBCL cells. The other four proteins were not detected in DLBCL cells of the study. SNAP25 was expressed in the cytoplasm of DLBCL cells, whereas LMO3 was expressed in the nucleus of DLBCL cells. LMO3 was positive in 35% (58/164) and SNAP25 was positive in 45% (74/164) of the patients examined. Thirty-three patients were positive for both antigens. For validation of our GEP data, we separately analyzed the 60 patients who were previously analyzed by GEP, and found that CD5 expression in DLBCL showed significant correlation with LMO3 (68%, P = 0.0002) and SNAP25 (73%, P= 0.032) expression.Analyses of clinical features at diagnosis according to each protein expression revealed that the LMO3+ group was characterized by poor performance status (> 1) (LMO3+ group, 36% vs. LMO3- group, 11%; P= 0.0004). There was no significant difference in clinical features between SNAP25+ and SNAP25- groups.The most prevalent first-line treatment in the 167 patients was CHOP chemotherapy (142 patients). Forty-four patients received rituximab during first-line chemotherapy. Patients with LMO3+ DLBCL and SNAP25+ DLBCL who attained complete response with the initial therapy accounted for 74% (43/58) and 80% (59/74), respectively, and patients with LMO3- DLBCL and SNAP25- DLBCL for 86% (91/106) and 84% (76/90), respectively. At a median follow-up of 84 months, the LMO3+ group also showed a significantly worse OS than the LMO3- group (P = 0.034, 5-year OS; 46% and 64%, respectively). There was no significant difference in OS between SNAP25+ and SNAP- groups. As the two genes were included originally in the CD5+ ABC DLBCL signature gene set, we divided the patients into two groups by CD5 expression and analyzed the OS of each group. Each protein expression had no significant difference in OS in patients with CD5+ DLBCL; however, LMO3 expression had a significant correlation with a shorter OS (P= 0.048) in the CD5- DLBCL group.The present study revealed the expressions of LMO3 and SNAP25 in DLBCL cells and an association between LMO3 expression and prognosis in patients with DLBCL. Further studies on LMO3 in DLBCL are warranted. (Supported in part by the National Cancer Center Research and Development Funds: #21-6-3, #23-A-17, and #26-A-4) DisclosuresNo relevant conflicts of interest to declare.

Upregulation of Cereblon Expression By the DNA Methyltransferase Inhibitor Azacytidine Strongly Enhances Lenalidomide Cytotoxicity in Germinal Center B-Cell-like (GCB) and Activated B-Cell-like (ABC) Diffuse Large B-Cell Lymphoma (DLBCL)

▪INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL.METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment.RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL.CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. DisclosuresNo relevant conflicts of interest to declare.