Expression In Diffuse Large B-cell Lymphoma Research Articles

Unmasking the suppressed immunopeptidome of EZH2-mutated diffuse large B-cell lymphomas through combination drug treatment

AbstractExploring the repertoire of peptides presented on major histocompatibility complexes (MHCs) helps identify targets for immunotherapy in many hematologic malignancies. However, there is a paucity of such data for diffuse large B-cell lymphomas (DLBCLs), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the enhancer of zeste homolog 2 (EZH2)-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon-γ (IFN-γ), restored MHC expression in DLBCL. In DLBCL, peptides presented on MHCs were identified via mass spectrometry after treatment with tazemetostat or decitabine alone or in combination with IFN-γ. Such treatment synergistically increased the expression of MHC class I surface proteins up to 50-fold and the expression of class II surface proteins up to threefold. Peptides presented on MHCs increased to a similar extent for both class I and class II MHCs. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells for 2 of 3 cell lines and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from the regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands that are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFN-γ can expand the repertoire of MHC ligands presented on DLBCLs by revealing suppressed epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets.

Significance of the Expression of pAKT1 and pSyk Activation Proteins in Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) amounts for 30-40 % of all adult non-Hodgkin's lymphomas. After R-CHOP immunochemotherapy 40 % of patients develop early relapsed or therapy-refractory disease. The conventional prognostic parameters in DLBCL are not always effective. Therefore, exploring further predictors of disease course remains an issue. Aim. To assess the prognostic value of pAKT1 and рSy< expression in DLBCL. Materials & Methods. The study enrolled 100 patients with newly diagnosed DLBCL treated with R-CHOP first-line immunochemotherapy. The relative count of pAKT1- and pSyk-expressing tumor cells was determined by immunohistochemical and morphometric methods. The expression cutoff of these proteins was calculated by ROC analysis. The relationship of protein expression with clinical parameters of DLBCL was analyzed by Fisher's exact two-tailed test. The 5-year overall (OS) and progression-free (PFS) survivals were estimated by Kaplan-Meier method (log-rank test). Results. High pAKT1 expression was associated with advanced DLBCL stages, International Prognostic Index > 2, serum lactate dehydrogenase concentration above normal, failures of R-CHOP therapy, as well as worse OS and PFS. No correlation between рSyk< expression and clinical lymphoma characteristics was found. The worst 5-year OS (27.6 %) was reported in cases of pAKT1 and pSyk co-overexpression (hazard ratio [HR] 5.2; 95% confidence interval [95% CI] 2.49-10.9; p < 0.001). A similar trend was observed for PFS (HR = 3.3; 95% CI 1.54-7.30; p = 0.002). Conclusion. Overexpression of pAKT1 is an informative indicator of a poor DLBCL prognosis. Co-overexpression of pAKT1 and рSyk< markers is associated with worse OS and PFS compared to their isolated expressions and other co-expression variants.

Phosphoglycerate Dehydrogenase Is Required for Germinal Center Formation and Is a Therapeutic Target in MYC-driven Lymphoma

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells.We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids.We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL.We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression.Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. DisclosuresGribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

SHP1 Loss Augments DLBCL Cellular Response to Ibrutinib: A Candidate Predictive Biomarker

BackgroundB-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK (Curr. Opin. Immunol.12, 307).Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive NHL. DLBCL demonstrates heterogeneity in clinical presentation, genomic alterations and cell signaling. BCR signature has been identified in a subset of DLBCL (Monti Blood 105, 1851). Chronic active BCR plays a particular role in the subtype of activated B-cell like (ABC) DLBCL by cell-of-origin subclassification. Based on the success of ibrutinib in other B-cell NHL, clinical trials of ibrutinib in DLBCL was conducted and the initial results showed a 37% response rate in ABC-lymphomas vs 5% in GCB-DLBCL (Wilson Nat Med 21, 922). However, a large phase 3 clinical trial of ibrutinib-containing regimen in >800 non-GCB patients did not find any substantial difference in response between those received ibrutinib vs placebo. Thus, biomarker based on cell-of-origin did not seem to help stratify patients as expected (Younes JCO 27, 1285). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib or other BCR-directed therapeutic agents.We hypothesized that SHP1 may play a role in defining the level of the activity of the BCR pathway. Suppressed SHP1 expression in various types of NHL has been reported. We hypothesized that lack of SHP1 may contribute to the hyperactivity of BCR in DLBCL; and tumors with low SHP1 may respond better to ibrutinib. In this work, we set out to explore the relationship between SHP1 & ibrutinib sensitivity in DLBCL.Methods and ResultsWe first reviewed the published literature on SHP1 in NHL using a meta-analysis to provide a quantitative and thus unbiased analysis. Seventeen articles were selected based on criteria including inclusion of primary tumors with normal benign controls. The analysis of the SHP1 protein expression by immunohistochemistry (IHC) revealed that lack of SHP1 expression is found in 178 of 242 (73.6%) NHL tissues vs. 32 of 264 (12%) normal/benign controls. The odds ratio derived by the meta-analysis was 119.58 with a 95% confidence interval of 37.60-380.15, suggesting loss of SHP1 is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL (OR 69.46, 95% CI 12.32-391.74).To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with 48 DLBCL tumors and 12 cell lines. By immunohistochemistry, we found a wide range of variability with regards to the expression levels and no substantial difference between the GCB and non-GCB subtypes.Using 12 cell lines together with resting B-cells from normal donors, we identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells.Using CRISPR, we knocked out SHP1 in both GCB and ABC cell lines. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones compared to the wild type ones. The results demonstrate that SHP1 depletion results in increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity of ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib.ConclusionsTogether, our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. DisclosuresWang: Incyte Research Institute: Ended employment in the past 24 months.

Upregulation of CD47 Expression in De Novo Diffuse Large B-Cell Lymphoma Is More Frequent in Activated B-Cell Type

CD47 is a marker of self that provides a “don't-eat-me-signal” through activation of signal-regulatory protein alpha (SIRPa), a cell surface receptor expressed on monocytes/macrophages and granulocytes. This interaction negatively regulates effector functions such as, phagocytosis, migration, and superoxide production. Upregulation of CD47 expression in cancer, including diffuse large B-cell lymphoma (DLBCL), has emerged as a mechanism to escape innate immune surveillance. Using conventional immunohistochemical detection, we assessed CD47 expression in DLBCL and interrogated its association with clinicopathologic features.Patients with de novo DLBCL were identified from two large institutions and were uniformly treated with R-CHOP and had sufficient material for study. Immunohistochemical stains (IHC) were performed on FFPE tissue (Hans algorithm, BCL2, MYC, and CD47) and scored semi-quantitatively from no reactivity (0) to strong (2 and 3; Figure 1). Mutational analysis using a 334 gene target sequencing panel, gene expression profiling using Lymph2Cx to determine the cell of origin (COO), and FISH analysis for MYC, BCL2, and BCL6 translocations, were performed. The Lymphgen tool (Wright et al, 2020) was also used to determine the DLBCL group. Fisher's exact test and Kaplan-Meier survival analysis for overall survival (OS) were performed and P <0.05 was considered significant.CD47 expression was assessed by IHC in a cohort of 152 cases of de novo DLBCL, including 107 cases of germinal center B-cell (GCB) type (70%), 37 cases of activated B-cell (ABC) type (24%), and 8 cases of intermediate type (5%). A total of 17 cases (11%) showed strong and diffuse CD47 expression with IHC scores of 2 or above (CD47hi). CD47hi cases were significantly more frequent in ABC DLBCL (24%, 9/37) than GCB DLBCL (6%, 6/107; P=0.003). The remaining 2 CD47hi cases were in the intermediate DLBCL group (25%, 2/8). ABC DLBCL with CD47hi showed more frequent mutations with TET2 (33% vs 7%; P=0.08) and ZFP36L1 (22% vs 0%; P=0.05) compared to cases with low expression of CD47 with IHC scores of less than 2 (CD47low). ABC DLBCL with CD47low showed more frequent mutations of NOTCH2 (18% vs 0%; P=0.31) and MYD88 (29% vs 11%; P=0.4) compared to CD47hi. GCB DLBCL with CD47hi showed frequent mutations of TP53 (67% vs 21%; P=0.026) and CCND1 (33% vs 0%; P=0.003) compared to CD47low. None of the 13 cases with double- or triple-hit for MYC, BCL2 and/or BCL6 showed CD47 expression. The Lymphgen tool showed that cases of DLBCL with CD47hi were mostly in the ‘other’ group (50%), with other groups represented such as ST2 (21%), EZB (14%), MCD and BN2 (1 case each). There was no difference in overall survival (OS) between CD47hi and CD47low DLBCL (5-year OS, 75% vs 72%; P=0.57), or with the GCB or ABC subtypes.Strong expression with CD47 is more frequent in ABC DLBCL and is seen in a subset of GCB DLBCL with mutations in TP53 and/or CCND1. The level of CD47 expression does not appear to predict OS in patients with DLBCL treated with R-CHOP. This study demonstrates that conventional immunohistochemical methods can readily identify DLBCL with high CD47 expression, and these patients may benefit from the use of anti-CD47 therapy. [Display omitted] DisclosuresHerrera: Gilead Sciences: Research Funding; Takeda: Consultancy; Tubulis: Consultancy; Karyopharm: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seagen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding.

Abstract 2443: APOBEC3G is a sex-specific determinant of post-transcriptional RNA processing and survival in diffuse large B-cell lymphoma

Abstract APOBEC3G is a cytidine deaminase with a well-characterized anti-viral role against HIV and other retroviruses. Its deaminase activity yields G to A edits in ssDNA to disrupt viral coding sequences and inhibits cDNA production during reverse transcription. However, APOBEC3G is also highly expressed in cells and tumors of B-lymphocyte lineage. In this study we aimed to evaluate the biological and clinical relevance of APOBEC3G in Diffuse Large B Cell Lymphomas (DLBCL), where it is expressed in absence of viral infection. We first confirmed the high expression of APOBEC3G in a panel of DLBCL cell lines. Interestingly, immunofluorescence of APOBEC3G in DLBCL cells demonstrated localization to p-bodies. SILAC mass-spectrometry of Flag-APOBEC3G further revealed its association with several RNA binding proteins (RBPs) involved in RNA metabolism. Using the enhanced crosslinking and immunoprecipitation (eCLIP) assay for identification of bound RNA species, we demonstrated that APOBEC3G binds to 3' UTR and exonic RNA sequences, mostly of genes encoding ribosomal components. To study the functional effects of APOBEC3G in DLBCL, we generated multiple DLBCL knockout (KO) lines for APOBEC3G loss using CRISPR-Cas9. Whole transcriptome sequencing in SC1 cells revealed that knockout of APOBEC3G led to a global reduction in the RNA transcripts for ribosomal proteins. Together, these results suggest a role for APOBEC3G in binding/regulating cellular RNA species and RNA processing in DLBCL, in the absence of viral infection. We then evaluated the clinical relevance of APOBEC3G expression in DLBCL using publicly available gene expression datasets. We noted a correlation between APOBEC3G and ribosomal protein mRNAs in DLBCL. Interestingly however the expression of APOBEC3G was linked to survival only in male patients of germinal center subtype DLBCL. On further study of our cell line knockout models (n=2 each of M/F) we noted a sex specific role of APOBEC3G in modulating cellular stress. In male cells, the loss of APOBEC3G led to decreased protein synthesis, differential expression of Y-chromosomal genes, endogenous retroviruses and interferon stimulated genes. These effects were not noted in female KO cells KO. As APOBEC3G loss did not affect viability following exposure to chemotherapeutics in-vitro, we postulate that the sex-specific association of APOBEC3G with survival is a function of possible immunomodulatory effects of its binding/processing complex RNA species. The biological significance of this sex-specific role of APOBEC3G in normal B-cell development and maturation is as yet unclear, and represents an important area for future research. Citation Format: Joanna D. Wardyn, Michal M. Hoppe, Allison S. Chan, Jia Li, Omer An, Shruthi Venguidessane, Dominic P. Sheng, Henry Yang, Gene W. Yeo, Daniel J. Hodson, Reuben S. Harris, Anand D. Jeyasekharan. APOBEC3G is a sex-specific determinant of post-transcriptional RNA processing and survival in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2443.

Prognostic significance of CD30 expression in diffuse large B-cell lymphoma: A systematic review with meta-analysis.

CD30 is variably expressed in diffuse large B-cell lymphoma (DLBCL), but its prognostic potential for the affected patients remains debatable and unclear. Therefore, we aimed to determine the frequency of CD30 expression in DLBCL and its potential for prognostic determination. An electronic systematic review was performed using multiple databases, followed by a quantitative meta-analysis to assess the frequency of CD30 expression with positivity cut-off values of >0% and >20%, and to determine its association with clinicopathological features and patients' survival. Using a cut-off value >0%, we observed that 3.5%-59.1% of the cases were considered positive for CD30. There was a significant association of the protein expression with a lower number of extra-nodal sites affected by the neoplasm, with Ann Arbor advanced stage, the absence of B-symptoms, the lack of MYC and BCL2 translocations, and a lower ECOG performance. Using a cut-off value >20%, we observed that 2.5%-36.7% of the cases were considered positive for CD30, being significantly associated with a lower number of extra-nodal sites affected by the neoplasm, Ann Arbor stages III/IV, non-GCB tumours, the lack of MYC and BCL2 translocations, and a lower ECOG value. CD30 expression was significantly associated with a better survival rate, regardless of what cut-off parameter was used. Despite variations in the cut-off values used to determine CD30 positivity in DLBCL, the expression of this protein seems to be associated with a higher survival rate and better prognosis.

Genetic Characterization and Clinical Impact of Loss of MHC Class I and II Expression in De Novo Diffuse Large B-Cell Lymphoma

Introduction: Immune recognition by T cells requires the presentation of processed peptides in association with major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Among the tumor immune escape mechanisms described to date, alterations in the expression of MHC molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Loss of MHC-I or -II expression in DLBCL can occur through multiple mechanisms, including deletion of the MHC-I and -II locus, loss of function mutations of B2M, and genetic alterations of CIITA ; however, the role of additional genetic and epigenetic mechanisms remains largely unknown. With regard to clinical significance, a number of studies have reported that low MHC-II expression on tumor cells is associated with shorter survival in DLBCL, while the prognostic effect of MHC-I expression has not been systematically evaluated in a large, homogeneously treated cohort. In addition, although the frequency of loss of MHC-II expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, the prognostic impact remains ill-defined. Here, we conducted a comprehensive phenotypic and genetic analysis in a population-based DLBCL cohort to investigate the genetic background and clinical relevance of loss of MHC-I and -II expression.Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analysis was performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 57 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by gene expression (Lymph2Cx assay) in 323 cases (183 GCB, 104 ABC and 36 unclassifiable).Results: We found the following MHC-I and -II staining patterns:membrane-positive (n=188 (57%) and n=233 (72%), respectively), cytoplasmic only-positive / membrane-negative (n=86 (26%) and 31 (10%), respectively) and negative staining for both membrane and cytoplasmic (n=55 (17%) and n=59 (18%), respectively). Overall, membranous MHC-I and -II expression was not detected in 141/329 (43%) and 90/323 (28%) cases, respectively. While the frequency of loss of MHC-I expression was not different between COO subtypes, loss of MHC-II expression occurred more often in ABC-DLBCL (36% vs 22%, p=0.026). This is consistent with previous observations showing that MHC-II expression is reduced during plasmacytic differentiation and in ABC-DLBCL. Loss of MHC-I expression was associated with B2M mutation and deletion as well as with HLA-C deletions (all p <0.001, Fisher's exact test). Interestingly, mutations of EZH2 and GNA13 were detected more frequently in cases that had loss of MHC-I expression (p <0.001 and p=0.004, respectively). Moreover, mutations of CIITA and RFXAP (components of the MHC-II enhanceosome), and mutations of PRDM1 and TNFAIP3, ABC-DLBCL-specific mutations, were significantly enriched in MHC-II negative cases. Of note, mutations of B2M and EZH2 were significantly associated with loss of MHC-II expression, as well (p=0.001 and p=0.016, respectively). The associations between EZH2 mutation and MHC-I and -II expression were also observed when the analysis was restricted to GCB-DLBCL (both p <0.0001).With a median follow up of 6.5 years for living patients, the loss of MHC-I expression was not associated with survival in the entire DLBCL cohort, as well as within each COO subtype. Although there was also no significant survival difference according to the MHC-II expression status in the entire DLBCL cohort, loss of MHC-II expression was associated with an inferior 5y-time to progression (TTP, 59% vs 79%, p=0.007) in the GCB subtype. Interestingly, in the ABC subtype, loss of MHC-II expression was associated with a superior 5y-TTP (64% vs 40%, p=0.03).Conclusion: Our approach using next generation sequencing and high resolution SNP arrays uncovered the genetic background, as well as the clinical impact of loss of MHC-I and-II expression in a uniformly R-CHOP-treated large population-based cohort of DLBCL patients. Our results warrant further studies investigating a potential mechanism of EZH2 mutations regulating MHC-I and -II expression in DLBCL. DisclosuresSavage:Bristol-Myers Squibb: Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche: Research Funding; Celgene: Consultancy; Merck: Honoraria. Sehn:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Morin:Epizyme, Inc: Consultancy. Connors:Genentech: Research Funding; Bayer Healthcare: Research Funding; Lilly: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Bristol-Myers Squibb: Research Funding; Cephalon: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding. Scott:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy.

Integrative Analysis of 1001 Diffuse Large B Cell Lymphoma Identifies Novel Oncogenic Roles for Rhoa

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy and is characterized by a striking degree of clinical and molecular heterogeneity. Gene expression profiling has long been used to define subtypes and understand the heterogeneity within DLBCL. Microarray-based gene expression studies have identified cell of origin subgroups (Alizadeh et al., 2000), as well as gene expression signatures derived from host inflammatory response (Monti et al., 2005), stromal tissues (Lenz et al., 2008) as well as other biological processes. However, the connections between these gene expression signatures and genetic alterations is largely unknown. In this study, we sought to define connections between gene expression signatures and genetic alterations to better understand the biology of DLBCL. We enrolled a total of 1001 DLBCL patients and comprehensively defined the landscape of genetic mutations, copy number alterations and expression through whole-exome and transcriptome sequencing. We identified 150 genetic drivers of DLBCL including many novel, clinically relevant genes (e.g. SPEN, SETD1B and KLHL14). We began the gene expression analysis with a comprehensive universe of nearly 9500 annotated gene sets from widely used gene set databases (Kegg, Reactome, MSigDB), several lymphoma-specific gene signature sources (Lenz et al. 2008, Monti et al. 2005, and Shaffer et al.) and other published sources. From this large universe, we identified 1228 gene sets that comprised genes with high intra gene set correlation. The vast majority of these 1228 gene sets also exhibited high inter-gene set correlation indicating a high degree of redundancy between these gene sets. We developed an approach to singular value decomposition to define 31 exemplar gene sets that essentially replicated the patterns of the 1228 described gene sets. Our results recapitulated a number of described patterns underlying DLBCL gene expression including distinct gene sets reflecting cell of origin, host response, BCR and Oxidative Phosphorylation (Monti et al., 2005), as well as the “Stromal 1” and “Stromal 2” signatures (Lenz et al., 2008) signatures. There was no association between the expression of stromal signatures and overall mutational burden (p=0.4). Our data identified the genetic underpinnings of each of these distinct gene sets that provide a better understanding of the underlying biology affected by different genetic mutations. Interestingly, genetic alterations in RHOA were strongly associated with the proliferation gene signature and poor survival in DLBCL. We sought to better define the role of RHOA mutations in DLBCL which are mostly unknown. We knocked out the expression of RHOA in vitro through the CRISPR-Cas9 approach and found that RHOA deletion was lethal in both ABC DLBCL (LY3, TMD8, HBL1) and GCB DLBCLs (Pfeiffer and SUDHL4), results were validated with shRNAs. Conversely, cells overexpressing R5Q mutant proliferated 30% faster than vector control cells and more than 10% faster than wildtype overexpressing cells, directly implicating these mutations and RHOA overexpression in the growth of DLBCL cells. We further investigated the effects of RHOA knockout in vivo using two complementary mouse models. We bred Rhoa conditional knockout mice with Mb1-Cre transgenic mice, in which Cre is restricted to the B cell lineage as well as AID-Cre transgenic mice in which Cre is restricted to germinal center (GC) B cells. While B cells represented the vast majority of cells in the lymphoid organs of Rhoa wildtype mice, we found a dramatic reduction in B cell population and a resulting increase in T cell percentages in Rhoa deleted mice (p Collectively, our data define the genetic basis for observed changes in gene expression in DLBCL. Our experimental data define RHOA as an oncogene with roles in B cell development, germinal center maintenance, and DLCBL growth. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Merck: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Research Funding. Flowers: Abbvie: Consultancy, Research Funding; Infinity: Research Funding; Clinical Care Options: Research Funding; National Cancer Institute: Research Funding; Spectrum: Consultancy; Onyx: Research Funding; OptumRx: Consultancy; Prime Oncology: Research Funding; Janssen Pharmaceutical: Research Funding; Acerta: Research Funding; Gilead: Consultancy; Seattle Genetics: Consultancy; Eastern Cooperative Oncology Group: Research Funding; Research to Practice: Research Funding; Burroughs Welcome Fund: Research Funding; Celgene: Consultancy, Research Funding; V Foundation: Research Funding; Bayer: Consultancy; Educational Concepts: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Genentech/Roche: Consultancy, Research Funding; Millennium/Takeda: Research Funding; National Institutes Of Health: Research Funding; TG Therapeutics: Research Funding. Gribben: TG Therapeutics: Honoraria; Kite: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Karyopharm: Honoraria; Genentech/Roche: Honoraria. Hsi: Cellerant Therapeutics: Research Funding; Abbvie: Research Funding; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Eli Lilly and Co.: Research Funding. Evens: Merck: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Millennium: Consultancy; AbbVie: Consultancy; Affimed: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy; Kite Pharma: Consultancy. Reddy: Abbvie: Consultancy; BMS: Consultancy; Celgene: Consultancy; Gilead: Speakers Bureau. Gordon: Janssen: Other: Data Monitoring Committee.

Clinical Significance of PD-1 and PD-L1 Expression and Ongoing Interaction in the Tumor Microenvironment in Diffuse Large B Cell Lymphoma (DLBCL) Treated with R-CHOP

Background: PD-1 blockade has demonstrated antitumor activity in diffuse large B-cell lymphoma (DLBCL). In order to select patients who are more likely to respond to PD-1 blockade, predictive biomarkers are being intensively pursued. PD-1 and PD-L1 expression in DLBCL treated with R-CHOP as standard-of-care has been examined in several studies. However, only overall PD-1+ cells but not T-cell specific PD-1 expression have been quantitated, and results of the prognostic significance of PD-L1 expression have been inconsistent in DLBCL patients. Whether ongoing PD-1/PD-L1 interaction in tumors contributes to the adverse prognostic effect of PD-1 and PD-L1 expression is unknown.Patients and Methods: Tissue microarray (TMA) sections ofdiagnostic samples from 414 patients with de novo DLBCL treated with R-CHOP were fluorescently stained with CD3 (T cell marker), PD-1, and PD-L1. Positive cells and PD-1/PD-L1 interaction were quantitatively assessed by the Automated Quantitative Analysis (AQUA) algorithms. To characterize PD-1/PD-L1 expression in each sample, CD3+, PD-1+, PD-L1+, and PD-1+CD3+ (double positive, i.e., PD-1+ T) cell numbers were divided by total nucleated cells (DAPI+) in the representative tumor areas. Ongoing PD-1/PD-L1 interactions were evaluated by the proximity of PD-1+ T cells to the PD-L1+ compartments and proportion of PD-1+ T cells co-localized with PD-L1, generating an interaction score for each sample. Biomarker expression and interaction scores were correlated with patient survival.Results: CD3+, PD-1+, PD-L1+, and PD-1+CD3+ cells were identified in 90%, 30%, 53%, and 18% of the cohort, respectively. The mean percentages of CD3+, PD-1+, PD-L1+, and PD-1+CD3+ cells in DLBCL tissues were 12.1%, 0.44%, 3.6%, and 0.19%, respectively. Activated B-cell-like DLBCL had a significantly higher mean percentage of PD-L1+ cells, but not PD-1+CD3+ cells, compared to germinal center B-cell-like DLBCL (p=0.0012). Three prognostic tiers were identified. Firstly, absence to very low percentage (0-3.5%) of tumor-infiltrating CD3+ T cells in the tissues was associated with poorer overall survival (OS) of DLBCL patients (p=0.028). CD3high DLBCL (>3.5% of all cells were CD3+) was associated with significantly higher percentages of PD-L1+ and PD-1+ cells (p<0.0001 and p=0.005, respectively). Secondly, in CD3high DLBCL, presence of PD-1+ T cells correlated with significantly poorer OS and progression-free survival (PFS) of patients (p=0.028 and p=0.0099, respectively). In contrast, presence/absence of PD-1+ non-T cells was not prognostic. Presence of PD-1+ T cells in CD3high DLBCL was associated with a higher mean percentage of PD-L1+ cells (p=0.019). However, PD-1/PD-L1 interaction scores did not add to the prognostic effect. This may suggest that phenotype of PD-1/PD-L1 interaction is not a biomarker to specify the immunosuppressive role of the PD-1/PD-L1 axis. Lastly, in CD3high DLBCL cases with PD-1−negative T cells, PD-L1 positivity correlated with significantly poorer OS and PFS of patients (p=0.0034 and p=0.0059, respectively), although PD-L1+ was not associated with non-T PD-1 expression either, suggesting PD-1−independent function of PD-L1. In the overall cohort, presence of PD-1+ T cells was associated with a trend of poorer PFS (p=0.079), whereas PD-L1 expression was not prognostic.Conclusions: PD-1 expression in T cells and lack of tumor-infiltrating CD3+ T cells are associated with significantly poorer survival of DLBCL patients. In addition, our data suggest that PD-1 can be a prognostic marker independent of PD-1/PD-L1 co-localization, and that PD-L1 has other PD-1-independent inhibitory functions. These results demonstrate the importance of both T-cell influx and antitumor function in prolonging the survival of DLBCL patients, and provide rationales for PD-1 blockade and immunotherapies activating T cell-mediated immune responses in DLBCL. DisclosuresTran:Genoptix, Inc: Employment. Adams:Navigate BioPhama Services, Inc: Employment. Roscoe:Navigate BioPhama Services, Inc: Employment. Hsi:Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Abbvie: Research Funding; Eli Lilly and Co.: Research Funding; Cellerant Therapeutics: Research Funding. Piris:Kura Oncology: Research Funding. Winter:Glaxo-Smith-Kline: Research Funding; Merck: Research Funding. Vaupel:Genoptix Laboratory: Employment. Dakappagari:Genoptix, Inc: Employment.

PD-L1 Expression and Gene Amplification in CD30+ Diffuse Large B-Cell Lymphoma (DLBCL): Significance of TP53 Genetic Impact and Clinicopathologic Characteristics

Introduction: Regulation of the PD-1/PD-L1 immune checkpoint has been under intense investigation in the era of immunotherapy, and PD-1 blocκade has shown promising clinical responses in several neoplasms including classical Hodgkin lymphoma (HL). Frequent amplification of the PD-L1 / PD-L2 gene locus (9p24.1) are found in most HLs, however, in other lymphoma types such as diffuse large B-cell lymphomas (DLBCL), genetic alterations of 9p24.1 locus are detected only in a small subset of cases (< 20%). Therefore, other mechanisms including transcriptional regulation of the PD-L1 gene are currently under investigation. Previous studies have shown that in another CD30+ lymphoma type, namely anaplastic large cell lymphoma of T-cell origin, PD-L1 is highly expressed despite the absence of gene amplification or other chromosomal alterations of 9p24.1 locus, and transcriptional regulation is biologically involved. However, the PD-L1 expression levels, the genetic basis of PD-L1 expression and their clinical significance in CD30+ DLBCLs are not yet studied.Methods: From a large database of 2110 DLBCLs, 78 patients with CD30+ DLBCL (44 GCB and 34 ABC-type) diagnosed and treated at the University of Texas M.D. Anderson Cancer Center (USA) and Karolinska University Hospital (Sweden) were identified. All patients were treated with R-CHOP regimen and had available clinical data. PD-L1 expression was assessed by immunohistochemistry using a monoclonal antibody and autostainer systems (Dako or Ventana). PD-L1 gene amplification and chromosomal aberrations of its gene locus were assessed by FISH. Mutations of TP53 gene were confirmed by sequencing. Statistical associations of the PD-L1 expression level (% of positive tumor cells) among various patient subgroups were based on the non-parametrical Mann-Whitney and Kruskall-Wallis tests. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. The DLBCL in vitro system included 3 cell lines (RC-K8, OCI-Ly3, U2932), which were treated with increasing concentrations of Nutlin 3A to stabilize and activate wild-type TP53. Western blot analysis was used to assess the protein levels of p53 and PD-L1 and quantitative RT-PCR was utilized to assess the PD-L1 mRNA level in the cell lines.Results: Using FISH, PD-L1 gene amplification, chromosomal gains and polyploidy were observed in 6 (7.7%), 3 (3.8%) and 7 (9%) cases, respectively. Expression of PD-L1 protein was found in 66 (85%) CD30+ DLBCLs and the percentage of positive tumor cells ranged from 5 to 100% (median 30%) among the positive cases. In this cohort, TP53 mutations were detected in 14 (18%) CD30+ DLBCL and were not statistically associated with PD-L1 gene amplification or expression levels. However, all PD-L1 negative tumors (n=12) were associated with a wild-type TP53 gene. High expression levels of PD-L1 correlated with ABC phenotype (p=0.04), B symptoms (p=0.03), high serum LDH (p=0.04), ECOG performance status>1 (p=0.015), and the presence of >1 extranodal sites (p=0.04) but not with Ann Arbor stage. Patients who achieved complete remission showed significantly lower PD-L1 expression level (median 20% vs. 40%, p=0.03). Given the significant survival benefit of CD30 expression in DLBCL, PD-L1 expression in this subset abolished advantage of failure-free (FFS) and overall (OS) survival after 48 months of median follow-up. The in vitro data showed that stabilization and activation of p53 in DLBCL cell lines carrying a wild-type p53 gene led to concentration-dependent increase of the PD-L1 mRNA and protein levels. As in silico analysis did not demonstrate a possible TP53 binding site on the PD-L1 gene promoter, an indirect mechanism of p53-dependent regulation of PD-L1 is suggested.Conclusions: PD-L1 is expressed in most CD30+ DLBCLs, but amplification of the PD-L1 gene locus is not a common mechanism unlike Hodgkin lymphoma. Activation of wild-type TP53 indirectly upregulates PD-L1 in DLBCL and the underlying mechanisms merit further investigation. DisclosuresSmedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

Association of AID and MUM1 by Immunohistochemistry in Diffuse Large B-Cell Lymphoma

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with dysregulation of somatic hypermutation (SHM) and class switch recombination (CSR) have been known to contribute for its lymphomagenesis. Activation-induced cytidine deaminase (AID) enzyme plays a vital role for both processes. Multiple myeloma oncogene 1 (MUM1) is known to upregulate the AID expression in normal and pathological conditions. However, both AID and MUM1 expression association in DLBCL is still unexplored using immunohistochemistry method. We examined DLBCL samples and then retrospectively tested its correlation with clinical findings.METHODS: A retrospective cohort study with 20 cases of DLBCL biopsy tissue with AID and MUM1 antibody was conducted. The samples were then classified into concordant (AID+/MUM1+ or AID-/MUM1-) and discordant group (AID-/MUM1+). The clinicopathological comparison was performed to observe any association between immunohistochemistry expression and clinical findings.RESULTS: Among 20 samples of DLBCL, concordant expression rate of AID and MUM1 was 80% with kappa Cohen’s of 0.578 (p=0.004). A significant association was observed between AID and MUM1 expression with a prevalence ratio of 2.25 (95% CI: 1.08-4.67; p=0.008). Clinical characteristics were not significantly different between each group. Restricted mean survival time was shorter in the concordant group compared with the discordant group but statistically insignificant (21.16 vs. 22.5 months; p=0.531).CONCLUSION: The result of this study showed the association between AID and MUM1 expression in DLBCL. However, whether the association may add further molecular heterogeneity of DLBCL is still to be confirmed by expanding the study.KEYWORDS: AID, CSR, DLBCL, MUM1, SHM

NFATc1 and Tumor Associated Macrophages Affect Progression of Certain Subsets of Diffuse Large B Cell Lymphoma Non-GCB Subtypes

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma among B cell lymphomas. The interaction of tumor cells with their microenvironment (tumor microenvironment, TME) leads to progressivity of malignancy. CD163 + macrophages known as components of TME. Nuclear factor of activated T cell (NFATc1) and MYC are important transcription factors in malignant transformation and progression. Therapeutic strategies were fast developed, nevertheless, efforts to decrease DLBCL morbidity and mortality are unsatisfied, therefore,new markers for prognosis and or therapeutic options of the patients are necessary. This study was aimed to investigate NFATc1 expression in DLBCL and its TME.&#x0D; Methods: Thirty-two paraffin blocks were selected then immunostained for expression of NFATc1, MYC, and CD163. Clinopathologic data i.e. ages, gender, and proliferation index Ki-67 were obtained. Data was analyzed by statistics&#x0D; Result: Positive expression of CD163 and NFATc1 was among 55% and 45% of cases respectively. All DLBCL cases in this study were non-GCB subtype and more patients were under 60 years (66%). Positive expression of CD163 was higher in males (69%) and in patients under 60 years (63%). Tissues positive for both NFATc1 and CD163 was observed higher among males and patients under 60 years.&#x0D; Conclusion: NFATc1 may affect development and or progression of certain subsets of DLBCL non-GCB subtype.

Discovery of novel targets for diffuse large B-cell lymphoma

The newly emerging CD19-targeted Chimeric Antigen Receptor T-cells (CAR-T) therapy has revolutionized the treatment of advanced diffuse large B-cell lymphoma (DLBCL) and achieved a functional cure in some patients. However, a majority of patients suffer relapse. A major pathway for tumor relapse is the loss of the target antigen, CD19, on the cancer cell surface, essentially making them invisible to the therapy. Consequently, new targets are required for the continued treatment of relapsed patients. Facing this dilemma, we hypothesized that a systematic search of the human genome may reveal more novel targets of DLBCL with characteristics similar to CD19. These include high expression in DLBCL and other B-cell tumors, but minimal expression in normal tissues. To test this hypothesis, we used several well-annotated public genomic and proteomic databases and carried out in-depth screening to pinpoint optimal new targets for DLBCL. Our study uncovered several new targets that have not been extensively pursued in the public domain, such as CD79A and TNFRSF13C. Based on our refined criteria for target evaluation, we finally concluded that CD79A represents the most ideal novel target for DLBCL and potentially other B-cell tumor types. This target should be explored as the antigen of next-generation CAR-T therapy that has the potential to save and extend the lives of patients.

RNA-Editing-Initiated MAVS Signaling is a Key Epitranscriptomic Alteration in Human B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancy. Genetically, it is a highly heterogeneous disease. Here, we explore the contribution of an epitranscriptomic modification (RNA editing) to DLBCL pathogenesis. We demonstrate that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumours can modulate pathway outcomes by altering sequence at either the genomic or the transcriptomic level. We further show that in DLBCL, RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as novel gene targets like MAVS, the essential downstream adaptor of RIG-I-like receptors and a central molecule in the innate response to RNA viruses. We next illustrate that ADAR1-mediated editing in the MAVS transcript correlates with increased MAVS mRNA and protein expression levels. Increased MAVS expression in DLBCL furthermore associates with increased interferon/NF-κB signaling and increased T cell exhaustion. We validate these findings in independent cohorts of DLBCL samples as well as another type of germinal center related B cell lymphoma, follicular lymphoma. Finally, using targeted RNA base editing tools to restore editing specifically within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is causal to an increase in downstream signaling, in the absence of activation by canonical nucleic acid receptor sensing. Overall, ADAR1-mediated RNA editing represents a new mechanism underlying human B cell lymphomagenesis.

Diffuse Large B Cell Lymphoma and Programmed Death-Ligand 1 Expression. a Clinical and Pathological Study of Patients Seen in Tawam Hospital UAE

Introduction: Diffuse Large B Cell Lymphoma (DLBL) is the most common type of Non-Hodgkin's Lymphoma (NHL) seen in Tawam hospital UAE. DLBL is a heterogenous disease arising either from a Germinal center (GC) or non- GCB cells. Molecular signature has identified 3 different groups of DLBL with significantly different prognosis (Primary mediastinal, Germinal center and Activated B Cell type). The therapy for DLBL has been without innovation since introduction of Rituximab more than 20 years ago and using R-CHOP. Attempts at improving the outcome has been uniformly unsatisfactory either by adding additional drugs, increasing the dose of drugs or deceasing the frequency of each cycle. New biomarkers and or targets are needed to improve the prognosis of patients with DLBL. Programmed Death-Ligand 1 (PDL1) has been shown to protect tumor cells by inducing T cell apoptosis (Dong et al., 2002). PDL1 targeting agents have been approved for many different types of malignancies including Hodgkin's Lymphoma. The role of PDL1 in DLBL is still under intense study. We report our findings of PDL1 expression in DLBL patients in UAE. Methods: Patients diagnosed with DLBL between December 1, 2019 till July 15th 2020. Patient records were reviewed in this study. Data was collected regarding patient demographics, clinical stage, International prognostic index (IPI), pathology and cell of origin {using immunohistochemistry (IHC) and Han's criteria}. PDL1 testing was performed by using FDA approved PharmaDx Dako Ab 22C3 kit for IHC on the gold standard Link 48 Dako platform. PDL1 expression was reported as Tumor proportion score (TPS). TPS in this assay was the percentage of viable tumor cells showing any perceptible partial or complete linear membranous (or membranous &amp; cytoplasmic) staining, in the large lymphoma cells. The specimen was considered PDL1 positive if the TPS was greater than or equal to 30%(Xu-Monette et al Blood 2018). All scores were reported by single pathologist Results Twenty-six patients were diagnosed with DLBL in the study period. The median age was 44 years (range 18-90 years). Majority of patients (75%) were less than sixty years of age. Advanced stage disease was seen in preponderance (66%) as compared to early stage (34%) while the LDH was elevated in 65 % patients (institutional normal &amp;lt; 225 IU per liter). Majority of patients (60%) were poor risk with IPI 3 or higher. Pathology review showed a uniformly elevated Ki 67 with the majority of specimens showing greater than 80 %. Molecular classification showed GCB type 50 % (n=13) as compared to non-GCB 42 % (n=11), data was not available for 2 patients. Epstein-Barr virus encoded small RNA (EBER) CISH was positive in only 3 patients (data was not available for 8 patients). PDL1 expression was tested in eighteen (GCB n=8, ABC n=10) patients. PDL1 expression was elevated in ABC type (90%) as compared GCB type (50 %) Fig 1. Conclusion The median age (44 years) is at least 2 decades less than the reported western cohort. Majority of the patients had high risk disease with advanced stage and IPI. Ki 67 was uniformly elevated (&amp;gt;80 % in 90 % of patients). There was a slight preponderance of GCB type (50 % vs 42%). PDL1 expression in ABC type DLBL was higher as compared to GCB. This observation needs to be validated in a larger cohort. Recent literature makes it very clear that ABC type DLBL has a poor outcome as compared to GCB type. PDL1 may offer an attractive target for treatment evaluation in ABC type DLBL. Figure Disclosures Alam: Pfizer:Honoraria;Roche:Honoraria.McCarthy:Karyopharm:Consultancy, Honoraria;Magenta:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Janssen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Takeda:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Juno Therapeutics, a Bristol-Myers Squibb Company:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board , Research Funding is to Roswell Park, Research Funding;AbbVie:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board;Genentech:Consultancy, Honoraria;Starton:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Overexpression of OCT-1 gene is a biomarker of adverse prognosis for diffuse large B-cell lymphoma (DLBCL): data from a retrospective cohort of 77 Brazilian patients

BackgroundOCT-1 gene is a member of the POU-homeodomain family of transcriptional regulators of B-lymphocyte differentiation by controlling expression of B-cell specific genes. BCL-2 gene is a potent inhibitor of apoptosis and it is essential during B-cell differentiation into germinal center. These genes may be expressed in diffuse large B-cell lymphoma (DLBCL), but the role of BCL-2 in its prognosis has been contradictory, and OCT-1 has yet to be tested.MethodsIn this study, we aimed to investigate the prognostic impact of OCT-1 and BCL-2 expression in DLBCL treated in the real world with immunochemotherapy in a single center. BCL-2 and OCT-1 genes were available in 78.5% (77/98) DLBCL patients, and the RNA for quantitative real-time PCR was isolated from formalin-fixed paraffin-embedded samples. The values obtained for gene expression were transformed in categorical variable according to their median.ResultsCohort median age was 54.5 years (15–84), 49 (50%) were male, 38/77 (49.4%) and 40/77 (51.9%) presented OCT-1 and BCL-2 expression ≥ median, respectively. The overall response rate (ORR) in all patients was 68.4% (67/98), 65,3% (64/98) of patients acquired complete response, and 3.1% (3/98) partial response, while 6.1% (6/98) were primary refractory. The median follow-up was 3.77 years (95% CI: 3.2–4.1), with 5.43 (95% CI: 2.2-NR) of overall survival (OS) and 5.15 years (95% CI: 2.9-NA) of progression free survival (PFS). OCT-1 ≥ median was associated with shorter OS at univariate analysis (p = 0.013; [HR] 2.450, 95% CI: 1.21–4.96) and PFS (p = 0.019; [HR] 2.270, 95%CI: 1.14–4.51) and BCL-2 gene overexpression presented worse PFS (p = 0.043, [HR] 2.008, 95% CI: 1.02–3.95). At multivariate analysis, OCT-1 overexpression was associated with poor PFS (p = 0.035, [HR] 2.22, 95% CI: 1.06–4.67).ConclusionIn this study, we showed that overexpression of OCT1 gene was an independent prognostic factor of adverse outcomes in DLBCL.

Quantitative analysis of tumor-specific BCL2 expression in DLBCL: refinement of prognostic relevance of BCL2

BCL2 overexpression has been reported to be associated with poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL). However, currently there is no consensus on the evaluation of BCL2 expression and only the proportion of BCL2 positive cells are evaluated for the determination of BCL2 positivity. This study aimed to define BCL2 positivity by quantitative analysis integrating both the intensity and proportion of BCL2 expression. BCL2 expression of 332 patients (221 patients for the training set and 111 patients for the validation set) with newly diagnosed DLBCL who received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) were analyzed using the tumor-specific automated quantitative analysis (AQUA) scoring method based on multiplex immunofluorescence. In the training set, high BCL2 AQUA score (N = 86, 38.9%) was significantly associated with poor prognosis (p = 0.01, HR 2.00; 95% CI [1.15–3.49]) independent of international prognostic index, cell of origin, and MYC expression. The poor prognostic impact of the high BCL2 AQUA score was validated in the validation set. AQUA scoring of BCL2 expression incorporating both the intensity and proportion of BCL2 positive cells was independently associated with survival outcomes of patients with primary DLBCL treated with R-CHOP.

Long Noncoding RNAs in Diffuse Large B-Cell Lymphoma: Current Advances and Perspectives.

Diffuse large B-cell lymphoma (DLBCL) is a complex and aggressive malignancy originating from B lymphocytes and characterized by extensive clinical, phenotypic and molecular heterogeneity. Although research conducted over the past decades has substantially improved our understanding of DLBCL, its pathogenesis has not yet been fully elucidated. The development of RNA sequencing technology has allowed the identification of numerous long noncoding RNAs (lncRNAs) that exhibit aberrant expression in DLBCL. These lncRNAs play crucial roles in DLBCL development and pathogenesis and are thus good candidates for use as diagnostic biomarkers or therapeutic targets. In this review, we describe the lncRNAs associated with DLBCL, summarize their characteristics and molecular functions, and discuss their relationships with clinical practice.

CD30 Expression in Germinal Center B-cell-like and non-Germinal Center B-cell-like Subtypes of Diffuse Large B-cell Lymphoma

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common and most heterogeneous type of non-Hodgkin lymphoma. With current therapeutic modalities, 30%–40% of DLBCL cases still experience recurrence. The discovery of CD30 expression in DLBCL in several studies has opened up alternative opportunities for new target therapies.&#x0D; AIM: This cross-sectional study aimed to determine CD30 expression in DLBCL and its difference in expression in germinal center B-cell such as (GCB) and non-GCB subtypes.&#x0D; METHODS: The sample consisted of 25 GCB and 25 non-GCB cases based on immunohistochemical examination performed at Cipto Mangunkusumo Hospital from 2014 to 2017. CD30 staining was carried out and assessed using tumor cells percentage with positive cutoff values of &gt;0%, &gt;10%, and &gt;20%.&#x0D; RESULTS: Positive CD30 expression was obtained in 8 (16%), 4 (8%), and 3 (6%), out of 50 DLBCL cases with positive cutoff values of &gt;0%, &gt;10%, and &gt;20%, respectively. We performed Fisher’s exact test to determine CD30 expression in the GCB and non-GCB subtypes and found no significant difference with p &gt; 0.05.&#x0D; CONCLUSION: Our study found no significant difference between CD30 expression in the GCB and non-GCB subtypes of DLBCL.