Targeted EZH2 Inhibitors in Diffuse Large B-Cell Lymphoma (DLBCL): Immunohistochemical and Mutational Profiles of Patients May Determine Candidates for Treatment

Abstract DLBCL is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling has identified two main subtypes: Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC). EZH2 plays an essential role in epigenetic regulation of DLBCL by specifically mono-, bi- and tri-methylating histone H3 lysine 27 (H3K27me1/-me2/-me3). Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, mostly affecting tyrosine 641 (Y641), inducing increased H3K27me3. Novel EZH2 inhibitors are currently being tested in phase 1 and 2 clinical trials in patients with and without EZH2 Y641 mutations, but no study has examined which patients would most benefit from this treatment. We studied a cohort of 100 patients with DLBCL with available biopsies (96 at diagnosis and 4 at relapse) and developed an immunohistochemical (IHC) assay based on antibodies specifically targeting EZH2, H3K27me3 or H3K27me2. Eighty-six biopsies (12 EZH2 Y641 mutant and 74 WT EZH2) were usable for IHC analysis. Biopsies were scored based on staining intensity and percentage of tumor cells stained, and a me3/me2 score (log of me3 to me2 ratio) was calculated for each patient. Sanger sequencing of EZH2was performed on all patients, GCB/ABC signature was determined by DASL technology based on the expression of 19 genes, and t(14;18) translocation was detected by karyotyping and FISH. The cohort was also extended to 15 patients with EZH2 Y641 mutations and 17 WT EZH2 patients for Next Generation Sequencing (NGS) analysis of a panel of 34 genes involved in lymphomagenesis. Among our cohort, 45 patients were ABC, 50 were GCB, and 5 were unclassified. Sanger sequencing identified 14 patients with EZH2 Y641 mutations (12 GCB, 1 ABC, 1 unclassified). The t(14;18) translocation was more frequent in patients with EZH2 Y641 mutations (9/14, 64%) (p<10-4). Three distinct IHC profiles emerged based on me3/me2 score: a me3-high/me2-low profile (me3/me2 score>0, n=12/86), a me3-low/me2-high profile (me3/me2 score<0, n=41/86) and an intermediate profile (me3/me2 score=0, n=33/86). Patients with EZH2 Y641 mutations mostly exhibit me3/me2 score>0 profiles (n=7/12), whereas patients with WT EZH2 are split between intermediate (n=29/74) and me3/me2 score<0 profiles (n=40/74) (p<10-5). Survival analysis was performed on patients with biopsies at diagnosis treated with Rituximab. ABC subtype is associated with both inferior OS and PFS within our cohort (p=0.03); among ABC patients, low EZH2 IHC expression is associated with superior OS (p=0.035) and PFS (p=0.02). No correlation was found between prognosis and IHC profile. All EZH2 mutations were confirmed by NGS along with their Variable Allele Frequency (VAF). Among GCB EZH2 Y641 mutant patients, a majority of clonal EZH2 mutations (n=11/14) and a minority of subclonal EZH2 mutations (n=3/14) were identified by comparing VAFs of EZH2 and of well-known DLBCL/Follicular Lymphoma driver mutations (including TNFRSF14,CREBBP and MYD88: figure). Among the 86 patients, a tendency toward a correlation between me3/me2 score and EZH2 VAF exists (p=0.09, r=0.51) and of the 5 patients with EZH2 Y641 mutations presenting a me3/me2 score ≤0, 3 exhibit a VAF inferior to the median. Two also present a subclonal EZH2mutation. These findings could potentially explain their unexpected IHC profile, and possibly decrease their response to EZH2 inhibitor treatment. Furthermore, 5 WT EZH2 patients present a me3/me2 score>0; 4 are of the ABC subtype, suggesting an EZH2 mutation bypass in ABC patients. NGS analysis also revealed remarkably similar mutational profiles for 2 of these patients, most notably mutations in PRDM1 and PIM1, potentially responsible for EZH2 upregulation by maintaining B cells in GC reaction and possibly justifying EZH2 inhibitor treatment. EZH2 inhibitors are currently being tested in clinical trials in DLBCL as novel and promising weapons in clinicians’ therapeutic arsenal. This study has shown that IHC and mutational profiles can identify patients most likely to benefit from EZH2 inhibitor treatment by highlighting their in vivo hyper-H3K27me3 status, pinpointing associated activating mutations, and determining EZH2mutation clonality. As such, analyzing these parameters could maximize EZH2 inhibitor benefit and potentially serve to grant access to patients who would otherwise not have been considered. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2417.2417 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2417-2417

Author(s):

Olga Ritz ◽

Jochen K Lennerz ◽

Karolin Rommel ◽

Karola Dorsch ◽

Elena Kelsch ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Proximal Promoter ◽

Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Identification of Genes Frequently Mutated In FL and DLBCL with Transcriptome, Genome and Exome Sequencing

Blood ◽

10.1182/blood.v116.21.804.804 ◽

2010 ◽

Vol 116 (21) ◽

pp. 804-804

Author(s):

Ryan D Morin ◽

Maria Mendez-Lago ◽

Andrew J Mungall ◽

Nathalie A Johnson ◽

Rodrigo Goya ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Somatic Mutation ◽

Germinal Center ◽

Rational Design ◽

High Throughput Sequencing ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

Histone Methyltransferase ◽

Driver Mutations

Abstract Abstract 804 Introduction: Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the two most common types of non Hodgkin lymphoma (NHL). It is widely accepted that DLBCL can be divided into two major subtypes using gene expression profiling: germinal center B-cell (GCB) and activated B-cell (ABC). Both FL and the GCB subtype of DLBCL derive from germinal center B cells and have been found to share some common mutational events such as translocations leading to the deregulation of the BCL2 oncogene and mutations affecting a single tyrosine (Y641) in the histone methyltransferase EZH2. In contrast, ABC DLBCL tumors are characterized by mutations leading to the constitutive activity of NFkB. The clear differences in treatment response between subtypes allow this distinction to be used as a prognostic indicator and may ultimately lead to therapies that target individual features of each subtype. However, besides the gene expression and mutational signatures that differentiate the DLBCL subtypes, there is a paucity of molecular prognostic markers in these NHLs. Further, there is limited knowledge about the genetic events that drive the GCB subtype of DLBCL, which, if better understood, may enable the design of targeted therapeutics. Methods: To identify mutations driving lymphomagenesis and in particular, aggressive cases of NHL, we applied Illumina second-generation sequencing technology to the analysis of tumor genomes and constitutional DNAs from a FL and a DLBCL tumor and the exomes from two additional DLBCLs. In these “omes”, we identified somatic protein-altering point mutations in more than 250 genes including genes known to be involved in cancer, for example TP53, FAS and TNFAIP3 (A20). Many of these mutations may represent passenger rather than driver mutations, the latter of which are involved in disease progression. To identify the likely driver mutations, we sought to identify the genes that are recurrent targets of somatic mutation in these cancers. To this end, we further analyzed the transcriptome sequences we generated using RNA-seq from 95 primary DLBCLs,13 FL cases and 10 DLBCL-derived cell lines. Results: 105 of the genes found mutated in the FL and DLBCL genomes were observed to be recurrent targets of somatic mutation in these diseases. Some of these were known targets of aberrant somatic hypermutation (SHM) including BCL2, PIM1, and IRF4 and others have been previously identified as targets of recurrent mutation in lymphoma, such as EZH2, CD79B and CARD11. One of the most frequently mutated genes was MLL2, a histone methyltransferase never before implicated in lymphomagenesis. MLL2 showed a pattern of mutation characteristic of a dosage-sensitive tumor suppressor gene. Another frequently mutated gene was MEF2B, a calcium-regulated transcriptional co-activator/repressor that cooperates with histone modifying enzymes to epigenetically regulate the expression of genes. We found that mutations affecting MEF2B occur in 11.7% of FL and 9% of DLBCL, with the majority (73%) of these mutations affecting three amino acids (K4, Y69, and D83). Analysis of these 105 recurrently mutated genes for prognostic signatures is ongoing. Conclusions: High-throughput sequencing platforms have enabled the identification of recurrent targets of somatic mutations never suspected to be involved in lymphoma. Some of these mutated genes may have prognostic value while others may represent targets for the rational design of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Cross-Species Investigations Reveal Role, Mechanism and Predictive Power of Paracrine, Secondary Senescence in B-Cell Lymphoma Therapy

Blood ◽

10.1182/blood.v120.21.3715.3715 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3715-3715

Author(s):

Jan R. Dörr ◽

Maja Milanovic ◽

Yong Yu ◽

Julia Kase ◽

Dido Lenze ◽

...

Keyword(s):

B Cell ◽

Predictive Power ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Brdu Incorporation ◽

Lymphoma Cells ◽

Long Term Outcome

Abstract Abstract 3715 Apoptosis and cellular senescence operate as anti-tumor safeguard mechanisms. Unlike apoptotic cells, senescent cells remain viable, and, hence, may crosstalk to other cells in their vicinity over extended periods of time. In fact, cells that entered oncogene-induced senescence or anticancer therapy-induced senescence (TIS) present with a senescence-associated secretory phenotype (SASP), a massive production of secretable factors, which reportedly reinforces senescence through an intracellular mechanism. Utilizing the Eμ-myc transgenic mouse lymphoma model, we provide evidence for an outcome-relevant paracrine, DNA damage-independent secondary senescence program (SecS) in vitro and in vivo. Apoptosis-blocked (bcl2-infected) lymphoma cells from different genetic backgrounds were treated with the DNA-damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in mice in vivo. TIS and SecS was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The secretome of senescent cells was analyzed by proteomics, gene expression and protein arrays. Overall and progression free survival in mice and patients was assessed by Kaplan-Meier analysis. Transcriptome and secretome analyses followed by functional studies found extracellular matrix proteins, especially small leucine-rich proteoglycans (SLRP), but not NF-kB-dependent cytokines and chemokines, to induce SecS in proliferating lymphoma cells in a paracrine fashion, and linked a “high secretor” status to stronger SecS induction. Dissecting senescence-mediating pathways in recipient cells by biochemical, genetic and pharmacological means unveiled an essential role for the LDL receptor-related protein 1 (LRP1), a receptor for SLRP and other SASP components, through the cell-cycle inhibitor p21CIP1 in SecS. Accordingly, mice harboring TIS-capable but genetically SecS-defective lymphomas (e.g. lacking LRP1 or p21CIP1 expression) experienced inferior long-term outcome to therapy. Not only the recipient cell-based LRP1 status but also the genetically and biologically distinct donor cell-based secretor gene signature stratified outcome in mice. Strikingly, humanized versions of both classifiers were predictive in a large cohort of diffuse large B-cell lymphoma (DLBCL) patients, where they identified – although composed of different gene sets – largely overlapping patient subgroups with superior prognosis, again suggesting SecS as the critical underlying treatment effector principle. Our study highlights the predictive power of senescence for treatment outcome in DLBCL, and provides functional examples (which will be discussed at the meeting) for SASP-related non-genotoxic pro-senescent therapies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Pilot Study of Lenalidomide-Rituximab Combination in Relapsed/Refractory Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.3695.3695 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3695-3695

Author(s):

Vadim Ivanov ◽

Diane Coso ◽

Thérèse Aurran ◽

Jean Marc Schiano ◽

Anne-Marie Stoppa ◽

...

Keyword(s):

Clinical Trials ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Induction Phase ◽

Significant Activity ◽

Intention To Treat ◽

Grade 3 ◽

Initial Results

Abstract Abstract 3695 Despite recent immunochemotherapy advances approximately 50% of DLBCL patients relapse. Data emerging from initial clinical trials demonstrated that Lenalidomide has a significant activity against different subtypes of relapsed/refractory aggressive B-cell lymphoma. Clinical responses are histology subtype-dependent and most prominent in mantle cell lymphoma. The results in DLBCL were less encouraging with ORR of 26%, CR of 9%, PFS of 2.7 mo. Concurrently targeting the tumor cell itself with monoclonal antibody and targeting the immune response and microenvironment with Lenalidomide may be a promising therapeutic strategy. Encouraging by our initial results of Lenalidomide-Rituximab (LR) combination in patient with refractory (R/R) DLBCL (Ivanov V. et al., 2010), Institutional Multidisciplinary Meeting proposed this combination for 17 patients with R/R DLBCL. All pts were refractory to three or more (range: 2–5) previous lines of conventional immuno-chemotherapy. All eligible pts, except 4 primary-refractory, were previously autografted. Median age for the whole group was 62,5 years, (range: 43–79), 5 pts are female. 65% of patients were younger than 65 y. Patients received combination of Rituximab 375 mg/m2 on day 1 or day 7; Lenalidomide (Revlimid), 15 mg/d for the first pt and 25 mg/d for other 16 pts, for 21/28 days. Dexamethasone 40mg, day 1–4 was given for first 7 pts. Initial decision on adding Dexamethasone was based on the extrapolation from the recommended regimen used in multiple myeloma, but it was abandoned in last 10 pts. Initially the treatment duration was established for 6 months, but it was prolonged to 7–11 months for patients in CR. Of 17 pts enrolled on study, 3 patients stopped the treatment during the first course: 1 pt because of grade 3 toxicity and 2 pts because of explosive disease progression. Both patients were switched to palliative care. In 14 pts, received more than 1 course of treatment, 7 (50 %) responded to LR combination, including 6 pts (43%) with CR and 1 (7%) patient with PR. One pt with clinical and PET-FDG scan improvement after 3 courses of LR was included into “auto-allo” tandem program and actually in CR at +12 months after PBSCT. Six pts progressed on LR treatment and were switched to palliative regimens. In intention to treat analysis the CR rate for the whole group was 35%. As regards the follow-up, all 7 pts in PR and CR are evaluable for evaluation. The patient in PR progressed after 5 courses of LR. Six patients in CR group received an average of 8 (range: 7–11) courses of LR treatment. Two patients relapsed after 5 and 26 months of CR and other 4 patients are actually in CR at +7, +17, +18 and +24 months. Adverse events were manageable and the most common toxicity included thrombocytopenia and neutropenia. In relapsed/refractory DLBCL modest initial results of Lenalidomide monotherapy emerge the use of new effective combinations. Recently the combination Lenalidomide-Rituximab (LR) was shown to be highly efficacious in phase 2 study in elderly (>65 y.o.) patients with DLBCL (Zinzani et al., 2011). Into the group of 23 pts the ORR rate at the end of 6-months induction phase was 35%. Our data confirm results of Bologna group in the younger group of patients. Given the poor prognosis of refractory DLBCL, enrolment in already running prospective clinical trials with Lenalidomide are underway and the investigation of the combination of Lenalidomide and Rituximab is further warranted. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Potential Synergic Effect of Lenalidomide-Rituximab Combination: Durable Complete Remissions in Refractory Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.4989.4989 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4989-4989

Author(s):

Vadim Ivanov ◽

Diane Coso ◽

Therese Aurran-Schleinitz ◽

Jean-Marc Schiano de Colella ◽

Anne-Marie Stoppa ◽

...

Keyword(s):

Clinical Trials ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Published Data ◽

Significant Activity ◽

Antitumor Effects ◽

Histologic Subtype ◽

Initial Results

Abstract Abstract 4989 Data emerging from initial clinical trials demonstrated that Lenalidomide has a significant activity against different subtypes of aggressive B-cell lymphoma. Clinical responses are histologic subtype-dependent and most prominent in mantle cell lymphoma. The results in DLBCL were less encouraging with ORR of 26%, CR of 9%, PFS of 2.7 mo. Concurrently targeting the tumor cell itself with monoclonal antibody and targeting the immune response and microenvironment with Lenalidomide may be a promising therapeutic strategy. By modulating the immune system through dendritic cells and NK cells, by changing the cytokine milieu, and by their anti-angiogenic effects, IMiDs in combination with rituximab resulted in augmented in vitro and vivo antitumor effects against B-cell lymphoma. Recently the combination Lenalidomide-Rituximab (LR) was shown to be highly efficacious in follicular NHL. Encouraging by our initial results of LR combination in patient with refractory (R/R) DLBCL (Leuk Lymphoma 2010), Institutional Multidisciplinary Meeting proposed this combination for other 8 patients with R/R DLBCL. All patients were refractory to three or more previous lines of conventional immuno-chemotherapy. All except 3 primary-refractory pts were previously autografted. Patients received combination of Rituximab 375 mg/m2, day 1 or day 7; Lenalidomide (Revlimid), 15 mg/d for the first pt and 25 mg/d for other 8 pts, for 21 days. Dexamethasone 40mg, day 1–4 was given for first 6 pts. Initial decision on adding Dexamethasone was based on the extrapolation from the recommended regimen used in multiple myeloma, but it was abandoned in last 3 pts. Of 9 pts enrolled on study, 8 received > 2 cycles of LR and all of them were evaluable for response. Median age for evaluable pts was 52 (range: 19–73), 3 pts are female. Of 8 evaluable pts, 5 (63%) responded to LR, including 3 pts (38%) with CR and 2 (25%) patients with PR. These two PR pts were primary refractory to chemotherapy before LR and both were grafted (1 auto and 1 allo) after three courses of LR. One pt with clinical and PET-FDG scan improvement after 3 courses of LR was included into “auto-allo” tandem program and actually in CR after PBSCT. Two pts progressed on LR treatment and were switched to palliative regimens. As regards the follow-up, 3 pts in CR are evaluable for evaluation. Two pts received 6 and one pt 8 courses of RL treatment. One patient relapsed after 24 mo of CR and other 2 patients are in CR at +11 and +6 months. In relapsed/refractory DLBCL modest initial results of lenalidomide monotherapy emerge the use of new effective combinations. Recently several phase II studies of LR efficacy in indolent NHL were proposed. For instance, there is no published data of long-term safety and efficacy of this combination in DLBCL. Given the poor prognosis of refractory DLBCL, enrolment in already running prospective clinical trials with lenalidomide are underway and the investigation of the combination of Lenalidomide and Rituximab is further warranted. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Early Macrophage and Mast Cell Responses in Diffuse Large B-Cell Lymphoma After Immunochemotherapy

Blood ◽

10.1182/blood.v118.21.5206.5206 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5206-5206

Author(s):

Minna Taskinen ◽

Anna Raunio ◽

Maria Pöyhönen ◽

Marja-Liisa Karjalainen-Lindsberg ◽

Sirpa Leppä

Keyword(s):

Mast Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Prognostic Impact ◽

Mrna Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Abstract 5206 Background: The prognostic impact of tumor microenvironment on the survival of lymphoma patients has recently been reported. However, early molecular and cellular responses to immunochemotherapy are unknown. Here, we have compared the tumor-associated macrophage (TAM) and mast cell (MC) contents in the lymphoma tissue in vivo before and after the first immunochemotherapy course in a small cohort of aggressive B-cell lymphoma patients. Patients and methods: The population of this pilot study consisted of seven diffuse large B-cell lymphoma (DLBCL) and three grade IIIB follicular lymphoma (FL) patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). Paired tumor samples were collected before and a day after the first course of therapy, and evaluated immunohistochemically for CD68+, CD163+ macrophages and tryptase+ MCs. Freshly frozen lymphoma tissue containing enough material for paired mRNA analyses was available from 8 patients. Results: Comparing pre- and post-treatment tissue samples, an increase in the number of CD68+ TAMs was observed (p=0.023), whereas no variation in MC contents was found. If the patients were grouped according to response, i.e. remission (n=7) vs relapse (n=3), the most significant increase after therapy was observed in M2-type CD163+ TAM content (p=0.001). In the exon array analyses, the mRNA levels of both CD68 (p=0.052) and CD163 (p=0.023) genes increased after therapy. Conclusions: Our preliminary data suggest significant changes in macrophage content and their relative subsets in the lymphoma microenvironment after the first course of immunochemotherapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Development of a Bruton's Tyrosine Kinase (Btk) Inhibitor, ONO-WG-307: Efficacy in ABC-DLBCL Xenograft Model – Potential Treatment for B-Cell Malignancies,

Blood ◽

10.1182/blood.v118.21.3731.3731 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3731-3731 ◽

Cited By ~ 4

Author(s):

Ryohei Kozaki ◽

Toshio Yoshizawa ◽

Shuji Tohda ◽

Tomoko Yasuhiro ◽

Shingo Hotta ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Xenograft Model ◽

B Cell Lymphoma ◽

Lymphoid Cells ◽

B Cell Malignancies ◽

Pharmacodynamic Marker ◽

Btk Inhibitor

Abstract Abstract 3731 Purpose: ONO-WG-307 is a small molecule inhibitor that covalently binds to Btk. Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis and new therapies, preferably chemo-sparing therapies, or as add-on to existing treatment regimens are required to help treat patients with ABC-DLBCL. Therefore, Btk constitutes an interesting therapeutic target, thus the activity of ONO-WG-307 was evaluated in an ABC-DLBCL xenograft model. Methods: Tumor cells (TMD-8) were implanted subcutaneously into female SCID mice. Tumors were allowed to grow to a volume of 100–200 mm3 before the mice were randomized into groups based on tumor size. ONO-WG-307 was administered orally at doses up to 10 mg/kg bid. Tumors were measured two or three times weekly after initiation of treatment, and tumor volumes were determined using the formula volume (=width2xlength)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3 or after a maximum period of 2 months. In parallel, an exploratory pharmacodynamic marker of Btk inhibition (Phosphorylated-Btk [P-Btk]) was also investigated in vivo. Results: Treatment with ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth in a TMD-8 xenograft model. Furthermore, parallel analysis of a pharmacodynamic marker, P-Btk, supported that Btk was inhibited and the level of P-Btk inhibition was correlated with the decreased tumor volumes observed in the TMD-8 model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in the ABC-DLBCL xenograft model, with Btk inhibition further supported using a PD marker. Given the need to treat and overcome disease resistance especially in ABC-DLBCL, the use of a Btk inhibitor is a novel, mechanistic approach to treating B cell malignancies. Additional work is underway, combining ONO-WG-307 with chemotherapy and other targeted agents. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Salmonella Enterica serovar Typhimurium As a Therapy for B-Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.4968.4968 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4968-4968

Author(s):

Sofia Grille ◽

María Moreno ◽

Jose Alejandro Chabalgoity ◽

Daniela Lens

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Control Group ◽

Solid Cancer ◽

Specific Response ◽

Systemic Level ◽

Facultative Anaerobic Bacteria

Abstract Abstract 4968 Patients with non-Hodgkin's lymphoma (NHL) respond well to treatment, but relapses are frequent after a period of months or years. This highlights the need for new therapeutic modalities for these patients. Since nineteenth century there are many reports that tumors may undergo spontaneous regression after a bacterial infection. There is great interest in developing anti-tumoral therapies based on living microorganisms because they show some selective replication or preferential accumulation in tumor microenvironment. Salmonella is a facultative anaerobic bacteria, which has the ability to colonize and replicate under both anoxia and oxygenated areas of tumors. Several therapies had evaluated the administration of Salmonella alone or used as a vector for relevant molecules in different solid cancer models. To date, many clinical trials have been conducted using live attenuated Salmonella and have demonstrated the safety of it use. Although many groups have studied the anticancer therapeutic effect of Salmonella, the mechanism responsible for its efficacy is unknown. However, it is known that anti-tumor properties of the bacteria mainly depend on activation of innate and adaptive immune responses. We previously developed a reproducible syngenic model of B-cell lymphoma in Balb/c mouse based upon the A20 NHL cell line. Using this model, we studied the in vivo antitumor effect of the administration of intratumoral attenuated Salmonella Typhimurium (LVR01). Mice were injected with A20 cells and, after tumor became palpable, were divided in groups and treated intratumorally with one of the following: 3 inoculations of 1×106Salmonella LVR01, 1 inoculation of 1×106Salmonella LVR01 and plus 2 of PBS or 3 inoculations of PBS as a control group. Mice were followed for survival and immune response was evaluated. Mice treated with 3 inoculations of Salmonella LVR01 showed a greater survival than others groups (p=0,0001). Prolonged survival was associated with a marked increase on the number of intratumoral CD8+ T cells (p=0.001) and neutrophils (p=0.012). Additionally, intratumoral CD8+ and NK cells showed a significantly higher percentage of activation antigen (CD69) expression. Further, we found a significant increase in the expression of IFN-g mRNA in tumor tissue of mice inoculated with 3 doses of Salmonella (p=0.03). At systemic level, we measured cytokine concentration in the supernatant of splenocytes stimulated with tumor cells and we found that only mice receiving 3 inoculation with Salmonella showed an tumor antigen specific response as evidenced by increased production of IFN-□ (p=0,004) and IL-12p70 (p=0,025). Overall, the results presented here indicate that inoculation with 3 doses of Salmonella induces in vivo recruitment of both innate and adaptative immune cells to the tumor site and elicit an anti-tumor immunity at systemic level that results in an extended survival. This approach promises to be an interesting strategy, with therapeutic value, to promote systemic immunity against human NHL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Viral Ortholog Complements Mir-155 Deficiency-Associated Abnormalities and Together with Additional Viral Mirnas Causes B Cell Hyperplasia and B Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.4866.4866 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4866-4866

Author(s):

Sang-Hoon SIN ◽

Dirk P Dittmer

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Conflicts Of Interest ◽

Human Tumor ◽

Human Herpesvirus ◽

B Cell Lymphoma ◽

Immunoglobulin Level ◽

Tumor Virus

Abstract Abstract 4866 MicroRNA-155 (miR-155) is overexpressed in many types of cancers including B cell lymphomas. It has also an essential function in GC development. These functions are conserved among species and miR-155 knockout (ko) mice are deficient in GC development in Peyer's patches (PP) and lymph nodes. The human tumor virus Kaposi sarcoma associated herpesvirus (KSHV) encodes an ortholog to miR-155, named miR-K12-11. MiR-155 and miR-K12-11 share 100% seed sequence homology (Nature. 2007 Dec 13;450(7172):1096-9, J Virol. 2007 Dec;81(23):12836-45). Another human herpesvirus, Epstein-Barr Virus, induces mir-155 upon infection and immortalization of naïve B cells in culture. To test the hypothesis that miR-K12-11 can complement the normal function of miR-155 in vivo, we generated transgenic mice, which express miR-K12-11, as well as the other 21 KSHV miRNAs. Using this mouse model, we show that KSHV latency locus which contains a viral ortholog of miR-155, KSHV miR-K12-11 complements B cell abnormalities associated with the lack of miR-155. Germinal center (GC) formation is rescued in spleen and PP and lowered immunoglobulin level is also augmented to normal in KSHV latency locus transgenic mice with miR-155 ko background. Furthermore, mature B cells were chronically activated, leading to hyperglobulinemia triggered by increased plasma cell frequency. Marginal zone (MZ) B cells developed hyperplasia and the mice had an augmented response to T-dependent antigen as well as the TLR4 ligand LPS in vivo. This suggests that by mimicking miR-155 (and other functions) this human tumor virus drives the expansion of infected B cells, which if left unchecked can accumulate secondary mutations leading to post-GC lymphoma and multicentric Castleman's disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Addiction of B Cell Lymphomas to microRNA-17-92 Is Mediated By a Single Target Gene

Blood ◽

10.1182/blood-2019-126483 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1258-1258

Author(s):

Katia Schoeler ◽

Teresa Mittermeier ◽

Andreas Villunger ◽

Klaus Rajewsky ◽

Verena Labi

Keyword(s):

B Cells ◽

B Cell ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

B Cell Lymphoma ◽

Messenger Rnas ◽

B Cell Lymphomas ◽

Apoptosis Pathway

Mature microRNAs (miRNA) are short non-coding RNAs that regulate gene expression by binding to messenger RNAs (mRNA) in a sequence-specific manner, causing translational repression and/or mRNA decay. The mammalian genome harbors thousands of miRNA genes, many of which are organized into transcriptionally co-regulated clusters such as miR-17-92. Knockout of the miR-17-92 cluster gene in mice blocked B lymphopoiesis, and ectopic miR-17-92 expression sufficed to initiate B cell lymphomas and autoimmunity. In humans, the miR-17-92 gene is commonly amplified or overexpressed via MYC-driven transcription in diffuse large B cell and Burkitt's lymphomas. A computationally predicted shared target of the miR-17-92 miRNAs is the pro-apoptotic BCL-2 family protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17-92:Bim interactions to the miR-17-92 knockout and overexpression phenotypes, we engineered a unique in vivo system of conditional mutagenesis of the nine Bim 3'UTR miR-17-92 binding sites. Instead of causing the predicted B cell developmental block, interruption of miR-17-92:Bim interactions produced a selective inability of B cells to resist cellular stress; and prevented lymphocyte hyperplasia caused by Bim haploinsufficiency. Surprisingly, partial genetic disruption of miR-17-92:Bim interactions was sufficient to fully prevent B cell lymphoma formation in two out of three mice using two independent pre-clinical MYC-driven cancer models. This protective effect could be attributed to an increased activity of the mitochondrial apoptosis pathway in pre-malignant B cells, as apoptosis was abolished by concomitant overexpression of an anti-apoptotic BCL-2 protein. MYC-driven B lymphoma cells are addicted to miR-17-92 function. Our data build on these results and strongly suggest that miR-17-92:Bim interactions are vital in this context as acute ablation of miR-17-92:Bim interactions effectively promoted lymphoma cell apoptosis, both in vitro and in vivo. In conclusion, among hundreds of putative miR-17-92 target mRNAs a single direct binding partner is vital for lymphoma development and maintenance, a discovery whose therapeutic exploitation is of major relevance. Disclosures No relevant conflicts of interest to declare.

Download Full-text