Germinal Center B-cell Lymphoma Research Articles

Protein Kinase C Delta in Diffuse Large B-Cell Lymphoma Post-Rituximab Era: Potential Prognostic Marker in Germinal Center B-Cell Lymphoma

Background: Diffuse Large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma. Acquirement of resistance in DLBCL is a major clinical problem. The identification of biomarkers of clinical behavior may aid in the implementation of novel therapeutic strategies. Protein Kinase C (PKC) delta is a class serine/threonine kinase among the PKC family and contributes to cell proliferation, survival and apoptosis. The role of PKC delta in apoptosis is still controversial. Previously studies reported that overexpressed PKC delta promoted apoptosis. However, recently data showed inhibition of PKC delta blocked proliferation and survival of cancer stem cells in vitro and in vivo . The value of PKC delta expression in lymphoma patients remains unclear. We conducted a retrospective study to investigate the impact of PKC delta expression in DLBCL patients. Methods: Histologically confirmed DLBCL patients (N=98) treated at Fudan University Shanghai Cancer Center with available diagnostic biopsy material were identified. Forty-three patients were treated with CHOP and fifty-nine patients with R+CHOP. PKC delta expression was determined and quantified by immunohistochemistry (IHC) with staining intensity or percentage of positive cells. Tumors were considered positive when at least IRS>9 of tumor cells expressed PKC delta. Kaplan-Meier method was used to calculate progression free survival (PFS) and overall survival (OS) and curves were compared with the long-rank test in both groups. Correlation between the PKC delta expression and clinical variables were tested by Pearson Chi Square test and a two-sided P value of <0.05 was considered to be statistically significant. Additionally, PKC delta was inhibited using a PKC delta inhibitor (Rotterin) in DBCL cell lines. Changes in cell viability following in vitro exposure to rotterin were determined by Presto Blue Assay. Result: In CHOP treatment DLBCL, PKC delta expression was detected in 10/41 (24.4%) DLBCL patients. PKC delta expression was associated with a worse progression free survival (PFS) compared to PKC delta negative patients (50% vs 77%, p=0.036) treated with CHOP. The overall survival (OS) of PKC delta positive patients was also worse compared to the negative group (50% vs. 80%, p=0.023). In rituximab-CHOP (R-CHOP) treated patients, PKC delta was present in 17/57 (29.8%) patients. Surprisingly, no differences between the PKC delta positive group and negative R-CHOP treated groups were observed in terms of PFS or OS. Of interest, a subset analysis of R-CHOP treated DLBCL patients base on the cell of origin (COO) demonstrated that PKC delta expression in germinal center B-cell lymphoma (GCB) DLBCL was associated with an inferior OS outcome. The OS was 67% for GCB-DLBCL expressing PKC delta vs 83% for GCB-DLBCL patients without PKC delta expression (p=0.01). To identify the contribution of PKC delta to the survival of B-cell lymphoma, we performed the in vitro viability assay in different subtype of lymphoma cell lines using Rotterin, a PKC delta inhibitor. We found that in vitro exposure of cell lines to rotterin resulted in cell death in vitro suggesting that PKC delta can serve as a targetable biomarker in DLBCL. Additional pre-clinical studies are underway. Conclusion: This is the first report demonstrating that, PKC delta expression is a negative predictor of clinical outcome. PKC delta is a potential novel target to treat lymphoma and further investigation of PKC delta need to be done in the near future. (Research supported by Shanghai Municipal Commission of Health and Family Planning Project (no.20134030), National Natural Science Foundation of China (no.81670177, no.81001048), Roswell Park Cancer Institute Alliance Grant) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-Clinical Development of Targeted Therapies for Double Hit (DH) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Gene expression profile classifies DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal B-cell lymphoma (PMBL). DH-DLBCL is a distinct subgroup, with a poor clinical outcome, harboring concurrent gene translocations involving the C-MYC and either/both BCL2 or BCL6 proto-oncogenes. DH-DLBCL has inferior response rates to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). In addition, pts with DH-DLBCL who are refractory to frontline chemoimmunotherapy fare dismally; a retrospective review at our institute yielded survival rates of zero and median OS of 6 months in these patients, highlighting the need for novel therapies. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, leading to rapid proliferation of neoplastic cells, and resistance to apoptotic stimuli. Upregulation of the anti-apoptotic protein Mcl-1 is implicated in chemotherapy resistance and poor clinical outcomes in ABC-DLBCL. Transcriptional regulation of MCL1 by c-MYC might further potentiate Mcl-1 induced resistance to chemotherapy in DH-DLBCL. Specific inhibitors targeting cell pro-survival proteins might provide a novel and alternative therapeutic approach in rituximab-chemotherapy resistant DH-DLBCL. We hypothesize that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 using readily available agents (Venetoclax and Proteasome inhibitors) is an effective strategy in inducing lymphoma cell death. Materials and Methods: We conducted pre-clinical in vitro and in vivo models targeting Bcl-2 with Venetoclax and Mcl-1 with either a c-Myc inhibitor (JQ-1) or Proteasome inhibitor (Carfilzomib) in DHL. Val, DOHH-2, and ROS-50 cells were exposed to Venetoclax (0-10 uM), JQ-1 (0-100 uM) and Carfilzomib (CFZ) (0-50 nM) for 24, 48 and 72 hours. IC50 drug concentrations were calculated for each agent. Subsequently, DHL cells were exposed to Venetoclax in combination with JQ-1 or CFZ for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) and changes in Bcl-2 family members and Caspase activation were determined by Western blotting. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including DHL were exposed ex vivo to Venetoclax +/- Carfilzomib. For in vivo experiments 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 ROS-50 cells via tail vein injection (IV). Subsequently, animals were treated with Venetoclax (100mg/kg/dose via gastric lavage on days 3-7 and 10-14); Carfilzomib (2mg/kg/dose IV days 3, 4, 10, 11), or both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro, Venetoclax, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both Venetoclax and CFZ and to a lesser degree with JQ-1. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Annexin/PI staining with the combination of Venetoclax and CFZ resulted in caspase dependent apoptosis. Western blotting confirmed changes in Bcl-2 family members favoring an apoptotic state as well as PARP cleavage following in vitro exposure to Venetoclax and CFZ. In vivo the combination of Venetoclax with Carfilzomib resulted in significant increased survival increase (P<0.05) when compared to single agent Venetoclax or CFZ. Conclusion: Our data suggests that Venetoclax exhibits strong synergistic activity with Carfilzomib in vitro and in vivo . Perhaps related to the synergy observed between Venetoclax and CFZ, is the effects of proteasome inhibition on Mcl-1 levels. Combination therapy targeting the proteasome inhibition and BCL-2 may provide an alternative therapeutic approach in DH-DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Implementation of a Public Lymphoma Clinic in a Private Hospital in Brazil: Challenges and Impact of Delayed Diagnosis in Patients with Diffuse Large B Cell Lymphoma

Introduction: Since 1990, Brazil has implemented a unified public health care, but access to specialized care remains a major challenge, especially due to delays in diagnosis and access to specialized centers. In order to help cancer care access, Hospital Israelita Albert Einstein, a private hospital, has implemented a public cancer clinic in Sao Paulo, only 11km apart. In an unprecedent situation in Sao Paulo, both public and private hospitals share most of the facilities, including laboratory, pathology, radiology, radiotherapy, pharmacy and medical team. Moreover, treatment protocols are the same and most drugs are also available in the public system. With 1 year of operation of the public clinic, we aim to analyze the outcome of patients with diffuse large B-cell lymphoma (DLBCL) compared with historical data from the private hospital. Moreover, since patients are only admitted after a confirmed cancer diagnosis, we aim to look at the impact of delayed diagnosis in the outcome of the patients. Method: This is a retrospective study comparing the clinical features, treatment and outcome of patients with DLBCL treated in the public system with historical data from the private system. In order to minimize the impact of treatment protocols, only patients treated between January 2016 and February 2020 were used for historical comparison. All biopsies were reviewed by the same hemepathologist. Time to treatment initiation (TTI) time was defined by the number of days between biopsy and the first day of treatment cycle. Time from symptom onset to medical consultation (TSTC) was also calculated. Survival rates were estimated by Kaplan-Meier and differences assessed by log-rank test. Results: In one year of operation, 23 patients with DLBCL were treated in the public clinic (PC) and were compared with 41 patients from the private hospital (PH), for a total of 64 patients. Clinical features were similar between PC and PH, and for all patients, median age was 61 years (range: 27-91), 76% with PS of 0-1, 78% with advanced stage and 45% with B-symptoms. Median R-IPI was 3 for both cohorts and approximately 50% of patients in both cohorts presented with R-IPI of 3 to 5. Interestingly, a difference in the cell-of-origin (COO) was observed between both cohorts, with a higher number of germinal center B-cell lymphoma in the PC (82% vs 52%, respectively, p=0.0041). TSTC was significantly higher in the PC compared to PH (160 vs 30 respectively, p&amp;lt;0.001), and it was mostly impacted by the delay in obtaining the biopsy result. Moreover, TTI was higher in the PC compared with PH (7 vs 39, respectively, p&amp;lt;0.0001) and was most impacted by the delay in receiving biopsy slides for review. With a median follow up of 18 months, 1 year-EFS was 76% and 1 year-OS was 86%for all cohort, with no statistical difference between public and private hospitals (p=0.40 and 0.20, respectively). Conclusion: In our cohort of patients from public and private settings treated equally by the same resources, there was no difference in the outcome of patients. Despite TSTC and TTI were higher for patients from the public setting, no difference in survival was observed, and this may be partially explained by an enriched cohort of GCB DLBCL in the public system. The extremely low number of ABC DLBCL (4/23) is uncommon and suspicious for mortality of these patients before access to specialized care. Figure Disclosures Perini: Janssen, Takeda: Honoraria; AbbVie, Janssen: Speakers Bureau.

A LYSA Phase Ib Study of Tazemetostat (EPZ-6438) plus R-CHOP in Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma (DLBCL) with Poor Prognosis Features

The histone-methyl transferase EZH2, catalytic subunit of the PRC2 complex involved in transcriptional regulation, is mutated in approximately 25% of germinal center B-cell lymphomas. Aberrant proliferative dependency on EZH2 activity can be targeted by the orally available EZH2 inhibitor tazemetostat (EPZ-6438). We report the results of the phase Ib tazemetostat plus R-CHOP combination (NCT02889523), in patients 60 to 80 years of age with newly diagnosed diffuse large B-cell lymphoma. The primary objective of this dose-escalation study was to evaluate the safety of the combination and to determine the recommended phase II dose (RP2D) of tazemetostat. A total of 17 patients were enrolled. During C1 and C2, two dose-limiting toxicities were observed: one grade 3 constipation at 400 mg and one grade 5 pulmonary infection at 800 mg. Grade 3 or more toxicities observed in more than 10% of the patients were constipation (24%), nausea (12%), and hypokalemia (12%). Grade 3 to 4 hematologic adverse events were recorded in 8 patients (47%): neutropenia (47%), leukopenia (29%), anemia (18%), and thrombocytopenia (12%). The tazemetostat RP2D was 800 mg. No organ-oriented toxicity increased with tazemetostat dosage escalation (severity and incidence). At 800 mg, AUC and Cmax of tazemetostat were similar compared with the single-agent study (E7438-G000-101). The RP2D of tazemetostat combined with R-CHOP is 800 mg twice a day. The association presents safety and PK comparable with R-CHOP alone. Preliminary efficacy data are encouraging and further investigations in phase II trial are warranted.

CREBBP and STAT6 co-mutation and 16p13 and 1p36 loss define the t(14;18)-negative diffuse variant of follicular lymphoma

The diffuse variant of follicular lymphoma (dFL) is a rare variant of FL lacking t(14;18) that was first described in 2009. In this study, we use a comprehensive approach to define unifying pathologic and genetic features through gold-standard pathologic review, FISH, SNP-microarray, and next-generation sequencing of 16 cases of dFL. We found unique morphologic features, including interstitial sclerosis, microfollicle formation, and rounded nuclear cytology, confirmed absence of t(14;18) and recurrent deletion of 1p36, and showed a novel association with deletion/CN-LOH of 16p13 (inclusive of CREBBP, CIITA, and SOCS1). Mutational profiling demonstrated near-uniform mutations in CREBBP and STAT6, with clonal dominance of CREBBP, among other mutations typical of germinal-center B-cell lymphomas. Frequent CREBBP and CIITA codeletion/mutation suggested a mechanism for immune evasion, while subclonal STAT6 activating mutations with concurrent SOCS1 loss suggested a mechanism of BCL-xL/BCL2L1 upregulation in the absence of BCL2 rearrangements. A review of the literature showed significant enrichment for 16p13 and 1p36 loss/CN-LOH, STAT6 mutation, and CREBBP and STAT6 comutation in dFL, as compared with conventional FL. With this comprehensive approach, our study demonstrates confirmatory and novel genetic associations that can aid in the diagnosis and subclassification of this rare type of lymphoma.

Mass cytometry defines distinct immune profile in germinal center B-cell lymphomas.

Tumor-associated macrophage and T-cell subsets are implicated in the pathogenesis of diffuse large B-cell lymphoma, follicular lymphoma, and classical Hodgkin lymphoma. Macrophages provide essential mechanisms of tumor immune evasion through checkpoint ligand expression and secretion of suppressive cytokines. However, normal and tumor-associated macrophage phenotypes are less well characterized than those of tumor-infiltrating T-cell subsets, and it would be especially valuable to know whether the polarization state of macrophages differs across lymphoma tumor microenvironments. Here, an established mass cytometry panel designed to characterize myeloid-derived suppressor cells and known macrophage maturation and polarization states was applied to characterize B-lymphoma tumors and non-malignant human tissue. High-dimensional single-cell analyses were performed using dimensionality reduction and clustering tools. Phenotypically distinct intra-tumor macrophage subsets were identified based on abnormal marker expression profiles that were associated with lymphoma tumor types. While it had been proposed that measurement of CD163 and CD68 might be sufficient to reveal macrophage subsets in tumors, results here indicated that S100A9, CCR2, CD36, Slan, and CD32 should also be measured to effectively characterize lymphoma-specific tumor macrophages. Additionally, the presence of phenotypically distinct, abnormal macrophage populations was closely linked to the phenotype of intra-tumor T-cell populations, including PD-1 expressing T cells. These results further support the close links between macrophage polarization and T-cell functional state, as well as the rationale for targeting tumor-associated macrophages in cancer immunotherapies.

Targeting MDM2 and XIAP By Idasanutlin in Diffuse Large B-Cell Lymphoma

Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

Molecular Background of Double Protein Expressor B-Cell Lymphomas

Introduction: Concomitant deregulation of MYC and BCL2, whether at the genomic or protein level, comprises clinically a significant biological high-risk feature in aggressive B-cell lymphomas. Yet, the molecular background of these lymphomas is not fully understood. We sought to comprehensively characterize BCL2 and MYC aberrations and to identify clinically feasible determinants of the recent genomic subtypes and clinical outcome in diffuse large B-cell lymphoma (DLBCL). Patients and Methods: We analyzed BCL2, BCL6 and MYC rearrangements and corresponding protein expression in the context of somatic exome-wide driver gene alterations, copy number annotations, transcriptome and clinical data in 181 patients with primary DLBCL. Results: We found that germinal center B-cell (GCB) and activated B-cell (ABC)-like DLBCLs utilized different mechanisms to deregulate MYC and BCL2 expression. While the structural variations of BCL2 were subtype-specific and specifically increased antiapoptotic BCL2 expression, MYC deregulation was associated with gene expression signatures including biosynthetic pathways and telomerase activity and depletion of JAK-STAT, cytokine and immune cell signatures. Genomic dissection of MYC translocations and protein overexpression revealed associations with other lymphoma drivers, of which we highlight a novel interplay between MYC deregulation and TP53 alterations. Double protein expression (DPE) arose from different molecular backgrounds in a subtype-dependent manner. In GCB-DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs. Importantly, we recognized more GCB lymphomas with double hit -like clinical behavior when BCL2 copy number alterations were considered. In the non-GCB DLBCLs in turn, concurrent alterations were rare and clinical behavior of DPE was dependent on the molecular context. Interestingly, BN2/C1 -like non-GCB lymphomas with BCL6 translocations had excellent outcomes regardless of the DPE status. Overall, DPE DLBCL defined a prognostic entity, which was independent of International Prognostic Index (IPI) and cell-of-origin, and together with TP53 alterations had synergistic dismal impact on survival. Conclusion: Our findings elucidate the pathogenesis and modern biological determinants of high-risk DLBCL and reveal subtype-specific and clinically feasible improvements to risk-stratification and predictors of outcome. Disclosures Leppa: Celgene: Consultancy; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding.

Clinical impact of the cell-of-origin classification based on immunohistochemistry criteria and Lymph2Cx of diffuse large B-Cell lymphoma patients in a South-east Asian population: a single center experience and review of the literature

ABSTRACTBackground: Previous studies in Western populations, using immunohistochemistry (IHC) methods to subtype diffuse large B-cell lymphoma (DLBCL), suggest that germinal center B-cell lymphomas (GCBs) have improved outcomes. However, data in Asians have been limited and conflicting. This study aims to evaluate the prognostic impact of cell-of-origin (COO) subtyping by IHC and Lymph2Cx in South-East Asian (SEA) DLBCL patients, and to summarize the existing literature.Methods: A single-center retrospective analysis of 384 DLBCL patients diagnosed 2013–2018 who received Rituximab-based chemotherapy was performed. Hans and Lymph2Cx were used to assign COO and correlated with outcomes.Results: International Prognostic Index (IPI) score was associated with overall survival (OS) and progression-free survival (PFS). The 5-yr-OS for non-GCB versus GCB for COO by Hans was 70% versus 71% p=0.39, while 5-yr-OS for ABC versus GCB for COO by Lymph2Cx was 74% versus 92% p=0.19. The 5-yr-PFS for non-GCB versus GCB for COO by Hans was 65% versus 70% p=0.26, while 5-yr-PFS for ABC versus GCB for COO by Lymph2Cx was 64% versus 86% p=0.07.Conclusions: IPI is reaffirmed to be relevant in the rituximab era. COO by Hans has no prognostic significance, while subtyping by Lymph2Cx trends toward GCBs having better PFS and OS.

Chromatin remodeling factor lymphoid-specific helicase links with Epstein-Barr virus associated the follicular germinal center B cell lymphomas.

We assessed the frequency of epigenetic lesions, including lymphoid-specific helicase (LSH), 5-hydroxymethylcytosine (5-hmC) and E2F1, and the possible correlations among molecular findings, phenotype, clinical features, and outcome. We investigated 181 paraffin-embedded B-cell lymphoma samples using immunohistochemistry and in situ hybridization. The levels of Ki67, LSH, 5-hmC, and E2F1 were all increased in germinal center B-cell lymphomas when compared with those in normal lymph nodes, and LSH was highly expressed in diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) that were positive for Epstein-Barr virus (EBV) infection, indicating that LSH is linked to EBV infection in DLBCL and BL. Interestingly, LSH was mainly localized in the germinal centers of lymph nodes whereas 5-hmC staining localized to areas surrounding the germinal centers. These findings indicate a critical role for LSH as a biomarker and therapeutic target in follicular germinal center B-cell lymphoma.

Utx Insufficiency Cooperates with Braf V600E in the Induction of Myeloma in Mice

Introduction:Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice.Methods:To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells.Results:Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis.Conclusion:Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents. DisclosuresOkuno:Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.

Lymphomas with pseudo–double-hit BCL6-MYC translocations due to t(3;8)(q27;q24) are associated with a germinal center immunophenotype, extranodal involvement, and frequent BCL2 translocations

High-grade B-cell lymphomas with MYC, BCL2, and/or BCL6 rearrangements, "double-hit" or "triple-hit" lymphomas (DTHL), are aggressive neoplasms associated with a poor prognosis. A t(3;8)(q27;q24) rarely occurs in B-cell lymphomas that results in a unique "pseudo-double-hit" BCL6-MYC fusion, indistinguishable by interphase fluorescence in situ hybridization (FISH) from more conventional DTHL with independent MYC and BCL6 translocations. Reports of t(3;8)(q27;q24) lymphomas are sparse, and to better characterize their pathologic, cytogenetic, and clinical features, 6 new cases from 2 institutions and 19 previously published cases were reviewed. All new cases displayed aggressive morphologic features, and most previously published cases were classified as aggressive lymphomas. Collectively, all t(3;8)(q27;q24) cases had a germinal center (GC) phenotype, and most had complex karyotypes (22/24, 92%), including frequent concomitant BCL2 rearrangements (17/24, 71%). When compared to two large published DTHL cohorts, t(3;8)(q27;q24) lymphomas less often expressed BCL2 (P < .01), had a greater likelihood of extranodal involvement (P < .01), and more frequently appeared triple-hit by FISH analysis (P < .01). Despite presenting with aggressive clinicopathologic features, 100% (6/6) of t(3;8;)(q27;q24) patients achieved complete remission after intensive induction regimens, and 2- and 3-year overall survival rates were 63% (10/16) and 57% (8/14), respectively. These findings suggest that lymphomas with t(3;8)(q27;q24) may represent a subset of GC B-cell lymphomas distinct from conventional DTHL. Our results further highlight the value of routine karyotype assessment in aggressive B-cell lymphomas, and the importance of recognizing the t(3;8)(q27;q24) so that its clinical significance can be more fully explored.

Gastric follicular lymphoma: A report of 3 cases and a review of the literature.

Follicular lymphoma (FL) occurs primarily in the gastrointestinal tract, with the stomach being one of the rarest sites. According to the literature, <20 cases of primary gastric FL have been reported, with the number of cases with detailed pathological descriptions being even less. The aim of the present study was to compare clinicopathological features of gastric FL with FL at alternative sites. A total of 3 cases of gastric FL were retrieved from among 3,216 cases in the databases of 4 university hospitals in South Korea: Seoul National University (SNU) Hospital, SNU Boramae Hospital, SNU Bundang Hospital (all Seoul, South Korea) and Dongtan Sacred Heart Hospital Hallym University (Dongtan, South Korea), including 2 primary cases and 1 case that was possibly secondary to nodal FL. The 2 primary gastric FL cases were incidentally detected in routine health check-ups. An endoscopy revealed a single polypoid submucosal mass and biopsies failed to confirm the diagnosis due to minimal mucosal involvement. Therefore, a partial gastrectomy was performed. The epicenters of the tumors were submucosal, with focal extension to the muscularis propria. However, 1 case exhibited an isolated FL nodule in the omentum. Histopathological examination revealed FL of grade 1–2 with a follicular pattern and with strong expression of germinal center markers and B-cell lymphoma 2 (BCL2). Rearrangement of BCL2 was not identified using fluorescence in situ hybridization studies in 2 cases. In contrast to these 2 cases, the remaining FL case was confirmed with an endoscopic biopsy. The endoscopy revealed multiple eroded polypoid lesions, and pathology revealed FL of grade 1–2 with a predominantly diffuse pattern, and with immunoglobulin heavy chain IGH/BCL2 translocation. In view of the extensive lymphadenopathy, the last case possibly presented as secondary involvement of nodal FL. It is challenging to diagnose FL in the stomach due to little mucosal involvement, as well as the unfamiliarity of the tumor due to its rarity. However, the results of the present study suggest that primary gastric FL may exhibit unique pathological features, including a predominantly follicular pattern and an absence of BCL2 rearrangement.

Regulatory myeloid cells: an underexplored continent in B-cell lymphomas.

In lymphomas arising from the germinal center, prognostic factors are linked to the myeloid compartment. In particular, high circulating monocyte or myeloid-derived suppressor cell counts are associated with poor prognosis for patients with high-grade B-cell lymphomas. Macrophages with an M2 phenotype are enriched within lymphoma tumors. However, the M1/M2 nomenclature is now deprecated and the clinical impact of this phenotype remains controversial. Across cancer types, myeloid cells are primarily thought to function as immune suppressors during tumor initiation and maintenance, but the biological mechanisms behind the myeloid signatures are still poorly understood in germinal center B-cell lymphomas. Herein, we describe the role and clinical relevance of myeloid cells in B-cell lymphoma and propose innovative approaches to decipher this complex cellular compartment. Indeed, characterization of this heterogeneous cell ecosystem has been largely accomplished with "low-resolution" approaches like morphological evaluation and immunohistochemistry, where cells are characterized using a few proteins and qualitative metrics. High-resolution, quantitative approaches, such as mass cytometry, are valuable to better understand myeloid cell diversity, functions, and to identify potential targets for novel therapies.

Spindle-Cell Variants of Primary Cutaneous Follicle Center B-Cell Lymphomas Are Germinal Center B-Cell Lymphomas by Gene Expression Profiling Using a Formalin-Fixed Paraffin-Embedded Specimen

Spindle-Cell Variants of Primary Cutaneous Follicle Center B-Cell Lymphomas Are Germinal Center B-Cell Lymphomas by Gene Expression Profiling Using a Formalin-Fixed Paraffin-Embedded Specimen

Pan‐lymphoma classification

Introduction: Cell of origin classification of diffuse large B-cell lymphoma (DL-BCL) using gene expression signatures identifies patients with distinct molecular features and survival outcomes. Whilst the identification of activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of DL-BCL is now routinely used in diagnostics, however, a significant proportion of patients display gene expression signatures that are intermediate between classes. Whilst some of these intermediate cases display genuine hallmarks of both ABC or GCB subtypes, leading to an intermediate DL-BCL COO-UNC type, others appear to display distinct molecular signatures. It is possible that that these signatures represent inherent noise in the expression profiling process, or biological noise resulting from, for example, high levels of T-cell infiltration in samples. A more intriguing possibility is that these cases represent intermediate types between DL-BCL and other types of lymphoma. The existence of intermediate molecular signatures raises important questions about which type of treatment is most applicable for these cases. Yet the identification of intermediate molecular signatures is challenging, because existing classification algorithms have tended to focus on a small subset of lymphoma types rather than across the pan-lymphoma spectrum. Moreover, it is not clear what features would be needed to identify the relationship between patients to diverse lymphoma classes. Methods: To address this issue, we have developed a pan-lymphoma classifier using a support vector machine (SVM) with an embedded recursive feature selection (RFS) algorithm. Our training and test dataset consisted of 431 samples, spanning eight different types of lymphoma: ABC (49), GCB (133), COO-UNC (40), Burkitt's (60), Hodgkin's (43), PMBL (35), PBL (39), and Plasmacytoma (32). Gene expression profiles for each sample were obtained using the Illumina DASL platform. We trained the SVM using 10-fold cross-validation with a 70:30 train/test split. On each iteration of the RFS, we removed the single least informative probe. We repeated this until a significant drop in average accuracy of the SVM was reached. Results: Initially training the SVM on 1125 probes, we obtained a pan-lymphoma average classification accuracy of 91%. We then used the SVM-RFS to obtain a list of just 23 probes that gave rise to an average classification accuracy of 86%. This allowed us to develop a between-class distance metric as a score of the relative association of any given sample to a lymphoma class. Retrospective classification of COO-UNC samples showed some share molecular features associated with non-DL-BCL classes. Conclusions: SVM-RFS appears to be a robust approach for identifying classes of lymphoma from underlying molecular phenotypes. A small number of most informative genes provide a separation of cases into subtypes that is in excellent agreement with laboratory diagnoses. Keywords: activated B-cell–like (ABC); GCB lymphoma subtype; gene expression profile (GEP)

Variegated RHOA mutations in human cancers.

RHOA is one of the most extensively investigated members of the Rho GTPase family of proteins and has long been implicated in malignant transformation as well as tumor invasion and metastasis. Recently, revolutionized sequencing platforms have revealed frequent RHOA mutations in a wide variety of human cancers, including angio-immunoblastic T-cell lymphoma, adult T-cell leukemia/lymphoma, germinal center B-cell lymphoma, diffuse-type gastric cancer, and other solid tumors. With their discrete positional distributions and types of amino acid substitution depending on cancer type, different RHOA mutations seem to have unique functional and biological properties. However, their impact on the development of different types of tumors remains controversial. Here, we present an overview of the current understanding of RHOA mutations in human cancers and highlight the unanticipated complexity of their genetic and biological properties.

Abstract 3038: Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL)

Abstract Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL. Materials &amp; Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. Citation Format: Priyank P. Patel, Alison Zeccola, Juan Gu, Cory Mavis, Sheila N. J. Sait, Vishala Neppalli, Francisco J. Hernandez-Ilizaliturri. Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3038.

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

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.