Germinal Center B-cell-like Research Articles

PATZ1 expression correlates positively with BAX and negatively with BCL6 and survival in human diffuse large B cell lymphomas.

Non-Hodgkin lymphomas (NHLs) include a heterogeneous group of diseases, which differ in both cellular origin and clinical behavior. Among the aggressive malignancies of this group, the diffuse large B-cell lymphomas (DLBCLs) are the most frequently observed. They are themselves clinically and molecularly heterogeneous and have been further sub-divided in three sub-types according to different cell of origin, mechanisms of oncogenesis and clinical outcome. Among them, the germinal center B-cell-like (GCB) derives from the germinal center and expresses the BCL6 oncogene. We have previously shown that Patz1-knockout mice develop B-cell neoplasias, suggesting a tumor suppressor role for PATZ1 in human NHLs. Here, by immunohistochemical analysis of a tissue-microarray including 170 NHLs, we found that PATZ1 nuclear expression is down-regulated in follicular lymphomas and DLBCLs. Moreover, consistent with our previous results showing a PATZ1-dependent regulation of BCL6 and BAX transcription, we show that low PATZ1 nuclear expression significantly correlates with high BCL6 expression, mainly in DLBCLs, and with low BAX expression, also considering separately follicular lymphomas and DLBCLs. Finally, by analyzing overall and progression-free survival in DLBCL patients that underwent rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy, low levels of PATZ1 were significantly associated to a worst outcome and demonstrated an independent prognostic factor in multivariate analysis, including known prognostic factors of DLBCL, IPI score and cell of origin (GCB/non-GCB). Therefore, we propose PATZ1 as a new prognostic marker of DLBCLs, which may act as a tumor suppressor by enhancing apoptosis through inhibiting and enhancing transcription of BCL6 and BAX, respectively.

Expression of CD40 is a positive prognostic factor of diffuse large B-cell lymphoma treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).

ObjectivesThe objective of this study was to investigate the expression level of CD40 and its role in the prognosis of patients with diffuse large B-cell lymphoma (DLBCL) who were treated with rituximab-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).Design and methodsThe immunohistochemical expressions of CD40 in 186 well-characterized DLBCL patients were evaluated by tissue microarrays, thereby revealing the relationship of the molecule CD40 with known tumor, patient-related variables, and survival rates.ResultsThe results showed that CD40 expressions were not statistically different between the germinal center B-cell-like (GCB) type and the non-GCB type. We also analyzed the relationships of CD40 expression with overall survival (OS) and progression-free survival (PFS) in DLBCL patients who were uniformly treated with R-CHOP. A low expression of CD40 compared to high expression is related to poor OS and PFS.ConclusionOur findings indicate that the CD40 level at onset acts as an independent prognostic predictor of DLBCL patients treated with R-CHOP.

Classification of diffuse large b-cell lymphoma (DLBCL) FFPE samples of the GELA LNH2003 program, using Lymph2Cx assay on the nCounter analysis system.

7547Background: DLBCL comprises two molecular subgroups defined according to gene expression profiling of frozen tissue samples: the Germinal Center B-cell-like (GCB) and Activated B-cell-like (ABC...

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

Overexpression of heme oxygenase-1 induced by constitutively activated NF-κB as a potential therapeutic target for activated B-cell-like diffuse large B-cell lymphoma.

There is an urgent requirement for a new therapeutic target for activated B-cell-like lymphoma (ABC-DLBCL), which is known to have dismal outcome and constitutive activation of NF-κB. Heme oxygenase-1 (HO-1) can inhibit apoptosis and promote proliferation in many cancers. To our knowledge, no studies have been performed on the correlation between HO-1 and DLBCL. In this study, immunohistochemical analysis of 31tumor tissues from DLBCL patients [20 of ABC subtype and 11 of germinal center B-cell-like (GCB) subtype] and 11normal lymph nodes revealed that HO-1 overexpression was characteristic of ABC-DLBCL. In addition, HO-1 mRNA expression levels were consistent with the immunohistochemistry results. High levels of HO-1 expression were significantly correlated with the involvement of more than 1 extranodal site (p=0.025), with a high positivity rate of Ki-67 (p<0.01). Similar to its anti-apoptotic role in other malignancies, HO-1 upregulation suppressed apoptosis of the ABC-DLBCL cell line OCI-ly10, whereas its downregulation sensitized the tumor cells to chemotherapeutic drugs. Further study demonstrated that the HO-1 overexpression was mediated by constitutively activated NF-κB which together played an anti-apoptotic role in ABC-DLBCL. Combination of the NF-κB inhibitor Bay11‑7082 and the lentivirus vector Lenti-siHO-1 significantly decreased HO-1 protein expression and increased apoptosis in OCI-ly10 cells. However, in GCB-DLBCL cells with low levels of NF-κB expression, the TNF-α-mediated activation of NF-κB leading to HO-1 upregulation rescued the cells from apoptosis caused by HO-1 silencing. These results indicated that HO-1 can be a potential target for the treatment of ABC-DLBCL.

Sphingosine-1-phosphate receptor 1 as a prognostic biomarker and therapeutic target for patients with primary testicular diffuse large B-cell lymphoma.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is produced during the metabolism of sphingolipid by sphingosine kinase. S1P has been implicated in the migration and trafficking of lymphocytes and several lymphoid malignancies through S1P receptors. Moreover, the overexpression of sphingosine-1-phosphate receptor 1 (S1PR1) has been correlated with the constitutive activation of signal transducer and activator of transcription (STAT)3 and poor prognosis of diffuse large B-cell lymphoma (DLBCL). Thus, in this study, we examined the expression of S1PR1 in 198 DLBCL samples collected from nodal and various extranodal sites and sub-classified formalin-fixed paraffin-embedded tissue samples into germinal centre B-cell-like (GCB) and non-GCB subgroups using immunohistochemistry. These analyses showed S1PR1 overexpression in 15·7% of all cases with DLBCL and in 54·2% of 24 cases with primary testicular (PT)-DLBCL; S1PR1 expression correlated with S1PR1mRNA expression and STAT3 phosphorylation in fresh samples. Analyses of data from a single institution suggested that S1PR1 overexpression was an independent negative prognostic marker in 68 patients with DLBCL of clinical stages I and II. The present high prevalence of S1PR1 overexpression warrants the consideration of PT-DLBCL as a distinct disease subtype and suggests the potential of the S1P/S1PR1 axis as a therapeutic target.

Treatment of Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) comprises a heterogeneous group with pathophysiological, genetic and clinical features. Many patients can be cured with R-CHOP therapy, which is the current standard regimen. Despite recent progress in improving patient survival, the 40% survival of DLBCL patients remains poor. Therefore, the most important issue for patients with DLBCL remains the development of a new front-line therapy. Several studies have reported that intensified chemotherapy with dose-adjusted EPOCH-R or R-ACVBP was superior to R-CHOP. Gene expression profiling has identified two distinct forms of DLBCL: activated B cell-like (ABC) and germinal center B-cell-like (GCB) types. ABC DLBCL exhibits a worse prognosis than GCB DLBCL by molecular diagnosis after R-CHOP therapy. Next-generation sequencing has identified unique oncogenic mechanisms and genetic complexity, which has provided rational therapeutic targets. There are also a number of biomarkers, including CD5, and prognostic factors. Efforts to distinguish many biomarkers will be crucial for individualized treatment in the future.

Immunohistochemical Profile and Prognostic Significance in Chinese Primary Central Nervous System Lymphoma: Analysis of 132 Cases

Primary central nervous system lymphoma (PCNSL) is aggressive and confined to the central nervous system, including the brain parenchyma, leptomeninges, spinal cord, eyes or cranial nervous. Morphologically, approximately 95% of these tumors are DLBCL according to the new World Health Organization (WHO) classification. However, PCNSL has treatment outcome distinct from those of systemic DLBCL, as well as dismal prognosis than systemic DLBCL. Our goal was to determine the immunohistochemical profile and prognostic significance for 132 Chinese PCNSL cases. The expression of CD20, CD10, BCL-6, MUM1, CD138, BCL-2, and Ki67 antigens were observed by immunohistochemical method. All cases expressed CD20. CD10, BCL-6, and MUM1 were positive in 15.2% (20/132), 86.4% (114/132), 90.2% (119/132). CD138 was negative in 100% (39/39). BCL-2 was positive in 89.3% (108/121). The Ki67 antigen, a proliferative index, ranging from 1% to 100% (median 85.3%) and 76.5% (101/132) PCNSLs showed Ki67 ≥ 90%. Among 132 cases, 25 (18.9%) were classified as germinal center B-cell-like (GCB); 107 (81.1%) were classified as activated B-cell-like (ABC). The Ki67 index in 25 GCB was similar to that in 107 ABC (p=0.663>0.05). No significant correlation was found between Ki67 index and BCL-2 (p=0.225>0.05). Significant positive correlation was found between Ki67 index and BCL-6 expression (p=.000<0.05). Among 132 cases, 43 had complete data of treatment that received chemotherapy regimens based on HD-MTX. GCB and ABC had similar OS (p=0.969) and PFS (p=0.070). These findings support that PCNSL predominantly express an ABC immunophenotype and express high Ki67 index, and suggest that the proliferative activity of GCB was similar to ABC and the expression of BCL-6 but not BCL-2 was positively correlated with the malignant degree of tumors.Table 1Clinical characteristics.CharacteristicsPatients, n (%)Age (years); n=132≥60 y, n=53; <60 y, n=79Median (range)57 (21-85)Gender; n=132Male69 (52.3)Female63 (47.7)ECOG; n=430-18 (18.6)2-435 (81.4)LDH; n=43Normal25 (58.1)Elevated18 (41.9)Numbers of lesions; n=132148 (36.4)>284 (63.6)Involvement of deep structures; n=132Absence43 (32.6)Presence89 (67.4)Table 2Hans classification.CD10BCL-6MUM1ImmunoprofilePCNSL, n (%)+++GCB16 (12.1)++-GCB3 (2.3)+-+GCB0 (0)+--GCB1 (0.7)-+-GCB5 (3.8)-++Non-GCB90 (68.2)--+Non-GCB12 (9.1)---Non-GCB5 (3.8)Table 3Chang classification.CD10BCL-6MUM1ImmunoprofilePCNSL, n (%)++-GCB (Pattern A)3 (2.4)+--GCB (Pattern A)1 (0.8)-+-GCB (Pattern A)5 (3.9)+++activated GCB (Pattern B)16 (12.6)+-+activated GCB (Pattern B)0 (0)-++activated GCB (Pattern B)90 (70.9)--+activated non-GCB (Pattern C)12 (9.4) [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Potent Preclinical Activity of Ponatinib in Germinal Center B-Cell-like Diffuse Large B-Cell Lymphoma (GCB-DLBCL) Models

IntroductionDiffuse large B-cell lymphoma (DLBCL), the most common type of Non-Hodgkin lymphoma (NHL), comprises 2 major molecular subtypes: germinal center B-cell-like (GCB) and activated B cell-like (ABC). Although standard therapy (rituximab+ chemotherapy [R-CHOP]) is effective in most patients (pts), a significant proportion do not achieve durable remissions. Treatment of relapsed and refractory DLBCL pts with targeted therapy, such as the BTK inhibitor ibrutinib, has shown some promise; however, responses are mostly restricted to the ABC subtype. Treatment options for pts with relapsed/refractory GCB, outside of stem cell transplantation, are especially limited.Ponatinib is a potent pan-BCR-ABL inhibitor approved for pts with refractory or T315I+ chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia. Initial characterization of the in vitro kinase activity of ponatinib demonstrated substantial activity against a number of additional oncogenic kinases, including KIT, RET, FLT3, and members of the FGFR, PDGFR, and SRC families. To obtain a broad, unbiased, assessment of the anti-proliferative effects of ponatinib, we screened a panel of 246 human tumor cell lines. Based on the novel finding that a GCB-DLBCL cell line was amongst those inhibited most potently by ponatinib, we conducted studies to further characterize the activity of ponatinib in NHL, and GCB-DLBCL in particular.ResultsA broad cell-based screen identified a small subset of cell lines (18/246; 7%) whose growth was potently inhibited by ponatinib (GI50<42 nM). A majority of these lines express activated variants of previously validated targets of ponatinib: ABL (N=5, GI50 <0.3 nM), FLT3 (N=1, GI50 1 nM), FGFR2 (N=2, GI50s 5-29 nM), and PDGFRα (N=1, 14 nM). In addition, ponatinib potently inhibited growth of the GCB-DLBCL cell line DoHH2 (GI50 8 nM). The cellular activity of ponatinib was next examined in a larger set of NHL cell lines enriched for the GCB subtype (Table 1). Ponatinib only exhibited modest activity (GI50 46-119 nM) against 2 mantle cell lymphoma (MCL) lines, but potently inhibited growth (GI50≤10 nM) of the one Burkitt's lymphoma (BL) line tested (Daudi). Most notably, ponatinib also potently inhibited growth of 5/9 GCB cell lines. In contrast, none of the GCB lines showed sensitivity to ibrutinib (GI50s >100 nM). Finally, we evaluated the in vivo potency of ponatinib in mice implanted with the GCB cell lines exhibiting the greatest (SU-DHL-4) and weakest (SU-DHL-10) in vitro sensitivity to ponatinib, using dosing regimens previously shown to be active in BCR-ABL models predictive of efficacy in patients. Once-daily oral administration of ponatinib resulted in a dose-dependent inhibition of SU-DHL-4 tumor growth, with 10 mg/kg inducing 78% tumor regression, and 30 mg/kg rapidly inducing complete regression that was maintained in all mice for an additional 2 weeks after ponatinib dosing was stopped. In contrast, ponatinib had much more modest effects on SU-DHL-10 tumors with 30 mg/kg only inhibiting tumor growth by 39%.ConclusionPonatinib has promising in vitro and in vivo activity against a substantial subset of GCB-DLBCL models tested, with potency similar to that observed in BCR-ABL models. These results provide support for evaluating ponatinib in GCB-DLBCL pts who have failed prior therapy. Studies to further characterize the molecular basis for the activity of ponatinib in NHL are ongoing.Table 1In vitro drug activity in 12 NHL cell linesCell lineTypePonatinib GI50 (nM)Ibrutinib GI50 (nM)SU-DHL-4GCB DLBCL1.3313DoHH2GCB DLBCL2.5114PfeifferGCB DLBCL62,074SU-DHL-6GCB DLBCL9.81,041WSU-NHLGCB DLBCL101,672FarageGCB DLBCL511,409U-2932GCB DLBCL79>10,000RLGCB DLBCL2126,939SU-DHL-10GCB DLBCL2382,827DaudiBL2.94,319MinoMCL46>10,000Jeko-1MCL1194,781GI50: the concentration that causes 50% growth inhibition. DisclosuresGozgit:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed). Song:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed). Wardwell:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed). Nadworny:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed). Ning:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed). Rivera:ARIAD Pharmaceuticals Inc.: Employment, Other: Full-time Employee & Shareholder (self-managed).

Cost-Effectiveness of Subtype-Based Treatment Strategies for Diffuse Large B-Cell Lymphoma Patients (DLBCL)

BACKGROUND Patients (pts) with DLBCL may have widely divergent outcomes despite harboring histologically similar tumors. Gene expression profiling (GEP) and immunohistochemistry (IHC) algorithms can assign pts to the germinal center B-cell-like (GCB) or activated B-cell-like (ABC) subtypes, with the latter carrying a less favorable prognosis. While IHC is widely available, GEP is more accurate in characterizing the exact subtype, however it is largely limited to academic institutions due to cost and feasibility. The addition of novel agents such as lenalidomide to the standard regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) demonstrates the potential to improve outcomes for ABC DLBCL compared to historical controls (Nowakowski et al. J Clin Oncol. 2015). We examined the cost-effectiveness of using subtype-specific treatment strategies compared to RCHOP with or without a novel agent.METHODS We developed a Markov model to compare cost and effectiveness of 3 treatment strategies for pts 18-65 years of age with newly diagnosed DLBCL: (1) administering RCHOP to all pts, (2) administering lenalidomide+RCHOP (R2CHOP) to all pts or (3) performing subtype testing and administering RCHOP to pts with GCB and R2CHOP to pts with ABC. We calculated the costs and effectiveness of each strategy based on a clinical scenario with data derived from Nowakowski et al. 2015. The model utilized GCB/non-GCB-specific overall survival (OS) and progression-free survival (PFS) data for historical controls treated with RCHOP (strategy 1), and data from pts treated with R2CHOP from the phase 2 study (strategy 2). Strategy 3 utilized RCHOP outcomes for GCB pts and R2CHOP outcomes for non-GCB pts. Next, we conducted an exploratory analysis comparing these strategies in a hypothetical scenario. We used composite PFS and OS survival curves from our previous systematic review (Read et al. CLML 2014) for GEP-defined GCB and ABC pts, respectively, with RCHOP (strategy 1), and assumed various hazard ratios (HRs) representing hypothetical improvements in PFS and OS by adding a novel agent to RCHOP for ABC pts and no benefit (HR=1) for GCB pts (strategy 2 and 3). In addition to the gold standard GEP test, we considered practical IHC testing with potential subtype misclassification. Health outcomes were measured in life years (LYs) and quality-adjusted life years (QALYs). Drug and administration costs were based on average wholesale price and 2015 Medicare physician fee schedule. Model robustness was addressed in probabilistic sensitivity analyses (PSAs).RESULTS. In clinical scenario analysis, Strategy 1 provided 5.2 QALYs (6.7 LYs) at a cost of $69,920; strategy 2 improved health outcomes by providing 6.5 QALYs (8.5 LYs) at a cost of $143,753; and the subtype-based strategy 3 provided the greatest benefit of 6.9 QALYs (8.8 LYs) at a cost of $97,200. The incremental cost-effectiveness ratio (ICER) for strategy 3 was $16,881/QALY ($13,340/LY) compared with standard RCHOP, and strategy 2 was dominated since subtype-based treatment was more effective and less costly. PSA demonstrated 99.7% probability that subtype-based treatment was the most cost-effective strategy, at a willingness-to-pay value of $50,000/QALY. In the hypothetical scenario, with HR of PFS and OS for ABC pts with novel treatment varying from 0.5 to 0.9, strategy 3 produced ICERs ranging from $16,971/QALY ($13,992/LY) to $123,509/QALY ($108,000/LY) when compared with RCHOP. Similar findings were observed for models involving IHC subtype testing (see Table).CONCLUSIONS. There are multiple subtype specific diagnostic and treatment strategies emerging for pts with DLBCL. The gains in QALYs and LYs, in addition to favorable ICERs suggest that subtype-specific treatment strategies for DLCBL can provide meaningful clinical benefit and value and should be explored in future trials.Table. Incremental cost-effectiveness of subtype-based treatment Table 1GEP test (sensitivity =specificity=1.00)IHC Hans algorithm (sensitivity=0.82, specificity=0.90)PFS/OS HR for ABCIncremental LYIncremental QALYICER($/QALY)Incremental LYIncremental QALYICER($/QALY)Nowakowski et al. 2015 HR(PFS)=0.35, HR(OS)=0.242.422.016,9712.201.8120,526HR=0.5 (OS/PFS)1.741.4920,2551.591.3525,047HR=0.7 (OS/PFS)0.970.8336,4750.880.7645,015HR=0.9 (OS/PFS)0.280.25123,5090.260.23148,391 DisclosuresFlowers:Gilead: Research Funding; Spectrum: Research Funding; Roche: Other: unpaid consultant; Biogen Idec: Other: unpaid consultant; Optum Rx: Consultancy; Algeta: Consultancy; Millennium/Takeda: Research Funding; Abbott: Research Funding; Celgene: Other: unpaid consultant, Research Funding; Genentech: Other: unpaid consultant, Research Funding.

Mutational Spectrum of Stromal Genes By Whole Exome Sequencing and Stromal-Cellular Interaction in Diffuse Large B-Cell Lymphoma

Introduction:Morphological sub-classification of DLBCL into germinal center B-cell-like (GCB) and activated B-cell like (ABC) lacks sufficient reproducibility for risk stratification and predicting survival based on International Prognostic Index (IPI). The importance of stromal cells and their cross-talk with lymphoma cells is important in lymphomagenesis and progression. We postulate that mutations in stromal genes may regulate the rate of tumor progression in DLBCL and understanding the stromal-cellular interaction in DLBCL may help evolve better markers of prognostication and identify novel therapeutic targets.Methods:The study was approved by the Institute ethics committee and patients were enrolled after an informed consent. Frozen samples from 6 cases which included 4 cases of de novo nodal DLBCL (2 cases each of GCB and ABC subtypes as per Han's algorithm) and two control cases of non-neoplastic reactive lymphoid hyperplasia were subjected to AmpliSeqExome - Whole Exome Sequencing(WES) using Ion TorrentTM and compared against human reference genome (hg19). Torrent suiteTM 4.4 version (Life Technologies) and Ion ReporterTM software was used to perform tumor vs normal analysis by choosing variants for missense, frameshift insertions/ deletions, stoploss and single nucleotide variations (SNP). Filtering for common SNPs were done by UCSC common SNPs followed by inclusion in dbSNP and COSMIC and further filtered by predictive functional scores of SIFT < 0.05 and Polyphen (0.15 - 1.0). Variants were visualized using Integrated Genome Viewer (IGV) software. Bioinformatics analysis was done using PANTHER, STRING and PCViz and data mined for mutations in extracellular matrix genes. Selected genes were validated in DLBCL cases using Sanger sequencing BigDye terminator v3.1 cycle sequencing kit (Life Technologies) and ABI PRISM 3130 analyzer (Life Technologies). Further, in-vitro experiments were carried out to evaluate the effect of the conditioned media of CD19- stromal cells on the CD19+ lymphoma cells. Fresh tissue from 5 cases of nodal DLBCL were collected in RPMI 1640, 20% FBS and 1% penicillin and streptomycin and subjected to primary culture at 37°C and 5% CO2 humidified atmosphere. After 24 hours, the cultured cells were sorted into CD19+ and CD19- cells (BD FACSAriaTM) which were re-cultured for 24 hours separately. After this, the conditioned media of CD19- cells (comprising stromal cells predominantly) was transferred onto CD19+ cells and incubated for further 48 hours. Thereafter, cytokine (IL-6, IL-10, TNF-α, IFN-γ, IL-2, IL-4, IL-17) analysis of conditioned media in comparison to mock controls was performed using BD THI/THII kit (560484).Results:Analysis of filtered mutated genes using Panther revealed thirty four genes coding for extracellular matrix mutated specifically in GCB subtype and 26 mutated genes in ABC DLBCL. These genes were then analyzed for association in STRING. STRING identified LAMC3, AGRN, LAMA2, LAMB2, COL5A2, COL13A1,FN1,TGFB3, LTBP1 andADAMTS16 in GCB phenotype whereas LAMB1,LAMA3,COL4A2, COL28A1,COL5A3, IBSP, FGA, MUC6, MUC2, MUC5B,SPARCL1, VWF, GAS6 and USH2A were mutated in ABC DLBCL (Figure 1A and B). Mutations of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B were further validated using Sanger sequencing in individual DLBCL cases. This advocated the role of collagen scaffolding and laminin cross-linking in DLBCL progression. In vitro experiments for effect of stromal cells on the secreted cytokine profile revealed increased IL-6, IL-10, TNF-α, IFN-γ, IL-4, IL-17 levels respectively in the media of CD19+ sorted lymphoma cells upon addition of conditioned media of CD19-stromal cells in comparison to controls.Conclusion:Mutational profile of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B stromal genes in DLBCL may help in refinement of risk stratification based on morphology. The cross-talk between neoplastic cells and interacting tumor microenvironment cells has implications for therapy in DLBCL.GCB Mutation ProfileABC Mutation Profile [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of MYC, BCL2 and BCL6 Rearrangement and Protein Expression in GCB and Non-GCB Type Diffuse Large B-Cell Lymphoma

Background:Diffuse large B-cell lymphoma, not other specified (DLBCL, NOS) is the most common type of malignant lymphoma and accounts for approximately one-third of all non-Hodgkin lymphomas. Translocation of MYC, BCL2, and BCL6 genes detected by fluorescence in-situ hybridization (FISH) were found in approximately 10%, 14%, and 20% DLBCL, respectively. MYC translocation is already reported to be an independent poor prognostic factor in DLBCL. Immunohistochemical(IHC) analysis has revealed that concurrent protein expression of MYC and BCL2 could be a predictive factor for overall survival (OS). However, the relationship between translocation and expression of MYC, BCL2 and BCL6 is still unknown, and it is not clear what proportion of MYC and BCL2 IHC is predictive for OS.Objectives:The purpose of this study was to clarify the clinical prognostic value of immunostaining and chromosomal translocation of MYC, BCL2 and BCL6 among the populations in whom these results were investigated.Patients and Methods:Sixty-one adult patients, newly diagnosed as DLBCL, NOS between October 2003 and October 2012 at Niigata University Hospital, were analyzed retrospectively. MYC, BCL2 and BCL6 rearrangements were detected by FISH, and the expression of MYC, BCL2, and BCL6 proteins were investigated by IHC. To assess the proportion of these proteins, we created a tissue microarray (TMA). The median age was 62 years (range: 17-85 years), and the median follow up period was 42 months (range: 2-127 months). All patients were treated with R-CHOP or R-CHOP-like regimens. OS was estimated by the Kaplan-Meier method. Multivariate Cox regression for OS was used to identify the independent prognostic factors.Results:According to univariate analysis, MYC rearrangement (10%) was a prognostic factor (P = 0.026); however, BCL2 and BCL6 translocation were not prognostic indicators (11%, P = 0.899; 13%, P = 0.819, respectively). On the other hand, the expression of MYC detected by IHC showed no statistical significance for OS, even if the cut-off level by MYC and BCL6 immunostaining was modified. However, if we divided the patients into two groups, i.e., those with 0-9% and those with ≥10% expression of BCL2 immunostaining, ≥10% expression of BCL2 may be a prognostic factor (P = 0.0087). We subsequently analyzed whether the concurrent expression of MYC and BCL2 or that of MYC and BCL6 would be prognostic factors for OS. In this study, patients with ≥30% expression of MYC and ≥30% expression of BCL2 showed poor prognosis compared to other patients (P = 0.00234, 5-year OS 42%, 84% respectively). Furthermore, we divided the patients in two groups i.e., the germinal center B-cell-like (GCB) type and non-GCB type as described by Hans et al., and the non-GCB type was observed to be a poor prognostic factor in both groups (P = 0.013). Further, we investigated whether these factors could be independent factors for OS. Multivariate analysis revealed IPI3-5 (HR, 3.1510 [range: 1.181-3.151), P = 0.022), MYC translocation (HR, 3.686 [range: 1.113-12.210], P = 0.033), and MYC (≥30%)/BCL2 (≥30%) double-expression (HR, 4.051 [range: 1.447-11.340], P = 0.0078) were independent poor prognostic indicators in newly diagnosed DLBCL patients treated with R-CHOP or R-CHOP like regimens.ConclusionsMYC translocation by FISH and MYC (≥30%)/BCL2 (≥30%) double-expression detected by IHC could be independent prognostic factors for OS. However, MYC expression is not a surrogate marker for MYC translocation by FISH. In conclusion, FISH analysis of MYC translocation and MYC and BCL2 co-expression are important for predicting the prognosis of DLBCL. These results indicate that further validation is required using another population. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Diffuse Large B-Cell Lymphoma Lacking Surface B-Cell Receptor Expression Are Germinal Center B-Cell (GCB)-Type and Lack Evidence of Known Downstream Activating Mutations

Background: Diffuse large B-cell lymphoma (DLBCL) typically express monotypic surface immunoglobulin (sIg) light chain as part of the B-cell receptor (BCR) complex. Occasional cases are, however, sIg-negative and the clinical significance of this remains unclear. Distinct subtypes of DLBCL, including germinal center B-cell-like (GCB) and activated B-cell (ABC) type, exhibit differential dependence upon BCR-mediated survival signals. The role of activating mutations downstream of the BCR signaling complex (i.e. CD79A/B or CARD11), or in parallel costimulatory pathways (i.e. MYD88 mutations), are now well recognized in promoting such survival mechanisms. Surprisingly, however, less is known about the importance of sIg expression, or lack thereof, on various subsets of DLBCL or other aggressive large B cell lymphomas.Design: We sought to further characterize the clinicopathologic and genetic features of primary human surface Ig-negative DLBCL-not otherwise specified (NOS). All cases of large B-cell lymphoma that had flow cytometry performed at the University of Rochester from 2010-2013 were screened for evidence of a surface Ig-negative B-cell population. Additional available immunophenotypic data and/or clinicopathologic features were recorded. PCR heteroduplex analysis and sequencing of fresh frozen tumor samples was performed to identify putative compensatory activating mutations in MYD88 and CD79A /B, respectively. In addition, using multiplexed PCR and sequencing analysis we attempted to identify the presence or absence of functional immunoglobulin heavy chain (IGH)gene rearrangements.Results: Among 209 diagnostic specimens analyzed, 13 cases (6.2%) were found to contain a discrete population of CD19/CD20-positive B-cells lacking sIg light chain expression. These included 8 DLBCL-NOS, nearly all of which were GCB-type, including 6 out of 7 cases, or 83%, which were CD10-positive. This proportion is significantly greater than the 42% of all DLBCL-NOS that express CD10 (based on data from a large publicly available on-line database; p < 0.025, based on binomial probability). Furthermore, of the other high grade sIg-negative B cell lymphomas identified, 4 out of 5 were also CD10-positive. These included 2 cases of B-cell lymphoma, unclassifiable (BCL-U), intermediate between DLBCL and Burkitt lymphoma, and 2 cases of transformed follicular lymphoma (FL) - therefore also histologically and immunophenotypically consistent with derivation from a germinal center B-cell. The only exception was a non-GCB primary CNS lymphoma. Among 5 cases of sIg- DLBCL available for analysis, all were negative for the MYD88 L265P activating mutation, while only a single case contained an activating mutation in CD79A/B (CD79B Y196 ITAM mutation). In the same set, only 2 (of 5) cases produced amplicons by IGH PCR, and sequencing revealed only one of these had an intact reading frame.Conclusion: DLBCL-NOS lacking definitive sIg light chain expression are infrequent (less than 10%), but these tumors are predominantly GCB subtype. This observation lends support to the model that these tumors rely less on BCR-mediated survival signals, which may have implications for choosing targeted therapy. The mechanism by which these lymphoma survive despite the apparent lack of functional BCR is unknown, but compensatory activating mutations known to be frequent among ABC-type DLBCL appear to be lacking. DisclosuresNo relevant conflicts of interest to declare.

Pevonedistat Sensitizes Diffuse Large B-Cell Lymphoma Cells to Death Receptor Ligand-Mediated Apoptosis

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in the U.S. DLBCL can be subdivided into the activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subgroups on the basis of gene expression profiling results. ABC-DLBCL is characterized by the constitutive activation of nuclear factor-κB (NFκB) transcription factor and high expression level of its target genes, including pro-survival BCL2 family members, cell cycles proteins, and chemokines. We and others have demonstrated that pevonedistat (MLN4924), an inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in DLBCL cell lines and primary chronic lymphocytic leukemia (CLL) cells. Pevonedistat has been shown to sensitize solid tumor cell lines to extrinsic apoptosis. Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists. By contrast, GCB-DLBCL and primary CLL cells are not sensitive to this combination.Methods We employed a panel of ABC- (OCI-LY3, U2932) and GCB-DLBCL (SUDHL6, SUDHL10, VAL) cell lines. CLL B-cells were obtained from patients in the hematology clinics at the Knight Cancer Institute and were subjected to in vitro co-cultures with CD40L-expressing stroma which we have previously shown to activate NFκB and thereby mimic the lymph node microenvironment. We used the Fas agonists Apo-1-L, CH-11 and SuperFasLigand as well as KillerTRAIL. Pevonedistat was provided by Millennium Pharmaceuticals Inc., a subsidiary of Takeda Pharmaceutical Company Limited.Results Either Fas or TRAIL agonists, as well as 0.5 µM pevonedistat alone induced modest apoptosis of ABC-DLBCL cells (<10%) upon a 24 hour incubation. Meanwhile, pre-treatment of OCI-LY3 cells with 0.5 µM pevonedistat overnight resulted in apoptosis of 72.6±6.2% cells following exposure to 100 ng/mL KillerTrail and 79.6±7.5% - 10 ng/mL SuperFasLigand (p<0.0001 compared with either agent alone). Similar findings were observed in U2932 cells. By contrast, pevonedistat had a minimal effect on death receptor ligand-mediated apoptosis in the GCB-DLBCL cell lines.Using immunoprecipitation, we confirmed that Fas engagement led to rapid assembly of the death-inducing signaling complex in OCI-LY3 cells (within 2 hours). This was accompanied by activation of caspase-8, as manifested by generation of its active forms (p41/43). Pre-treatment with pevonedistat promoted sustained and prolonged Fas-mediated cleavage of caspase-8 (for 6 hours, as opposed to transient cleavage following isolated Fas stimulation), accompanied by caspase-mediated PARP processing. We found that pevonedistat treatment downmodulated the transcript and protein levels of cFLIP in OCI-LY3 cells, an antagonistic caspase-8 homolog, whose expression is highly dependent on NFκB transcriptional activity. This is consistent with earlier findings where abrogation of NF-κB transcriptional activity was the dominant event preceding pevodinestat-induced apoptosis in ABC-DLBCL cells. By contrast, in GCB-DLBCL cells, targeting neddylation resulted in accumulation of Cdt1, DNA re-replication and cell cycle arrest. Thus, preferential sensitivity of ABC-DLBCL to death receptor ligands in combination with pevonedistat may be dependent on the effects of the latter on NFκB signaling and cFLIP.We previously reported that, like in ABC-DLBCL, pevonedistat abrogates NFκB in CD40-stimulated primary CLL cells. We found that CD40 upregulated Fas and TRAIL receptors, as well as cFLIP, in CLL cells. While treatment with pevonedistat downregulated cFLIP in CLL cells, it did not sensitize CLL cells to Fas or TRAIL; caspase-8 cleavage and Bid processing were not induced. Conversely, neutralizing antibodies against TNFα and TRAIL-R1/2 did not modulate the pro-apoptotic activity of pevonedistat in CLL. Hence, distinct from observations in normal monocytes, pevonedistat did not enhance death receptor-mediated apoptosis, consistent with previous reports indicating that such signaling is dysfunctional in CLL cells.Conclusions Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists by a mechanism which includes downmodulation of cFLIP. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies. DisclosuresDanes:3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment. Spurgeon:Gilead sciences: Honoraria, Research Funding; Bristol Meyers Squibb: Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria; Janssen: Research Funding; Genentech: Honoraria. Berger:3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment.

GENO-27GENOMIC CHARACTERIZATION OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA REVEALS PREDOMINANT NONSYNONYMOUS SOMATIC MUTATIONS OF MYD88 AND PIM1 GENES

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to CNS. Despite low tendency of systemic dissemination, its prognosis is poor with median overall survival of 4 years when treated with standard of care high-dose methotrexate-based chemoradiotherapy. Despite majority of PCNSL is diffuse large B-cell lymphoma (DLBCL), its response to therapy and biological behavior differ from the systemic disease, necessitating detailed investigation of its pathogenesis and genetic alterations. Here we performed whole-exome sequencing (WES) on 41 PCNSL and paired normal specimens as well as RNA sequencing. Seven cases were classified as germinal center B-cell-like (GCB) subtype (17%), while 34 cases were non-GCB. Progression-free survival was significantly longer in cases with GCB subtype as in systemic DLBCL (p = 0.027). WES yielded sequencing data with a mean coverage of 158Χ with 98% of the targeted bases covered by ≥20 independent reads. We identified a total of 17,385 somatic mutations, of which 62% were nonsynonymous single nucleotide variations. The predominant nucleotide substitution was a C > T transition (57.8%), especially in the context of the sequence GCG. WES revealed a substantial extent of aberrant somatic hypermutation (aSHM) with a high frequency of nonsynonymous somatic mutations especially in PIM1 (90.2%) and BTG2 (65.9%). Genes mutated other than aSHM include MYD88 (85.4%), most of which being L265P activating mutation. These mutations were not associated with GCB/non-GCB subtypes. Notably, mutations in a number of genes related to the NF-κB signaling pathway were also prominent. Copy number aberration analysis identified gains in chromosomes 1q, 7, 12, and 18q, and a loss of 6q. These results suggest that MYD88 and PIM1 mutations may play an important role in PCNSL development regardless of GCB subtypes, and NF-κB pathway could be the therapeutic targets of this intractable disease.

High expression of REV7 is an independent prognostic indicator in patients with diffuse large B-cell lymphoma treated with rituximab

REV7 is a multifunctional protein involved in DNA damage tolerance, cell-cycle regulation, gene expression, and carcinogenesis. Although its expression is reportedly associated with poor prognosis in human solid tissue cancers, the significance of REV7 expression in hematopoietic malignancies is unclear. This study evaluated the prognostic significance of REV7 expression in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-combined chemotherapy. Using immunohistochemistry, we analyzed 83 specimens of de novo DLBCL [38 germinal center B-cell-like (GCB) and 45 non-GCB DLBCLs] treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone for REV7 expression. Aberrant REV7 expression was detected in DLBCL cell nuclei. High REV7 expression was associated with significantly shorter overall survival (OS) and progression-free survival (PFS) using Kaplan-Meier analysis and log-rank tests (P<0.01 and P<0.01, respectively). Multivariate analysis revealed that REV7 expression is an independent prognostic factor for both OS and PFS. Additionally, when patients were divided into four groups using a combination of REV7 expression and international prognostic index (IPI) or Bcl-2 expression, REV7(High)/IPI(Poor) and REV7(High)/Bcl-2(High) patients showed the poorest outcome. These results indicate that REV7 may be a useful biomarker to predict the prognosis of patients with DLBCL treated with rituximab.

Evaluation of NF-κB subunit expression and signaling pathway activation demonstrates that p52 expression confers better outcome in germinal center B-cell-like diffuse large B-cell lymphoma in association with CD30 and BCL2 functions

Nuclear factor-κB (NF-κB) is a transcription factor with a well-described oncogenic role. Study for each of five NF-κB pathway subunits was only reported on small cohorts in diffuse large B-cell lymphoma (DLBCL). In this large cohort (n=533) of patients with de novo DLBCL, we evaluated the protein expression frequency, gene expression signature, and clinical implication for each of these five NF-κB subunits. Expression of p50, p52, p65, RELB, and c-Rel was 34%, 12%, 20%, 14%, and 23%, whereas p50/p65, p50/c-Rel, and p52/RELB expression was 11%, 11%, and 3%, respectively. NF-κB subunits were expressed in both germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, but p50 and p50/c-Rel were associated with ABC-DLBCL. p52, RELB, and p52/RELB expressions were associated with CD30 expression. p52 expression was negatively associated with BCL2 (B-cell lymphoma 2) expression and BCL2 rearrangement. Although p52 expression was associated with better progression-free survival (PFS) (P=0.0170), singular expression of the remaining NF-κB subunits alone did not show significant prognostic impact in the overall DLBCL cohort. Expression of p52/RELB was associated with better overall survival (OS) and PFS (P=0.0307 and P=0.0247). When cases were stratified into GCB- and ABC-DLBCL, p52 or p52/RELB dimer expression status was associated with better OS and PFS (P=0.0134 and P=0.0124) only within the GCB subtype. However, multivariate analysis did not show p52 expression to be an independent prognostic factor. Beneficial effect of p52 in GCB-DLBC appears to be its positive correlation with CD30 and negative correlation with BCL2 expression. Gene expression profiling (GEP) showed that p52+ GCB-DLBCL was distinct from p52− GCB-DLBCL. Collectively, our data suggest that DLBCL patients with p52 expression might not benefit from therapy targeting the NF-κB pathway.

Abstract 108: Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy

Abstract Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL) representing more than 30% of new cases diagnosed in the United States. DLBCL comprises multiple biologically and clinically distinct subtypes including germinal center B-cell-like (GCB) and activated B-cell like (ABC) DLBCL. Despite improvement in current therapies, nearly 40% of patients die from the disease. Recent studies of the epigenome and the methylome in particular, have revealed widespread epigenetic changes in DLBCL. Aberrant methylation of core histone tails occur with deregulation of methylase enzymes such as Ezh2 in DLBCL. In contrast, the role of JMJD3 and UTX, H3K27(me)3 demethylases in DLBCL remains unknown. In this study, we have evaluated the activity of the selective JMJD3 inhibitor GSK-J4 in DLBCL and investigated the mechanism of its action. Methods Panel of DLBCL cell lines: DB, KARPAS 422, Pfeiffer, Toledo, Sudhl2, Sudhl6, Sudhl8, TMD8, OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly8, OCI-Ly4, OCI-Ly10, and primary DLBCL cells were analyzed for the expression of lysine demethylases (KDMs) using RT-PCR and Western blot analysis. Sub-G1 analysis was performed to analyze drug-induced apoptosis. Chemical inhibitor GSK-J4 and siRNAs were used for specific inhibition of the JMJD3. Results JMJD3 inhibitor, GSK-J4 induced the killing of DLBCL cell lines of both GC and ABC subtypes. Cell sensitivity to GSK-J4 could not be predicted from the expression of prognostic markers in DLBCL such as Bcl6, IRF4 and Ezh2. For further studies, we selected a cell line Sudhl6 (Bcl6 dependent), sensitive to the killing effect of GSK-J4. Treatment with GSK-J4 reduced the Bcl6 levels along with concomitant increase in IRF4 and FBXO11, negative regulators of Bcl6. Knockdown of JMJD3 by siRNAs also reduced the levels of Bcl6, implicating Bcl6 in the mechanism of action of GSK-J4. Additionally, GSK-J4 synergized in killing with vincristine, doxorubicin as well as proteasome inhibitors bortezomib and carfilzomib. Conclusion We demonstrate the potent effects of inhibition of H3K27(me)3 demethylase JMJD3 by GSK-J4 on proliferation and apoptosis of DLBCL cells. Our results clearly show synergistic effects of inhibition of demethylase with standard drugs in elimination of DLBCL cells. Note: This abstract was not presented at the meeting. Citation Format: Rohit Mathur, Lalit Sehgal, Zuzana Berkova, Sattva S. Neelapu, Felipe Samaniego. Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 108. doi:10.1158/1538-7445.AM2015-108

Abstract 3893: Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations

Abstract Primary central nervous system lymphoma (PCNSL) is defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system. Although PCNSL cannot be histologically distinguished from extracerebral DLBCL, its prognosis is quite poor. To gain insights into the transforming mechanism of PCNSL, we conducted whole-exome sequencing for 44 PCNSL specimens. Genomic DNA was extracted from tumors and their matched normal samples. Exome fragments were captured by using SureSelectXT Human All ExonV5+lncRNA, and subjected to deep sequencing with the HiSeq 2000 system. There were 8 cases with germinal center B-cell-like (GCB) subtype and 34 cases with non-GCB subtype. Whole-exome sequencing of tumor and paired normal yielded sequencing data with a mean coverage of 156X and 80X, with 98% and 99% of the targeted bases covered by ≥20 independent reads, respectively. Of 17,832 somatic mutations identified in the PCNSL specimens, 11,611 (65%) were nonsynonymous single nucleotide variations. Predominant nucleotide substitution was a C&amp;gt;T transition (57%) especially in the context of the sequence GCG. We also identified 798 insertions/deletions. Interestingly, MYD88 mutations were detected in 35 of 44 patients (79.5%), and PIM1 mutations were detected in 42 patients (95.5%). Also, a number of genetic aberrations were shown to activate the NF-κB pathway. These results suggest that MYD88 and PIM1 mutations have important roles in the development of PCNSL and could be the therapeutic targets of this intractable disorder. Citation Format: Motoo Nagane, Kazutaka Fukumura, Toshihide Ueno, Jeunghun Lee, Yukiko Shishido-Hara, Mitsuaki Shirahata, Kazuhiko Mishima, Koichi Ichimura, Akitake Mukasa, Yoshitaka Narita, Ryo Nishikawa, Hiroyuki Mano. Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2015-3893

Gene expression profiling of diffuse large B-Cell lymphomas supervised by CD5 expression.

CD5-positive (CD5(+)) diffuse large B-cell lymphoma (DLBCL) has a poor prognosis and high incidence of central nervous system (CNS) relapse, even in the rituximab era. To determine the gene expression profile of CD5(+) DLBCL, total RNA from 90 patients with DLBCL, including 33 CD5(+) DLBCL and 57 CD5-negative (CD5(-)) DLBCL patients, was examined using Agilent human oligo microarrays. These cases were separated into 78 activated B-cell-like (ABC) DLBCLs and 12 germinal center B-cell-like (GCB) DLBCLs. All cases of CD5(+) DLBCL were classified as ABC DLBCLs. The classifier based on gene expression used in a supervised analysis correctly identified CD5 expression in the DLBCL and ABC DLBCL samples. The gene most relevant to CD5 expression was SH3BP5. Enriched GO categories in the CD5(+) ABC DLBCL signature gene set included multicellular organismal signaling, transmission of nerve impulse, and synaptic transmission. The present study, which includes the largest reported number of patients with CD5(+) DLBCL, confirmed that most CD5(+) DLBCLs are ABC DLBCLs, suggesting that therapeutic strategies for ABC DLBCL may be effective for the treatment of CD5(+) DLBCL. Our CD5(+) ABC DLBCL signature gene set may provide insights into the cause of the high frequency of CNS relapse in CD5(+) DLBCL.