Aggressive Diffuse Large B-cell Lymphoma Research Articles

UCH-L1 is induced in germinal center B cells and identifies patients with aggressive germinal center diffuse large B-cell lymphoma

AbstractGene expression profiling has identified 2 major subclasses of diffuse large B-cell lymphoma (DLBCL). Cases resembling germinal center (GC) B cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-GC cells (activated B-cell DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here, we show that UCH-L1 reflects GC lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in GC B cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. We also find that UCH-L1 cooperates with BCL6 in a mouse model of GC B-cell lymphoma, but not with the development of multiple myeloma derived from post-GC cells. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biological diversity in this subset of disease. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in GC B cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL.

Targeting of BCL2 Family Proteins with ABT-199 and Homoharringtonine Reveals BCL2- and MCL1-Dependent Subgroups of Diffuse Large B-Cell Lymphoma.

To investigate the roles of BCL2, MCL1, and BCL-XL in the survival of diffuse large B-cell lymphoma (DLBCL). Immunohistochemical analysis of 105 primary DLBCL samples, and Western blot analysis of 18 DLBCL cell lines for the expression of BCL2, MCL1, and BCL-XL. Pharmacologic targeting of BCL2, MCL1, and BCL-XL with ABT-199, homoharringtonine (HHT), and ABT-737. Analysis of DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL. Immunoprecipitation of MCL1 complexes in selected DLBCL cell lines. Experimental therapy aimed at inhibition of BCL2 and MCL1 using ABT-199 and HHT, single agent, or in combination, in vitro and in vivo on primary cell-based murine xenograft models of DLBCL. By the pharmacologic targeting of BCL2, MCL1, and BCL-XL, we demonstrated that DLBCL can be divided into BCL2-dependent and MCL1-dependent subgroups with a less pronounced role left for BCL-XL. Derived DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL, as well as the immunoprecipitation experiments, which analyzed MCL1 protein complexes, confirmed these findings at the molecular level. We demonstrated that concurrent inhibition of BCL2 and MCL1 with ABT-199 and HHT induced significant synthetic lethality in most BCL2-expressing DLBCL cell lines. The marked cytotoxic synergy between ABT-199 and HHT was also confirmed in vivo using primary cell-based murine xenograft models of DLBCL. As homoharringtonine is a clinically approved antileukemia drug, and ABT-199 is in advanced phases of diverse clinical trials, our data might have direct implications for novel concepts of early clinical trials in patients with aggressive DLBCL.

Elucidation and Pharmacological Targeting of Novel Molecular Drivers of Follicular Lymphoma Progression.

Follicular lymphoma, the most common indolent subtype of non-Hodgkin lymphoma, is associated with a relatively long overall survival rate ranging from 6 to 10 years from the time of diagnosis. However, in 20% to 60% of follicular lymphoma patients, transformation to aggressive diffuse large B-cell lymphoma (DLBCL) reduces median survival to only 1.2 years. The specific functional and genetic determinants of follicular lymphoma transformation remain elusive, and genomic alterations underlying disease advancement have only been identified for a subset of cases. Therefore, to identify candidate drivers of follicular lymphoma transformation, we performed systematic analysis of a B-cell-specific regulatory model exhibiting follicular lymphoma transformation signatures using the Master Regulator Inference algorithm (MARINa). This analysis revealed FOXM1, TFDP1, ATF5, HMGA1, and NFYB to be candidate master regulators (MR) contributing to disease progression. Accordingly, validation was achieved through synthetic lethality assays in which RNAi-mediated silencing of MRs individually or in combination reduced the viability of (14;18)-positive DLBCL (t-DLBCL) cells. Furthermore, specific combinations of small-molecule compounds targeting synergistic MR pairs induced loss of viability in t-DLBCL cells. Collectively, our findings indicate that MR analysis is a valuable method for identifying bona fide contributors to follicular lymphoma transformation and may therefore guide the selection of compounds to be used in combinatorial treatment strategies.

Abstract B29: Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies

Abstract Class I family enzymes, histone deacetylases 1 and 2 (HDAC1,2), are targets of FDA-approved small molecule inhibitors currently used in cancer therapy. HDAC inhibitors impair DNA repair and replication in cancer cells to trigger genotoxic stress and cause cell death. How HDAC1,2 function in genome maintenance processes, DNA replication and repair, in mammalian cells is far from fully understood. Using small molecules to selectively inhibit HDAC1,2 activities, we have systematically defined their direct functions in DNA replication and repair. Our studies show that HDAC1,2 inhibition reduces replication fork velocity and activates the replication stress associated DNA damage independent of its effect on transcription. HDAC1,2 inhibition increases histone H4K16 acetylation (H4K16ac) and nuclease sensitivity of nucleosomes associated with newly replicated DNA, suggesting that HDAC1,2 are required for chromatin compaction during DNA replication. Thus, mechanistically, HDAC1,2 activities are required for maintaining nascent chromatin structure and dynamics required for efficient progression of the replication fork. In addition to H4K16ac, we have found that HDAC1,2 inhibition activities increases H3K27ac and H4K91ac present on the histone tail and core regions, respectively. Increase in these histone acetyl marks perturbs the acetyl-methyl or the acetyl-ubiquityl switch during DNA repair leading to impaired EZH2-mediated H3K27me3-dependent DNA repair signaling and the H4K91ubiquityl-dependent 53BP1-mediated DNA repair signaling in chemoresistant EZH2 gain-of-function diffuse large B-cell lymphoma cells. We are currently investigating the therapeutic benefits of HDAC1,2 inhibition in mouse models of aggressive chemoresistant diffuse large B-cell lymphoma and in primary patient samples. Overall, our studies have uncovered novel functions for HDAC1,2 in genome maintenance processes and provide insight into the mode-of-action of HDAC1,2 inhibitors in overcoming chemoresistance in cancer cells (via targeting the DNA repair process) and in inducing DNA damage and death in the rapidly cycling cancer cells (via targeting DNA replication). Note: This abstract was not presented at the conference. Citation Format: Danielle Johnson, Simon Jones, Steven Quayle, Jeff Shearstone, Srividya Bhaskara. Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B29.

Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice.

ABSTRACTHistone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). Here, we report that the Eμ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eμ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.

UCH-L1 Cooperates with BCL6 and Identifies Patients with Aggressive Germinal Center Diffuse Large B-Cell Lymphoma

Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. DisclosuresGalardy:Mission Therapeutics: Research Funding.

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

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

ABC, GCB, and Double-Hit Diffuse Large B-Cell Lymphoma: Does Subtype Make a Difference in Therapy Selection?

Personalized therapy for the treatment of patients with cancer is rapidly approaching and is an achievable goal in the near future. A substantial number of novel targets have been developed into therapeutic agents. There is a substantial variability to antitumor activity by novel therapeutics because of the unique heterogeneity and biology that exists both between and within lymphoma subtypes. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). Approximately 40% of patients have refractory disease or disease that will relapse after an initial response, and the majority of patients with relapsed DLBCL will succumb to the disease. There are two major biologically distinct molecular subtypes of DLBCL: germinal center B-cell (GCB) and activated B-cell (ABC). ABC DLBCL is associated with substantially worse outcomes when treated with standard chemoimmunotherapy. In addition to GCB and ABC subtypes, double-hit lymphomas (approximately 5% to 10% of patients) and double-expressor lymphomas, which overexpress MYC and BCL2 protein, are aggressive DLBCLs and are also associated with a poor prognosis. Double-hit lymphomas have concurrent chromosomal rearrangements of MYC plus BCL2 (or less likely, BCL6). Advances in molecular characterization techniques and the development of novel agents targeting specific subtypes of DLBCL have provided a foundation for personalized therapy of DLBCL based on molecular subtype. A number of early clinical trials evaluating combinations of novel targeted agents with standard chemotherapy (R-CHOP) have been completed and have demonstrated the feasibility of this approach with encouraging efficacy. As such, molecular classification of DLBCL is not only important for prognostication, but moves to center stage for personalization of therapy for DLBCL.

Clinical Characteristics and Outcome of 41 Patients with Upper GI Diffuse B-Cell Non-Hodgkin Lymphoma. Results of Brazilian-Italy Lymphoma Registry

Background: Gastrointestinal lymphoma represents only 1-4% of the malignant tumors in the gastrointestinal tract and is the most common extranodal site among all types of Non- Hodgkin Lymphomas (NHL). Only 3% of gastric neoplasms are primary lymphomas.Aim: Identification of factors with a potential prognostic impact among all cases with aggressive gastric diffuse large B-cell lymphoma - the clinical characteristics, the response to therapy and overall survival in the Brazilian-Italy Lymphoma registry.Patients and Methods: Between January 1st 2010 and May 1st 2014, 267 patients with DLBCL were recorded of which 41 (15%) had gastric involvement. The following parameters were evaluated to establish correlations with the response to treatment and survival data: clinical symptoms, serum albumin, serum lactate dehydrogenize (LDH), b2 microglobulin, presence of local regional adenopathies, bulky tumor, stage of disease, international prognostic index (IPIa), type of treatment – surgery plus chemotherapy.Results: A total of 41 patients, with a median age of 69 (28-86) years, were enrolled in this study, with a female: male ratio of 1.7:1. Their ECOG performance status was distributed in 44% grade 0; 34% grade 1; 14.6% grade 2 and 7.4% grade 3, whereas IPIa classification was 5 (12.2%) patients with high risk; 14 (34.2%) high-intermediate risk and 22 (53.6%) low- intermediate risk.Stage III-IV was presented in 66% of cases. LDH values were elevated in 17 patients (41.5%), b2 microglobulin value were elevated in 31 patients (75.6%). The test for identification of Helicobacter Pillory (HP) was available in 33 patients and was positive in 9 patients (27%). The most common symptoms included abdominal pain and weight loss (both 86%), dysphagia (80%) and nausea, vomiting (74%). Bulky were found in 27%, plus anemia (44%) and bleeding (25%). Endoscopic examination was realized in 94%, however, only 8% was submitted to surgery. 39 patients received chemotherapy (84% R-CHOP), one Brazilian case died early and one Italian case was lost follow up. Overall 17.5% (7/40) received radiotherapy. The median follow-up time was 14 (1-71) months, with 1-, 2- and 5-year survival rates of 73, 63 and 58%, respectively. It was found the 5-year overall survival (OS) rate differed significantly according to Anemia (p= 0.02) and LDH at the diagnosis (p< 0.0001). Among Brazilian and Italian patients the difference clinical characteristics were B symptoms (p=0.02); weight loss (p= 0.003); nausea/vomiting (p= 0.002); pain and dysphagia (p< 0.0001); showing a wider severity in Brazilian group. Although it did not mean a worst outcome. There was no statistically significant differences in terms of OS according to gender (p=0.19); B symptoms (p= 0.12); bleeding at the diagnosis (p= 0.08) and clinical stage (p= 0.14). Furthermore, the only confirmed factor that influenced OS applying Cox Regression was the LDH (HR 8.74, 95% CI: 2.32-32.8; p= 0.001).In conclusion, the median follow-up time of DLBCL group with gastric involvement does not differ in relation to overall DLBCL patients; nevertheless, it was found negative factors for these patients: anemia and increased LDH at the diagnosis. Furthermore, in the Brazilian patients the HP researching is necessary due the high frequency in this population. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Transplantation with CCR5 Δ32/Δ32 Cord Blood Hematopoietic Cells in a HIV-Infected Patient

Allogeneic HCT from a CCR5 Δ32/Δ32 unrelated donor by Hutter et al provides the only evidence to date of long term control of HIV infection, and a compelling argument in favor of homozygous CCR5 Δ32reconstitution as the key mechanism driving resistance to infection 6 years after stopping antiretroviral therapy (ART). Low prevalence of Δ32/Δ32 genotype (<1%) and stringent HLA criteria for conventional donors made the search for Òpatient number 2Ó unsuccessful for years. Here, we describe a case of allogeneic HCT using CCR5 Δ32/Δ32 cord blood (CB) cells in a patient with diffuse large B-cell lymphoma (DLBCL) and HIV infection.A 36 year-old man with HIV-1 infection from 2009 was diagnosed with DLBCL stage IIA in 2012. The patient had no illnesses associated with AIDS, good disease control and adherence to ART, with <50 HIV-1 RNA copies/mL and 360 CD4 T-cells/μL at DLBCL onset. Prior to HCT, the patient received CHOP-R (x6), ESHAP-R (x3), an autologous transplant with BEAM conditioning, GEMOX (x3), and abdominal radiotherapy. At HCT the patient had a refractory lesion in the left psoas, no B symptoms and good clinical performance. Baseline HIV-1 latent reservoir tests showed DNA copies in bulk and resting CD4 T cells and in GALT, CD4 T cell-associated HIV-1 RNA, free HIV-1 RNA in cerebrospinal fluid, replication competent viral size of 1.2 copies/107 CD4 T cells, and SCA (single copy assay) of 303 copies/mL. Patient’s HIV-1 was CCR5 tropic by both genotypic and phenotypic analyses.No HLA-matched sibling donors were available. Two CCR5 Δ32/Δ32 CB units (CBU) identified at the StemCyte inventory (Covina, CA) were HLA-compatible with the patient at the 4/6 level. A myeloablative CB alloHCT was performed using the single CCR5 Δ32/Δ32 CBU with higher cellularity (#1; Table 1) plus CD34+ cells from a haploidentical brother. The patient provided informed consent and IRB approved the alloHCT and research protocol. During HCT, ART combined abacavir, lamivudine and raltegravir. Neutrophil engraftment, 100% derived from the haploidentical donor, occurred on day +11. Post-thawing analysis of CBU#1 showed very poor clonogenic capacity, and donor switch to CB-origin failed, with persistently low CB-chimera <5% for up to 7 weeks (Figure 1). CBU#2 was infused on day +52, leading to a very rapid increase in CBU#1 chimerism, to 20% by day +59, 83% by day +66 and 100% by day +73. At this point, the patient’s hematopoiesis, including CD4 T-lymphocytes, became CCR5 Δ32/Δ32. Ultrasensitive plasma viral load analysis decreased right after HCT, reaching minimal levels at the time of full CB-chimera. No HIV-1 DNA was detected using droplet digital PCR quantification or semiquantitative tests of amplification. In addition, although recipient CD4 T cells responded to proliferation and activation stimuli, they were resistant to infection by the patient’s viral isolate as well as laboratory-adapted HIV-1 strains. Regretfully, our patient developed an aggressive DLBCL progression, and following very rapid clinical deterioration died from disease progression three months after HCT.Long term monitoring and target organs and tissues analysis of viral reservoir will not be possible in this case. However, our data suggest that CCR5 Δ32/Δ32 CB HCT can successfully eliminate HIV-1 and render the recipient’s T cells resistant to HIV-infection. Thus, they strongly support the use of CB as a platform for a broader application of this curative technology to other HIV-1 infected individuals.Abstract 1261. Table 1Patient, Donor and Cell Product CharacteristicsRecipientHaploidentical BrotherCBU #1CBU #2Year of birth or collection1975197120082008CCR5 genotypeWT / WTWT / Δ32Δ32 / Δ32Δ32 / Δ32HLA typing- HLA A01:01 / 68:0111 / 6801:01 / 03:0101:01 / 02:XX- HLA B08:01 / 07:0235 / 0708:01 / 07:0208:01 / 07:02- HLA DR03:01 / 13:0101 / 1303:01 / 14:0103:01 / 14:01Cell products contentPre-HCT ControlsPost Thawing #Pre-HCT ControlsPost Thawing #CD34 (x105/kg)−40 ¤0.70.520.730.22TNC (x107/kg)−−2.532.051.981.64CFU-GM (x104/kg)−−1.02 *0.110.12 *NilTotal CFU (x104/kg)−−2.13 *0.210.42 *Nil¤ Purified by Miltenyi positive selection to include <1 x104/kg CD3+ cells; * Quality controls performed prior to HCT at the transplant center’s laboratory in aliquot samples from segments attached to the units, as data were not available in the reports from the cord blood bank; # Subsequent quality controls in the final cell product after thawing the CBU for infusion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 81: Deregulated expression of HDAC9 in B cells leads to the development of lymphoproliferative disease and lymphoma

Abstract Aberrant epigenetics leading to changes in chromatin structure and patterns of gene expression is an important factor in cancer pathogenesis. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme that, within the haematopoietic system, is preferentially expressed in the B-cell lineage. Furthermore, HDAC9 expression is deregulated in B-lymphoid malignancies. To examine the role of HDAC9 in malignant B-cell transformation, we generated an in vivo mouse model that constitutively expresses human HDAC9 from early stages of B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eµ). In this study, we demonstrate that from an average of six months of age, Eµ-HDAC9 transgenic mice displayed splenomegaly and immunophenotypic features that resemble lymphoproliferative disease (LPD) in spleen. Later on in life, up to twenty-three months of age, 24% (19 out of 78) of Eµ-HDAC9 mice developed mature B-cell neoplasms such as splenic marginal zone lymphoma (SMZL) and aggressive diffuse large B-cell lymphoma (DLBCL) that could represent a progression stage of the observed lymphoproliferative disease (LPD) and monoclonal B-cell lymphocytosis (MBL). In contrast, a low background incidence of these malignancies was observed in control mice, 2% (1 out of 46) (p = 0.0008). The molecular pathogenesis of the Eµ-HDAC9 B-cell tumors seems to involve activation-deactivation of the BCL6-p53 axis by HDAC9 influenced deacetylation thus affecting DNA repair and cell cyle progression in germinal centre (GC) during immune response. Consistently, ZFN-mediated HDAC9-knockout results in cell cycle changes leading to strong inhibition of cell growth in vitro. In summary, aberrant expression of HDAC9 leads to the development of lymphoproliferative disorders, including GC and post-GC lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells and highlighting its importance as a therapeutic target. Citation Format: Veronica S. Gil. Deregulated expression of HDAC9 in B cells leads to the development of lymphoproliferative disease and lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2014-81

High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial.

The dosage of soluble programmed cell death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed in a prospective patient cohort. We evaluated the clinical impact of sPD-L1 level measured at the time of diagnosis for newly diagnosed diffuse large B-cell lymphoma (DLBCL). Soluble PD-L1 was measured in the plasma of 288 patients enrolled in a multicenter, randomized phase III trial that compared R-high-dose chemotherapy with R-CHOP. The median follow-up was 41.4 months. A cutoff of 1.52 ng/ml of PD-L1 level was determined and related to overall survival (OS). Patients with elevated sPD-L1 experienced a poorer prognosis with a 3-year OS of 76% versus 89% (P<0.001). Considering clinical characteristics, the multivariate analysis retained this biomarker besides bone marrow involvement and abnormal lymphocyte-monocyte score as independently related to poor outcome. sPD-L1 was detectable in the plasma and not in the serum, found elevated in patients at diagnosis compared with healthy subjects and its level dropped back to normal value after CR. The intention-to-treat analysis showed that elevated sPD-L1 was associated with a poorer prognosis for patients randomized within the R-CHOP arm (P<0.001). Plasma PD-L1 protein is a potent predicting biomarker in DLBCL and may indicate usefulness of alternative therapeutic strategies using PD-1 axis inhibitors.

Antigen expression patterns of MYC-rearranged versus non-MYC-rearranged B-cell lymphomas by flow cytometry

B-cell lymphomas with MYC rearrangement are a heterogeneous and unique group of neoplasms that may behave aggressively, especially in conjunction with other oncogenic alterations that cooperate with MYC, warranting early and intensive treatment in a subset of patients. Identification of MYC-rearranged B-cell lymphomas by immunophenotypic analysis could help guide cytogenetic work-up, as well as expedite therapeutic interventions. Using flow cytometry we analyzed the expression of CD10, CD19, CD20, CD38 and CD45 in 53 patients with three distinct neoplasms with MYC rearrangements: Burkitt lymphoma (n = 12), double hit lymphoma (n = 17) and diffuse large B-cell lymphoma (DLBCL) with MYC rearrangement (n = 8). The expression profile of these commonly used antibodies was similar among these three groups. The antigenic pattern of bright CD38 and dim CD45 was unique to the MYC-rearranged neoplasms when compared to non-MYC-rearranged aggressive DLBCL (n = 16), with a combined sensitivity of 67% and specificity of 100%, suggesting a shared underlying biology among these MYC rearranged B-cell lymphomas.

Oncogenic Y641 mutations in EZH2 prevent Jak2/β-TrCP-mediated degradation

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase β-TrCP (FBXW1). β-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs β-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of β-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind β-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that β-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Acquisition of MYD88 L265P mutation during treatment of diffuse large B cell lymphoma of the parotid gland

Dear Editor, Although the identification of mutations as an initial event is very difficult, the use of next-generation sequencing and high coverage in sequencing data have made it possible to distinguish early mutations from late events, based on the allelic frequency of the mutations. Since recurrently observed among certain subtypes of B cell malignancies, L256P mutation in MYD88 is regarded as one of the initial molecular events [1]. Ten years before death, the patient, a 68-year-old Japanese man, had a gradually growing mass in the left side of the neck. Pathological analysis of an excisional biopsy of the mass led to the diagnosis of primary malignant lymphoma in the parotid gland (the pathological diagnosis at that time was high-grade mucosa-associated lymphoid tissue (MALT) lymphoma that was retrospectively diagnosed as diffuse large B cell lymphoma (DLBCL) according to the 2011 WHO classification: CD10 negative, Bcl-6 positive, MUM1 negative, and 5 % of Ki67 labeling index; Fig. 1a–d). The patient received three courses of chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)) combined with radiotherapy. Although the combined therapy was effective at first, the patient suffered from exacerbation of preexisting chronic hepatitis B during the last course of chemotherapy. Anti-hepatitis B viral therapy with lamivudine was administered for 6 months. After being clinically recurrence-free of malignant lymphoma for 8 years, the patient again developed a mass in the left side of the neck. Biopsy revealed the presence of the non-germinal center B (GCB) subtype of DLBCL (CD10 (−), Bcl-6 (−), MUM1 (+)). From the clinical record, we confirmed that except for BCL2 amplification at the relapsed phase, neither translocations nor breaks were evident in BCL6, IgH, and MYC genes. Three courses of chemotherapy (rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP)) were ineffective, however, and the patient died of multiple organ failure. Autopsy revealed aggressive DLBCL infiltrating virtually all the organs, especially the liver (Fig. 1e–h) and the spleen. B cell monoclonality of DLBCL at the parotid K. Fujiishi :R. Kitazawa :Y. Nagai : T. Watanabe :R. Haraguchi : S. Kitazawa (*) Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan e-mail: kitazawa@m.ehime-u.ac.jp

Blood Soluble PD-L1 Protein In Aggressive Diffuse Large B-Cell Lymphoma Impacts patient's Overall Survival

PurposeProgrammed death 1 (PD-1) protein is a key immune-checkpoint receptor expressed by activated T cells and it mediates immunosuppression. It has been recently shown that the blockade of PD-1 or its ligand, PD-L1, by monoclonal antibodies may lead to significant antitumor effects. In diffuse large B-cell lymphoma PD-L1 has been reported expressed by tumor cells and PD-1 by tumor-associated T cells. The dosage of soluble programmed death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed during a clinical trial. We evaluated the clinical impact of PD-L1 level measured at the time of diagnosis for newly diagnosed aggressive diffuse large B-cell lymphomas. MethodsTranslating a previous study (NEJMed2004; 350: 1287) in the rituximab era, the Groupe Ouest-Est des Leucemies et des Autres Maladies du Sang (GOELAMS) 075 study is a multicenter randomized trial in which adults 18 to 60 years old with untreated histologically proved CD20 positive diffuse large B-cell lymphoma were randomly assigned to receive 8 courses of R-CHOP or, high-dose chemotherapy associated to rituximab followed by autologous stem cell support (clinicaltrials.gov: NCT00561379). Patients were required to have an Ann Arbor stage of I or II with bulk greater or equal to 7 cm or, III or IV with 0 to 3 adverse prognostic factors as defined by the age-adjusted International Prognostic Index. The population of interest consisted of 288 of the 340 consecutively enrolled patients with available plasma collected at the time of diagnosis before any treatment. Soluble PD-L1 was measured using an ELISA assay. The median follow-up was 41.4 months. ResultssPD-L1 levels were found significantly increased in the plasma collected at diagnosis for patients compared to healthy -gender and age matched- subjects' levels (P<0.0001). The optimal cut-off point for PD-L1 measured at diagnosis was found to be equal to 1.52 ng/mL. Eighty-nine (30.9%) patients had a PD-L1 level greater than the cut-off point as compared to two (3%) controls. The high-level sPD-L1 group was significantly associated with bone marrow involvement, more than one extranodal localization and, 2-4 performance ECOG status. None of the other patients' characteristics, including the assigned arms of randomization and, the positive vs. negative FDG-PET scan response at the intermediate evaluation, was associated with high PD-L1 level. The PD-L1 immunohistochemical analysis showed that 55 out of the 73 interpretable tissue microarrays presented at least 5% of PD-L1-positive tumor cells. No association was found between sPD-L1 and tumor PD-L1 expressions (p=0.5). The 73 patients were representative of our 288 patients' cohort, with significantly decreased overall survival for patients with a high level of sPD-L1 compared to those with low sPD-L1 levels (64.6 vs. 86.7%, P=0.007). Cell-of-origin analysis using Hans's criteria on the tissue microarrays did not shown any correlation between elevated sPD-L1 and GC or non-GC origin.Patients with elevated PD-L1 experienced a poor prognosis with a three-year overall survival of 76 vs. 89 percent (P<0.001).There was no difference of overall survival rates between the two arms of treatment. Univariate analysis identified age greater than 50 years old, high-intermediate and, high age-adjusted International Prognostic Index score, bone marrow involvement, more than one extranodal localization, Ann Arbor stage after FDG-PET scan of III or IV, abnormal lymphocyte-monocyte score and, high level of sPD-L1 as factors associated with poor survival. High level of sPD-L1, bone marrow involvement and, abnormal lymphocyte-monocyte score were found after multivariate analysis to be the only factors remaining significant after adjustment with a P-value <0.05. Soluble PD-L1 was detectable in the plasma and not in the serum, found elevated in lymphoma patients at diagnosis compared to healthy subjects and its level dropped back to normal value for patients in complete remission. The intention-to-treat analysis showed that elevated PD-L1 was associated with a poorer prognosis for patients randomized within the R-CHOP arm (P<0.001). [Display omitted] ConclusionSoluble PD-L1 protein expression in peripheral blood at the time of diagnosis is a potent predicting biomarker in diffuse large B-cell lymphoma and may indicate usefulness of alternative therapeutic strategies using PD1 axis inhibitors. Disclosures:Fest:Roche SA France: Research Funding.

Novel Targeting Of BCL2 and MYC DNA Secondary Structures In Diffuse Large B-Cell Lymphoma (DLBCL)

BackgroundThe most aggressive diffuse large B-cell lymphomas (DLBCL) display concurrent over-expression of the B-cell lymphoma gene-2 (BCL2) and cellular-myelocytomatosis (MYC) oncogenes. DLBCL with these features are nearly uniformly fatal due to the lethal combination of resistance to apoptosis and high proliferation. Currently, there are no effective strategies to treat these patients. We propose directly targeting BCL2/MYC transcription through DNA secondary structures to induce chemosensitization in BCL2/MYC overexpressing DLBCL cell lines. DNA secondary structures are proposed to act as molecular switches, turning gene expression on or off. In support of this, genome-wide analyses have detected DNA secondary structure-forming sequences within close proximity of transcriptional start sites in 43% of promoter regions, which were mostly oncogene promoters including BCL2 and MYC (Huppert et al., Nucleic Acids Res 2007). Directly upstream of the BCL2 and MYC promoters are guanine and cytosine (GC)-rich elements capable of transitioning to G-quadruplex and i-motif secondary structures, respectively.Previous high-throughput fluorescence resonance energy transfer (FRET) screening assays yielded a steroid derivative (NSC59276) and an ellipticine analog (NSC338258) from the NCI diversity library of small molecules. Compound NSC59276 targets the BCL2 i-motif and subsequently decreases mRNA and protein levels that leads to increased sensitivity in etoposide-resistant Burkitt's and mantle cell lymphoma cell lines in vitro and in vivo (Kendrick et al., Nature Chemistry, under revision 2013). Similarly for the MYC G-quadruplex, NSC338258 down-regulates MYC expression and induces cytotoxicity in Burkitt's lymphoma cell lines (Brown et al., J Biol Chem. 2011). Here, we assess the efficacy of the NSC59276 and NSC338258 as proof of principle for the targeting of BCL2/MYC concurrently in DLBCL, which has clinical implications for improving treatment for the most aggressive DLBCL. MethodsWe evaluated the cytotoxic and chemosensitizing effects of the BCL2 and MYC inhibitors using the MTS cytotoxcity assay in a DLBCL cell line model that consists of varied BCL2/MYC expression levels and mechanisms of regulation (Table 1). The cytotoxicity assays were conducted with either NSC59276 (BCL2i) or NSC338258 (MYCi) alone or concurrently in combination with standard chemotherapy agents for DLBCL (cyclophosamide and doxorubicin). ResultsThe simultaneous targeting of BCL2/MYC with BCL2i and MYCi in combination with cyclophosphamide treatment had a potent effect on decreasing cell viability by 23–64% compared to cyclophosphamide alone. This effect was observed in DLBCL cell lines that over-express BCL2/MYC regardless of mechanism of deregulation and was negligible in the HT cells that do not express detectable levels. Interestingly, the most significant decrease in cell viability was seen in cell lines with BCL2/MYC gene amplification. Furthermore, BCL2i and MYCi had a synergistic effect on lowering cyclophosphamide IC50 concentration by 30- to a 100-fold in cell lines, particularly of the ABC subtype. Cell viability and cyclosphosphamide IC50 were also decreased when cells were treated with BCL2i or MYCi; however, BCL2i alone had a more significant effect than MYCi. A similar, but less pronounced effect was observed with doxorubicin combination experiments. ConclusionThese data support our hypothesis that modulation of BCL2 and MYC with transcriptional repressors in combination with standard chemotherapy agents will improve the efficacy of chemotherapy alone. Further investigations that are ongoing will determine if the combination treated DLBCL cells are more significantly undergoing apoptosis compared to cyclophosphamide or doxorubicin alone. This is a novel, alternative approach of using small molecules that target BCL2 and MYC DNA secondary structures to down-regulate expression and act as chemosensitizing agents to improve current chemotherapy in refractory patients. Disclosures:Hurley:TetraGene: Consultancy, Equity Ownership.

A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G2/M arrest and apoptosis and resistance by reversible G1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

MicroRNA Profiling in Ocular Adnexal Lymphoma: A Role for MYC and NFKB1 Mediated Dysregulation of MicroRNA Expression in Aggressive Disease

Ocular adnexal lymphoma (i.e., lymphoma with involvement of the orbit, eyelids, conjunctiva, lacrimal gland, and lacrimal sac), although rare, is common among malignant tumors involving the ocular adnexal region. The main subtypes are low-grade extranodal marginal zone lymphoma (EMZL) and aggressive diffuse large B-cell lymphoma (DLBCL). In rare cases, low-grade EMZL are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal disease. Using LNA-based arrays from Exiqon, we performed global microRNA (miRNA) expression profiling of 18 EMZLs and 25 DLBCLs involving ocular adnexal sites to investigate changes in the miRNA expression in low- versus high-grade disease. Findings were confirmed by real-time quantitative PCR (RTq-PCR). Our analysis revealed 43 miRNAs with altered expression profiles in DLBCL compared to EMZL. Seven of the miRNAs down-regulated in DLBCL relative to EMZL showed enrichment for a direct transcriptional repression by the oncoprotein MYC. We also report a possible loss-of-regulation of NFKB1 and its downstream miRNAs. In addition, our analysis identified a group of DLBCLs whose expression profiles resembled that of EMZL. Although transformation of EMZL to DLBCL in the ocular adnexal region is rare, we hypothesize that the intermediate group potentially may derive from transformation of EMZL that was not recognized by histology. We conclude that fundamental differences in miRNA expression exist between ocular adnexal EMZL and DLBCL, mainly due to differences in MYC and NF-ĸB regulatory pathways.

Plasmablastic lymphoma of the oral cavity in a human immunodeficiency virus-negative patient: A case report with literature review

Plasmablastic lymphoma (PBL) is an aggressive diffuse large B-cell lymphoma variant that is most frequently observed in the oral cavity of human immunodeficiency virus (HIV)-infected individuals. However, in recent years, some cases have emerged in patients without HIV infection and involve other sites like stomach, lung, nasal cavity, and jejunum. We report a rare case of PBL in the maxillary anterior area of a 62-year-old man without HIV infection. The tumor cells were characterized by non-cohesive round or oval shape cells with eccentrically-placed nuclei with a prominent perinuclear halo. Immunohistochemical studies showed that the tumor cells were strongly positive for MUM1, VS38c, VMT, and κ light chain, focally positive for LCA and CD79a, and negative for CD3, CD20, CD56, λ light chain, CK-pan, EMA, and HMB45. The patient was treated with chemotherapy using cyclophosphamide, doxorubicin, vincristine, and prednisone. The lesion showed partial remission.