Activated B-cell-like Research Articles

Abstract IA09: Therapy of lymphoma inspired by functional and structural genomics

Abstract Diffuse large B cell lymphoma (DLBCL) is a heterogeneous diagnostic category that is comprised of two prominent molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB). These DLBCL subtypes are now viewed as molecularly distinct diseases since they arise from distinct stages of normal B cell development, require distinct recurrent genetic abnormalities to become malignant, have distinct cure rates with current chemotherapy regimens, and respond differentially to targeting agents. We defined a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as the kinase BTK or components of the BCR itself. Over one fifth of ABC DLBCLs have mutations affecting the CD79B or CD79A subunits of the BCR that augment BCR signaling. To attack chronic active BCR signaling therapeutically, we initiated clinical trials in relapsed/refractory DLBCL of ibrutinib, an irreversible and highly selective inhibitor of BTK. Ibrutinib monotherapy induced a high rate of complete and partial responses in ABC DLBCL, while GCB DLBCL tumors rarely responded. Genetic analysis of responding tumors demonstrated an enrichment for those with CD79B mutations, but most responses were observed in tumors with wild type BCR subunits. This observation allowed us to define a non-genetic mechanism of BCR activation that depends on reactivity of the lymphoma BCR to self antigens. In addition to chronic active BCR signaling, the signaling adapter MyD88 is activated by oncogenic mutations in 40% of ABC DLBCL cases. Since these mutant MyD88 isoforms act in parallel to BCR signaling to activate the NF-kB pathway in ABC DLBCL, they might have been predicted to mitigate the clinical effects of ibrutinib. On the contrary, ABC DLBCL tumors with both CD79B and MYD88 mutations were highly responsive to ibrutinib, leading us to demonstrate that the BCR and MyD88 pathways positively regulate one another. To extend the efficacy of ibrutinib, we have identified additional therapeutic targets in the oncogenic signaling pathways in ABC DLBCL including: 1) ubiquitin ligases, such as LUBAC, that are required to assemble active signaling complexes downstream of the BCR; 2) the kinase IRAK4, which mediates oncogenic MyD88 signaling; 3) the PI3 kinase pathway, which is activated by chronic active BCR signaling; 4) BCL2, which is expressed highly in most ABC DLBCL tumors and is the target of recurrent amplifications. Several synergistic, mechanism-based drug combinations that exploit these redundant survival pathways will be presented. Citation Format: Louis M. Staudt. Therapy of lymphoma inspired by functional and structural genomics. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr IA09.

Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34(+) ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.

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.

CD5-positive DLBCL: molecular basis and treatment strategies

CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) comprises 10% of DLBCL and is one of the immunohistochemical subgroups in the 2008 WHO classification. It shows many distinct clinical characteristics including elderly onset, advanced stage at diagnosis, high serum lactate dehydrogenase level and frequent involvement of extranodal sites. CD5+ DLBCL has a poor prognosis and a relatively high incidence of central nervous system (CNS) relapse even in the rituximab era. Eighty-three percent of patients who experienced CNS relapse had brain parenchymal disease. Immunohistochemically, 82% of CD5+ DLBCLs are classified as the non-germinal center B-cell type. BCL2 protein is positive in more than 70% of patients. P-glycoprotein, which is associated with multidrug resistance, was positive in 59% of patients with CD5+ DLBCL tested at Mie University Hospital. Based on gene expression profiling, most patients with CD5+ DLBCL are classified as activated B-cell-like (ABC) DLBCL. A more effective first-line therapy for CD5+ DLBCL needs to be explored. In Mie University Hospital, four patients with newly diagnosed stage IV CD5+ DLBCL were successfully treated with dose-adjusted (DA)-EPOCH-R combined with hi-dose methotrexate (HD-MTX). Based on these observations, a phase II study of DA-EPOCH-R/HD-MTX for newly diagnosed CD5+ DLBCL (PEARL5 trial) is ongoing in Japan.

IX. Is it only about MYC? How to approach the diagnosis of diffuse large B-cell lymphomas.

Keywords: diffuse large B-cell lymphoma; cell of origin; genetic alterations; double hit; double expresser; MYC; BCL2; BCLU

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.

Insights into the Molecular Pathogenesis of Activated B-Cell-like Diffuse Large B-Cell Lymphoma and Its Therapeutic Implications.

Within the last couple of years, the understanding of the molecular mechanisms that drive the pathogenesis of diffuse large B-cell lymphoma (DLBCL) has significantly improved. Large-scale gene expression profiling studies have led to the discovery of several molecularly defined subtypes that are characterized by specific oncogene addictions and significant differences in their outcome. Next generation sequencing efforts combined with RNA interference screens frequently identify crucial oncogenes that lead to constitutive activation of various signaling pathways that drive lymphomagenesis. This review summarizes our current understanding of the molecular pathogenesis of the activated B-cell-like (ABC) DLBCL subtype that is characterized by poor prognosis. A special emphasis is put on findings that might impact therapeutic strategies of affected patients.

Cell-of-Origin in Diffuse Large B-Cell Lymphoma: Are the Assays Ready for the Clinic?

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide and consists of a heterogeneous group of cancers classified together on the basis of shared morphology, immunophenotype, and aggressive clinical behavior. It is now recognized that this malignancy comprises at least two distinct molecular subtypes identified by gene expression profiling: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) groups-the cell-of-origin (COO) classification. These two groups have different genetic mutation landscapes, pathobiology, and outcomes following treatment. Evidence is accumulating that novel agents have selective activity in one or the other COO group, making COO a predictive biomarker. Thus, there is now a pressing need for accurate and robust methods to assign COO, to support clinical trials, and ultimately guide treatment decisions for patients. The "gold standard" methods for COO are based on gene expression profiling (GEP) of RNA from fresh frozen tissue using microarray technology, which is an impractical solution when formalin-fixed paraffin-embedded tissue (FFPET) biopsies are the standard diagnostic material. This review outlines the history of the COO classification before examining the practical implementation of COO assays applicable to FFPET biopsies. The immunohistochemistry (IHC)-based algorithms and gene expression-based assays suitable for the highly degraded RNA from FFPET are discussed. Finally, the technical and practical challenges that still need to be addressed are outlined before robust gene expression-based assays are used in the routine management of patients with DLBCL.

Research progress of CARMA1 gene in activated B cell-like subtype of diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive lymphoma. Activated B cell-like (ABC) subtype of DLBCL patients are characterized by high recurrence rate, low survival rate and poor prognosis. Continued excessive activation of Nuclear factor (NF)-κB signaling pathway is the most significant feature of ABC subtype of DLBCL which differentiate it from other subtypes of DLBCL, and it is also one of the key signaling pathways which lead to ABC subtype of DLBCL. In recent years, many research results confirmed that CARMA1 plays an important role in ABC subtype of DLBCL occurrence through the regulation of NF-κB signaling pathway. The article reviews literatures on CARMA1 function, gene mutation and post translation modification impact on ABC subtype of DLBCL. Key words: Lymphoma, large B-cell, diffuse; CARMA1 protein; NF-kappa B

Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Dysregulated CXCR4 expression promotes lymphoma cell survival and independently predicts disease progression in germinal center B-cell-like diffuse large B-cell lymphoma.

Abnormal expression of the chemokine receptor CXCR4 plays an essential role in tumor cell dissemination and disease progression. However, the significance of CXCR4 overexpression in de novo diffuse large B cell lymphoma (DLBCL) is unknown. In 743 patients with de novo diffuse large B cell lymphoma (DLBCL) who received standard Rituximab-CHOP immunochemotherapy, we assessed the expression of CXCR4 and dissected its prognostic significance in various DLBCL subsets. Our results showed that CXCR4+ patients was associated with male, bulky tumor, high Ki-67 index, activated B-cell-like (ABC) subtype, and Myc, Bcl-2 or p53 overexpression. Moreover, CXCR4+ was an independent factor predicting poorer progression-free survival in germinal-center B-cell-like (GCB)-DLBCL, but not in ABC-DLBCL; and in patients with an IPI of ≤2, but not in those with an IPI>2. The lack of prognostic significance of CXCR4 in ABC-DLBCL was likely due to the activation of p53 tumor suppressor attenuating CXCR4 signaling. Furthermore, concurrent CXCR4+ and BCL2 translocation showed dismal outcomes resembling but independent of MYC/BCL2 double-hit DLBCL. Gene expression profiling suggested that alterations in the tumor microenvironment and immune responses, increased tumor proliferation and survival, and the dissemination of CXCR4+ tumor cells to distant organs or tissues were underlying molecular mechanisms responsible for the CXCR4+ associated poor prognosis.

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

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

Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis.Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. [Display omitted] Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Strand-Specific Total RNA Sequencing Establishes the Complete Transcriptome and Alternative Splicing Repertoire in Diffuse Large B Cell Lymphoma

Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL.Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors.Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL.Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. DisclosuresMann:Quiagen: Research Funding.

The Bruton’s Tyrosine Kinase Inhibitor CC-292 in Diffuse Large B-Cell Lymphoma (DLBCL), T-Cell Lymphoma (TCL), and Hodgkin Lymphoma (HL): Induction of Cell Death and Examination of Rational Novel/Novel Therapeutic Combinations

Abstract Background: Recent clinical trials have highlighted the therapeutic benefit of BTK inhibitors in lymphoma with varying degrees of activity dependent in part on morphology and genetic origin. There appears to be a proclivity of activity in activated B-cell-like (ABC) DLBCL, while the efficacy appears more limited in germinal center (GC) DLBCL. In addition, the activity of BTK inhibitors is not clear in TCL or HL. We sought to determine the in vitro effects of the investigational BTK inhibitor CC-292 as a single agent and in combination with several targeted small molecule inhibitors in DLBCL, TCL, and HL. Methods: DLBCL cell lines (GC: SUDHL6, SUDHL10, Farage, and OCI-LY19; and ABC: OCI-LY3) were treated with increasing concentrations of CC-292 (0.1-20μM) alone and in combination with AKT inhibitor (AZD5363) and dual PI3K/mTOR inhibitor (BEZ235) for 24-72 hours. We analyzed cell viability with MTT assay and expression of NFκB, AKT, mTOR, MEK, and PARP by Western blot analysis. Calcusyn (Biosoft, Ferguson, MO) software program was used to analyze synergistic effects with drug combinations, based on isobologram and the method of Chou and Talay, a combination index (CI) value &lt;1 indicates synergism. We also examined the efficacy (cytotoxicity by MTT) of CC-292 (0.1-10μM) in L428 HL and in Jurkat and HH TCL cell lines. Results: Treatment with CC-292 resulted in a dose dependent decrease in cell viability in all DLBCL lines. The 50% inhibitory concentration (IC50) for Farage, OCI-LY19, SUDHL6, and SUDHL10 were 2.2μM, 6.3μM, 7.2μM, and 3.3μM respectively. Furthermore, treatment with 2.5 to 10μM of CC-292 effectively reduced phosphorylation of mTOR, AKT, and MEK, while it increased phosphorylation of NFκB (p65) and cleaved PARP in Farage, OCI-LY19, and SUDHL10. Treatment with CC-292 in combination with BEZ235 (PI3K/mTOR inhibitor) showed synergistic cell death in Farage cells (CI value at IC50, 75, and 90: 0.842, 0.731, and 0.661, respectively). Further, CC-292 treatment in combination with AZD5363 (AKT inhibitor) also resulted in synergistic cell death in Farage cells (CI value at IC50, 75, 90: 0.772, 0.771, 0.698) (Figure 1). In addition, CC-292 in combination with AZD5363 (AKT inhibitor) significantly down regulated phosphorylation of AKT and mTOR downstream targets, p70 S6K, and eIF4E by Western blotting compared with either drug as a single agent. Finally, in HL and TCL cells, CC-292 was highly effective in reducing cell viability with the following IC50 values in the presence of CC-292: 2.8μM and 2.2 μM for HH and Jurkat (TCL), respectively, and 7.1μM for L428 (HL) cells (Figure 2). Conclusion: In summary, we demonstrated in this preclinical investigation the efficacy of the BTK inhibitor, CC-292, as a single agent as well as in synergistic combinations with an AKT small molecule inhibitor and a dual PI3K/mTOR inhibitor at clinically relevant doses in ABC-DLBCL and multiple GC-DLBCL cell lines. Furthermore, these synergistic combinations decreased mTOR and AKT dependent phosphorylation to a significant extent. Additionally, we demonstrated the cytotoxic effect of CC-292 in HL and TCL cell lines. Collectively, these results suggests that CC-292 is active as a single agent in TCL and HL and novel/novel combinations with PI3K/mTOR or AKT inhibitors may have potential therapeutic value in the treatment of DLBCL. Figure 1: Farage lymphoma cells treated with CC-292, AKT inhibitor (AZD5363), or both and cell viability measured with MTT assay (72 hours). Figure 1:. Farage lymphoma cells treated with CC-292, AKT inhibitor (AZD5363), or both and cell viability measured with MTT assay (72 hours). Figure 2: T-Cell Lymphoma (HH and Jurkat) and Hodgkin Lymphoma (L428) cell lines treated with CC-292 and cell viability measured by MTT assay (72 hours). Figure 2:. T-Cell Lymphoma (HH and Jurkat) and Hodgkin Lymphoma (L428) cell lines treated with CC-292 and cell viability measured by MTT assay (72 hours). Disclosures No relevant conflicts of interest to declare.

Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues

Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues

ONO-4059—a Potent and Selective Reversible Bruton’s Tyrosine Kinase (Btk) Inhibitor: Single Agent, Twice Daily (BD) Dosing and Dosing with Food Results in Sustained, High Trough Levels of ONO-4059, Translating into 100% Tumour Remission in a TMD-8 Xenograft Model

Purpose: Bruton’s tyrosine kinase (Btk) is a key regulator of the BCR signaling pathway and abberant BCR signaling has been implicated in the survival of malignant B-cells. The activated B-cell-like (ABC) sub-type of Diffuse Large B-Cell Lymphoma (DLBCL) correlates with poor prognosis. There is still a high unmet, medical need for new therapies, preferably chemo-sparing, to help treat patients with ABC-DLBCL and CLL as well as other B-cell malignancies. ONO-4059 is a highly selective, orally bioavailable inhibitor of Btk kinase activity with a potency (IC50) of 2.2 nM. ONO-4059 reversibly blocks BCR signaling and B-cell proliferation and activation. Data from the ongoing Phase 1 study (ONO-4059POE001), where ONO-4059 is administered as monotherapy (QD) demonstrated a best overall response rate of 47% (7/15) in non-GCB DLBCL patients (Walter et al, ASCO 2014; Rule et al, EHA 2014). We hypothesized that the efficacy of ONO-4059 could be further enhanced by increasing the drug trough concentration. To address this, we examined different dosing regimens of ONO-4059 in an ABC-DLBCL xenograft model.Methods: TMD-8 tumour cells (ABC-DLBCL cell line) were implanted sub-cutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 100-200 or 400-450 mm3. ONO-4059 was administered orally or mixed in food at doses up to 20 mg/kg/day and animals were dosed QD or BD. Tumour volumes were measured twice a week after initiation of treatment, and tumour volumes were determined using the formula volume (= width2 x length)/2. Animals were euthanized when the tumours reached a maximum volume of 3,000 mm3.Results: For the100-200 mm3 tumour groups, tumour growth inhibition at the final treatment day was 23% in QD, 72.9% in BD and 100% in dose mixed in food, groups respectively. For the 400-450 mm3 tumour groups, no growth inhibition was observed in the QD group and, growth inhibitions of 27.5% in BD and 100% in dose mixed in food were observed. Interestingly, the treatment with ONO-4059-containing diets resulted in tumour remission in 10/10 animals, in both 100-200 and 400-450 mm3 treatment groups, whereas no tumour free animals were observed in the other treatment groups. The PK concentration and phosphorylated Btk (pBtk) inhibition levels of those animals whose dose was mixed in with food were higher than that of other treatment groups.Conclusion: Our previous study demonstrated that 100% tumour remission can be achieved partially with an ONO-4059 and GA101 or rituximab combination (Yoshizawa et al, ASH 2013). However, to date, no orally bioavailable targeted-agent administered as monotherapy has demonstrated 100% tumour remission in an advanced ABC-DLBCL xenograft model. Although the clinical data for ONO-4059 given as monotherapy (QD) is very encouraging in both relapsed and refractory CLL/NHL patients, this data indicates that a more frequent dosing regimen such as BD may be a more effective treatment, especially for non-GCB DLBCL and warrants further investigation in the clinical setting, along with food effect studies. DisclosuresYoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Yasuhiro:Ono Pharmaceutical Co., Ltd.: Employment. Honda:Ono Pharmaceutical Co., Ltd.: Employment. Kawabata:Ono Pharmaceutical Co., Ltd.: Employment.

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

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

BET Bromodomain Inhibitor OTX015 Affects the Expression of Micrornas Involved in the Pathogenesis of Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for 30%-40% of all cases. Despite a major improvement in the cure rate, a large number of DLBCL patients lack therapeutic options. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA, including microRNA (miRNA), contribute to DLBCL pathogenesis and represent potential therapeutic targets. OTX015 targets bromodomain and extra-terminal (BET) proteins, which are epigenetic readers contributing to gene transcription. It has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) and promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). To better understand the mechanism of action of OTX015, we studied molecular changes induced by this compound in DLBCL cell lines.Methods. Total RNA was extracted from 2 DLBCL cell lines, the germinal center B-cell (GCB) type DOHH2 and activated B-cell-like (ABC)-type SU-DHL-2, following treatment with 500 nM OTX015 or DMSO for 4h or 8h. RNA samples were labeled with cyanine-3 dye using the Agilent microRNA Complete Labeling System & Hyb Kit and hybridized to the Agilent Human microRNA microarray v.3. Raw expression values were obtained with Agilent Feature Extraction Software, log-transformed and normalized by the quantile method. Data were filtered to exclude relatively invariant features and those below the detection threshold. Limma (Linear Models for Microarray data analysis) was employed using R/Bioconductor and the filtered dataset. Baseline miRNA profiling was obtained from 22 DLBCL cell lines with the Nanostring nCounter Human v2 miRNA Expression Assay kit. Baseline gene expression profiling (GEP) was obtained in these cell lines with the Illumina HumanHT-12 v4 Expression BeadChip. Selected miRNA changes were validated by real-time PCR. Validated miRNA targets were retrieved using the miRWalk database (Dweep et al, 2011). Gene Set Enrichment Analysis (GSEA) software was used to assess enrichment of miRNA targets in the GEP datasets.Results. miRNA profiling of the GCB and ABC DLBCL cell lines exposed to OTX015 identified four downregulated miRNAs and eight which were upregulated. Among them, the oncomirs miR-92a-1-5p (log2 FC, -2.01; P=0.004) and miR-21-3p (log2 FC, -0.37; P=0.0045) were downregulated, while the tumor suppressor miR-96-5p (log2 FC, 0.39; P=0.041) was upregulated. Interestingly, changes of these miRNAs matched GEP variations of validated target genes (e.g., miR-92a-1-5p: CDKN1A, log2 FC, 0.81, CDKN2A, log2 FC, 0.81; miR-96-5p: MYC, log2 FC, -0.57, MYD88, log2 FC, -0.35). We then evaluated if these three miRNAs play a role in OTX015-sensitivity by obtaining baseline miRNA and GEP profiling data in 22 DLBCL cell lines. Compared to 8 cell lines with lower sensitivity to OTX015 (IC50 >500 nM), the 14 sensitive cell lines (IC50 <500 nM) presented lower miR-96-5p expression levels (log ratio, 2.12; P=0.026) and their GEPs were significantly enriched for validated miR-96-5p targets (normalized enrichment score, 1.4; P=0.026), suggesting miR-96-5p levels may predict response to OTX015.Conclusions. Changes in the expression levels of biologically relevant miRNAs may contribute to response to OTX015. miR-92a-1-5p, the oncomir which was most strongly downregulated by OTX015, is a member of the MYC target MIR17HG (mir-17-92 cluster), involved in the pathogenesis and chemo-resistance of lymphomas, mainly contributing to PI3K/AKT/mTOR pathway activation. Since the cell cycle transcriptional regulator E2F1 is targeted by mir-17-92, OTX015 may contribute to cell cycle arrest and to downregulation of the E2F1 target gene reported with BRD inhibitors in DLBCL cell lines. miR-21-3p, also downregulated by OTX015, is a well-known oncomir, and forced miR-21-3p expression in transgenic mice results in the development of leukemias and lymphomas. miR-96-5p, upregulated by OTX015, targets oncogenes such as RAS or MYC, and low expression has been reported in mantle cell lymphoma. Interestingly, low miR-96-5p baseline levels were associated with higher sensitivity to OTX015, an observation meriting validation in other tumor models and evaluation in clinical studies. DisclosuresStathis:Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

NFκB expression is a feature of both activated B-cell-like and germinal center B-cell-like subtypes of diffuse large B-cell lymphoma

The activation of nuclear factor kappa B (NFκB) transcription factor family is considered to have a key role in diffuse large B-cell lymphoma (DLBCL) pathogenesis and is associated with a specific molecular subtype, the activated B-cell-like (ABC) subtype. We evaluated the expression of NFκB by immunohistochemistry in a large series of DLBCL cases. The five different NFκB family members (NFκB1, NFκB2, RELA, RELB, and REL) showed a heterogeneous expression pattern with the vast majority of cases being positive for at least one factor. Two independent series of tumor samples were classified into germinal center B-cell-like (GCB) or ABC subtypes using different approaches, immunohistochemistry, or gene expression profiling, and the expression of NFκB family members was assessed. Notably, no significant differences regarding the expression of the different NFκB members were detected between the two subtypes, suggesting that NFκB signaling is a prominent feature not only in the ABC subtype, but also in the GCB tumors. Of the five transcription factors, only REL expression had a significant clinical impact on R-CHOP-treated diffuse large B-cell lymphoma, identifying a subgroup of patients with superior clinical outcome.