Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice

Blood ◽  
2021 ◽  
Author(s):  
Miguel A Galindo-Campos ◽  
Nura Lutfi ◽  
Sarah Bonnin ◽  
Carlos Martínez ◽  
Talia Velasco-Hernandez ◽  
...  
Keyword(s):  
Dna Damage ◽  
B Cells ◽  
B Cell ◽  
Immune Escape ◽  
Cell Lymphoma ◽  
Tumour Progression ◽  
Cancer Therapeutics ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Parp 1

Dysregulation of the c-Myc oncogene occurs in a wide variety of haematologic malignancies and its overexpression has been linked with aggressive tumour progression. Here, we show that Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphomas. PARP-1 and PARP-2 catalyse the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA-strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphomas, while PARP-1-deficiency accelerates lymphomagenesis in the Em-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in pre-leukemic Em-Myc B cells resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1-deficiency induces a proinflammatory response, and an increase in regulatory T cells likely contributing to immune escape of B-cell lymphomas, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centred therapeutic strategies with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumours.

Profiles Of De Novo CD25-Positive Mature B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4308-4308
Author(s):  
Shin-ichiro Fujiwara ◽  
Raine Tatara ◽  
Kiyoshi Okazuka ◽  
Iekuni Oh ◽  
Ken Ohmine ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
High Expression ◽  
B Cell Lymphomas ◽  
Cns Involvement ◽  
Cd25 Expression

Abstract Background Interleukin 2 (IL-2) is an important cytokine that controls the proliferation and differentiation of not only T- but also B-lymphocytes. Recently, we reported that CD25 (IL-2 receptor alpha chain, IL-2R) is expressed in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and high expression of CD25 in the two types of lymphoma is correlated with a poor prognosis following chemotherapy regimens containing rituximab (ASH annual meeting, 2011 118:2666, 2012 120:1543). We evaluated the clinical significance of CD25 expression in a larger series of different mature B-cell lymphomas (BCL). Patients and Methods Four hundred and thirty-seven newly diagnosed patients who were admitted to our hospital between 2002 and 2013 were retrospectively evaluated. Lymph node or related tissue biopsy samples of BCL were analyzed using flow cytometry, as follows: 182 patients, DLBCL; 92, FL; 48, chronic lymphocytic leukemia (CLL); 21, mantle cell lymphoma (MCL); 23, marginal zone lymphoma (MZL); 8, Burkitt lymphoma (BL); 18, B-cell lymphoma unclassifiable with features intermediate between BL and DLBCL (BL/DLBCL); 5, lymphoplasmacytic lymphoma (LPL); and 39, reactive lymphadenopathy with sufficient B-cells. CD25-positivity was defined as >20% of clonal B-cells in a gated region. Results CD25 expression in patients with MCL, CLL, MZL, and DLBCL was significantly higher than that in patients with reactive lymphadenopathy (P<0.001,<0.001, =0.019, and <0.001, respectively). BL and FL, which were derived from germinal center B-cells, did not express CD25. These results indicate that pre- or post- germinal center-derived B-cells, activated by IL-2/IL-2R signaling, may give rise to CD25+ BCL such as CD25+ MCL, CLL, MZL, and DLBCL. The highest median CD25 expression (41.5%) was observed in MCL. CD25 expression was higher in MCL than CD5+ BCL (CLL and CD5+ DLBCL) (median, 41.5 vs. 16.9%, respectively; P<0.001). With a cut-off value of 60% CD25-positivity, patients with CD25-high (>60%) MCL (n=9) were not treated with aggressive chemotherapy regimens such as Hyper-CVAD due to their age and characteristics, compared with those with CD25-low (<60%) MCL (n=12) (11.1 vs. 72.7%, respectively, P=0.021). In patients with CLL, the range of CD25 expression was wide (0.4-90.7%), and 29 patients (60%) showed CD25-positivity (CD25+ CLL). CD25+ CLL showed higher soluble IL-2R (sIL-2R) levels and an inferior overall survival (OS) than CD25- CLL (median sIL-2R, 2,195 vs. 706 U/ml P=0.047; 5-year OS, 62.7 vs. 100%; P=0.037). There was a significant correlation between levels of CD25 and sIL-2R (r=0.53, P=0.0053). It is clinically important to distinguish between DLBCL and BCL involving MYC oncogene rearrangement (BL and BL/DLBCL, MYC+ BCL). The former showed higher CD25 expression than the latter (median, 10.2 vs. 2.1%, respectively, P=0.04). The progression-free survival rate (PFS) after rituximab containing chemotherapy was inferior in patients with CD25+ DLBCL (n=72) than those with CD25- DLBCL (n=110) and MYC+ BCL (5-year PFS, 49 vs. 70.4, 66.3%, respectively). In patients with DLBCL, central nerve system (CNS) involvement was observed in 15 patients (7 at diagnosis and 8 at relapse). CD25+ DLBCL showed a higher frequency of CNS involvement than CD25– DLBCL (13.8 vs. 4.5%, respectively, P=0.049). Regarding MZL, CD25 was highly expressed in nodal MZL, but it showed a low expression in splenic MZL. Regarding the sites of extranodal MZL, CD25 expression was lower in the thyroid than at other sites (median, 5.1 vs. 21.2%, respectively, P=0.37). There were some differences between CD25+ (n=9) and CD25- (n=14) MZL concerning the presence of B symptoms (33.3 vs. 0%, respectively) and advanced stage (66.6 vs. 35.7%, respectively). Conclusion CD25 expression using flow cytometry can potentially provide diagnostic and prognostic implications on BCL patient. The high expression of CD25 in MCL and CLL suggests the possibility of targeted anti-CD25 immunotherapy. These findings may shed light on the role of CD25 expression in B-cell lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous development of B-cell lymphomas in vivo

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4926-4933 ◽  
Author(s):  
Yuri Kasama ◽  
Satoshi Sekiguchi ◽  
Makoto Saito ◽  
Kousuke Tanaka ◽  
Masaaki Satoh ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Infection ◽  
Mouse Serum ◽  
B Cell Lymphomas ◽  
Α Subunit

AbstractExtrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%-70% of HCV-infected patients. B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. The mechanism by which HCV infection of B cells leads to lymphoma remains unclear. Here we established HCV transgenic mice that express the full HCV genome in B cells (RzCD19Cre mice) and observed a 25.0% incidence of diffuse large B-cell non-Hodgkin lymphomas (22.2% in males and 29.6% in females) within 600 days after birth. Expression levels of aspartate aminotransferase and alanine aminotransferase, as well as 32 different cytokines, chemokines and growth factors, were examined. The incidence of B-cell lymphoma was significantly correlated with only the level of soluble interleukin-2 receptor α subunit (sIL-2Rα) in RzCD19Cre mouse serum. All RzCD19Cre mice with substantially elevated serum sIL-2Rα levels (> 1000 pg/mL) developed B-cell lymphomas. Moreover, compared with tissues from control animals, the B-cell lymphoma tissues of RzCD19Cre mice expressed significantly higher levels of IL-2Rα. We show that the expression of HCV in B cells promotes non-Hodgkin–type diffuse B-cell lymphoma, and therefore, the RzCD19Cre mouse is a powerful model to study the mechanisms related to the development of HCV-associated B-cell lymphoma.

scholarly journals Cannabinoid Receptor Signaling As a Target for Personalized Therapy in Aggressive B Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4181-4181
Author(s):  
Lan Pham ◽  
Juan Chen ◽  
Archie Tamayo ◽  
Jerry Bryant ◽  
David Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cells ◽  
Cell Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
The United States ◽  
Cell Growth Inhibition

Abstract Non-Hodgkin Lymphoma (NHL) is the most common hematological malignancy, with B-cell lymphoma (NHL-B) accounting for 85% of all lymphomas. In the United States, there are ~500,000 lymphoma patients currently living with this disease and ~20,000 lymphoma-related deaths occur annually. The current overall cure rate for B-cell lymphoma is estimated at ~30%, indicating that new innovative therapeutic approaches are needed to significantly reduce the high mortality rate, particularly of relapsed/refractory (r/r) NHL-B. The poor quality of life in patients suffering from chronic diseases like cancer has forced many patients to pursue alternative treatment options, including medicinal cannabinoids (CB), in order to improve their clinical prospect/outcomes. Medicinal cannabinoids have been legalized in 23 states and DC for several medical conditions such as cachexia, chronic pain, epilepsy and other similar disorders characterized by seizures, glaucoma, HIV- AIDS, Multiple Sclerosis, muscle spasticity and GI enteritis. Lately however, cannabis has been shown to have a broader biologic activity spectrum with various cannabis compounds functioning as ligands binding the two principle cannabinoid-specific G protein-coupled receptors (GPCR) CB1 (in neural cells), and CB2, in immune lymphoid, particularly B cells, but have also been identified, showing aberrant expression in a wide variety of important human cancers. This suggests not only a wider spectrum of cellular usage of cannabinoids and their cognate receptors, but also their potential utility as novel therapeutic targets. Gene expression profiling data has demonstrated, however, that B-cell lymphoma is one of the top three cancers (glioma and gastric are the other two) showing high expression of CB1 and CB2 receptors. Our studies showed that CB1 receptor is highly expressed in aggressive NHL-B, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cells in comparison to normal unstimulated (G0) B cells, and that targeting CB1 using an siRNA approach leads to cell growth inhibition. Furthermore, pharmacological approaches targeting CB1 with small molecule antagonists (Rimonabant and Otenabant) inhibited lymphoma cell viability, leading to the induction of apoptosis and G2M cell cycle arrest. Using proteomic approach via reverse-phase protein array (RPPA), we have demonstrated that lymphoma cells treated with the CB1 antagonist Rimonabant showed a robust effect on apoptosis (increases in caspase 3 and 7, Bad, and bak), cell cycle (increases in p27 and cyclin D1), DNA damage (increases in gH2AX), and autophagy (increases in LC3A) associated proteins. In addition, Rimonabant treatment also inhibited several growth and survival pathways, including STAT3, SRC, and b-catenin, while enhancing the PI3K/ATK pathway. Of note, Rimonabant treatment also activated the DNA damage response (DDR) pathway through stimulating two checkpoint kinases (Chk1 and Chk2). Blocking Rimonabant-induced Chk1 and Chk2 with a selective ATP-competitive inhibitor of Chk1 and Chk2 leads to a robust synergistic effect on cell growth inhibition and apoptotic induction, suggesting that blocking the DDR pathway with Chk kinase inhibitors prevents cells recovering from rimonabant-induced DNA damage. These findings suggest that targeting the cannabinoid receptors and the DDR pathway represents a new therapeutic strategy against resistant r/r NHL-B cells. Disclosures Pham: Vyripharm Biopharmaceuticals: Research Funding. Bryant:Vyripharm Biopharmaceuticals: Equity Ownership. Yang:Vyripharm Biopharmaceuticals: Employment.

scholarly journals Genomic and Transcriptional Characterization of Primary Mediastinal Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2398-2398
Author(s):  
Rebecca J Leeman-Neill ◽  
Devang Thakkar ◽  
Sarah L. Ondrejka ◽  
Eric D. Hsi ◽  
Amy Chadburn ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Immune Escape ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Dna And Rna ◽  
Large B Cell Lymphoma ◽  
Recurrent Mutations ◽  
Large B Cell

Abstract Introduction: Primary mediastinal large B-cell lymphoma (PMBL) is a rare non-Hodgkin lymphoma subtype that occurs predominantly in young adults, with an overall favorable prognosis. The cell of origin is presumed to be thymic medullary B-cells and the gene expression profile of PMBL is similar to classic Hodgkin lymphoma. Recent studies have begun unravelling the genomic alterations underlying PMBL. Frequent, recurrent mutations (e.g. B2M, TNFAIP3, SOCS1, STAT6, GNA13) have been reported, but most of the studies have analyzed a small number of cases. To gain further insights into disease biology, we recruited 63 cases of PMBL as part of the Atlas of Blood Cancer Genomes (ABC-G) initiative, a consortium consisting of 25 institutions. Methods: Formalin-fixed paraffin-embedded (FFPE) biopsies and clinical data were collected. All cases were subjected to centralized review by an experienced panel of hematopathologists to ensure accurate diagnosis. Whole-exome DNA and RNA sequencing was performed using the Illumina platform and the DNA and RNA reads aligned to the GRCh38 genome and transcriptome respectively. Exonic variants were filtered using a set of paired normal samples and population-based databases to identify putative driver mutations, which were then aggregated at the gene level. Mutational analysis was performed on 56 samples that passed quality filtering and expression analysis on 45 samples. RNAseq data was normalized using DESeq2. Results: The cohort included samples from 16 males and 24 females, with a median age of 33 years (range 16 - 72) at the time of diagnosis. The majority of patients were treated with R-CHOP (47%) or R-EPOCH (43%), with 93% of patients surviving through the end of follow-up (median follow-up: 60.1 months). Besides the known recurrent mutations involving the JAK-STAT (STAT6 -21%, SOCS1 - 26%), NFKB (TNFAIP3 - 27%, NFKB1A - 7%), immune escape (B2M - 20%, LTB - 11%, IRF8 - 9%, IRF4 -9%), and chromatin modification (ZNF217 - 16%, CREBBP - 11%, KMT2D -11%) pathways , we discovered recurrent somatic variants in novel candidate driver genes in this disease, including NOTCH4 (7%), DICER1 (11%), MCL1 (7%), amongst others. EZH2, EP300, and XPO1 mutations were not detected. CIITA mutations and fusions were observed in 14% and 11% of cases, respectively, with novel partner genes (IGHA2, IGHG1, CDC6) detected in 67% of the fusion positive cases. Copy number alterations included gains at 2p16.1 (REL - 20%) and 9p24.2 (JAK2/PDL1/PDL2 - 24%), as well as loci not previously implicated in PMBL, 8q24.3 and 9q34.3 (each in 20%). Of note, CIITA alterations and 9p24 gains were virtually mutually exclusive, highlighting diverse mechanisms of immune escape in this entity. The transcriptomes of cases harboring CIITA alterations demonstrated differential enrichment of genes involved in protein glycosylation. The PMBLs in our series showed significant enrichment of the reported PMBL genetic classifier score, compared to nodal diffuse large B cell lymphoma (DLBCL) (p=0.0003). Finally, the gene expression profile of thymic B cells was more similar to that of PMBL than nodal DLBCL (p=0.0144). Conclusions: Our study, representing one of the largest comprehensive genomic and transcriptomic analyses of PMBL, expands the mutational landscape of PMBL, provides evidence for biologically distinct disease subsets and suggests an origin of PMBLs from thymic B-cells. Disclosures Hsi: AbbVie: Research Funding; Eli Lilly: Research Funding; Cytomx: Honoraria; Seattle Genetics: Honoraria. McKinney: BTG: Consultancy; Celgene: Consultancy, Research Funding; Epizyme: Consultancy; Genetech: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Molecular Templates: Consultancy, Research Funding; Nordic Nanovector: Research Funding; Novartis: Research Funding; Pharmacyclics: Consultancy; Verastem: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau. Jaye: Stemline Therapeutics: Honoraria. Cohen: Genentech, Takeda, BMS/Celgene, BioInvent, LAM, Astra Zeneca, Novartis, Loxo/Lilly: Research Funding; Janssen, Adaptive, Aptitude Health, BeiGene, Cellectar, Adicet, Loxo/Lilly, AStra ZenecaKite/Gilead: Consultancy. Behdad: Lilly: Speakers Bureau; Roche/Foundation Medicine: Speakers Bureau; Thermo Fisher: Speakers Bureau. Dave: Data Driven Bioscience: Current equity holder in publicly-traded company.

Spatial Signatures Identify Immune Escape via PD-1 as a Defining Feature of T-cell/Histiocyte-rich Large B-cell Lymphoma

Blood ◽  
2020 ◽  
Author(s):  
Gabriel K. Griffin ◽  
Jason L. Weirather ◽  
Margaretha Roemer ◽  
Mikel Lipschitz ◽  
Alyssa Kelley ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Immune Escape ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Spatially Resolved ◽  
Large B Cell Lymphoma ◽  
Immune Signatures ◽  
Large B Cell

T-cell/histiocyte-rich large B cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbor PD-L1/PD-L2 copy gain or amplification in 64% of cases, which is associated with increased PD-L1 expression (p = 0.0111). By directed and unsupervised spatial analyses of multi-parametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune 'neighborhoods' in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1-positive T cells. Further, unbiased clustering of spatially-resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in three of five patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies, including two complete responses and one partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL, and also support the potential utility of spatially-resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.

A Novel NF-kB:NFAT Enhanceosome Synergistically Regulate CD154 Gene in Aggressive B Cell Lymphomas.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 28-28 ◽  
Author(s):  
Lan Pham ◽  
Archito Tamayo ◽  
Linda Yoshimura ◽  
Yen-Chiu Lin-Lee ◽  
Richard J. Ford
Keyword(s):  
Transcriptional Regulation ◽  
B Cells ◽  
B Cell ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cd40 Ligand ◽  
B Cell Lymphomas ◽  
Incidence And Mortality

Abstract Non-Hodgkin’s Lymphoma (NHL) causes many deaths world wide, and is one of the few cancers that have a continual increase in incidence and mortality rates over the last few decades. Diffused large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma that accounts approximately 40% of all NHL-B cells. We have previously shown that dysregulated CD40 ligand (CD40L/CD154) expression in DLBCL maintains lymphoma cell growth and cell survival. CD40 ligand is a member of the TNF superfamily of proteins that has a wide variety effects throughout the immune system and is critical for both cellular and humoral immunity. It was originally thought that CD154 expression was restricted to activated CD4+ T lymphocytes. However, expression of CD154 has also been demonstrated in non-lymphocytic leukocytes such as mast cells, basophils, eosinophils, dendritic cells and macrophages. More importantly, dysregulated CD154 expression has been noted in a number of diseases, including systemic lupus erythematosus (SLE), Alzheimer’s, and in B cell lymphomas. Although the functional analysis of CD154 has been extensive, little is known about the mechanisms controlling CD154 expression in activated T cells, let alone in normal and malignant B cells. In this study, we show that constitutive NF-kB and NFAT activation in aggressive lymphoma B cells directly interact and synergistically regulate CD154 gene expression. Immunoprecipitation and confocal analyses indicate that NFATc1 and NF-kB (p65 and c-rel) complex and colocalize in lymphoma B cells. Chromatin Immunoprecipitation (CHIP) assays analysis demonstrate that NFATc1, p65 and c-rel bind to the CD154 promoter, that is enhanced by HDAC inhibitor (TSA or SAHA) treatments. HDAC inhibitors also enhance CD154 promoter activity, providing evidence that CD154 transcriptional regulation involves histone acetylation. Over-expression studies show a synergistic effect by NFATc1 and NF-kB (c-rel) on the CD154 promoter. Promoter deletion and site direct mutagenesis studies on the CD154 promoter reveal that the transcriptional regulation of CD154 requires two sites, the distal kB site at −1180 and a proximal NFAT site at −250. These sites bind to both, NFAT and NF-kB proteins, as wells as the DNA architectural protein HMG-1, indicating that the CD154 promoter DNA may loop and form an enhanceosome-like complex. Studies using siRNA to p65, c-rel, or NFATc1 repress CD154 promoter activity, indicating that these transcription factors are necessary and sufficient for CD154 gene transcription. These findings strongly suggest a novel control mechanism for CD154 gene transcription, therefore, providing potential treatment modalities for lymphoma B cells as well as other disorders involving dysredulated CD154 expression.

Inactivation Of BANK1 By a Novel IGH-Associated Translocation and 5’ Hypermethylation In B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2497-2497 ◽  
Author(s):  
Kui Nie ◽  
Taotao Zhang ◽  
Jiong Yan ◽  
Leonardo Boiocchi ◽  
Shuhua Cheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Methylation Status ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Intron 1 ◽  
Large B Cell

Abstract A novel IGH-associated reciprocal translocation, t(4;14)(q24;q32), was identified, along with trisomy 9, in 20 of 20 metaphases by conventional karyotyping in a case of malignant gastric post-transplant lymphoproliferative disorder (PTLD). Cloning of the translocation site by inverse PCR identified BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific adaptor protein with putative functions in B-cell receptor and CD40 signaling, as a novel IGH translocation partner. The breakpoints were located at the Sα region of IGH and intron 1 of BANK1. The translocation juxtaposed the two genes in opposite orientations, and surprisingly, resulted in transcriptional inactivation of BANK1 as a result of dissociation of the major BANK1 promoter. While BANK1 isoforms were expressed in all tonsillar B-cells, with lower levels (∼ 5 fold) in the germinal centers (GC) compared to naïve and memory B-cells, transcription from the major promoter in the tumor was absent and transcription from the minor promoter was reduced 50% relative to GC B-cells, suggesting that the non-translocated BANK1 allele was also inactivated. The total BANK1 expression was very low (∼10% of normal GC B cells) and crytic promoter activation was not identified. Several genes (PPP3CA, MIR1255A, FLJ20021 and SLC39A8), located 180 to 440 kb away from BANK1, were analyzed for mRNA expression; there is no significant activation in any of these genes, further supporting that BANK1is indeed the target gene affected by the translocation. Interphase FISH using break-apart BANK1 probes confirmed breakpoint in the index case but did not identify translocations in additional 15 PTLDs and 68 diffuse large B-cell lymphomas (DLBCL), implying that BANK1 translocation may be a rare event. To determine if BANK1 inactivation may occur in B-cell lymphomas by other mechanisms, 23 B-cell lymphoma cell lines, including 8 Burkitt lymphoma (BL), 9 diffuse large B cell lymphoma (DLBCL), 3 primary effusion lymphoma (PEL), and 3 classical Hodgkin lymphoma (cHL) were bisulfite sequenced to assess the methylation status of 37 CpG dinucleotides in a 436 base-pair region at the 5’ end of BANK1, which extends across exon 1 into the 5’ portion of intron 1. High level of methylation (>60% methylation on average among all CpGs) was seen in all 3 cHL and 2 of 3 PEL cell lines. Regional methylation was seen in 3 of 8 BL lines and 1 of 3 PEL lines. No hypermemethylation was identified in the DLBCL lines or in normal tonsils. Hypermethylation was associated with almost complete silencing of BANK1 transcription. In the DLBCL lines and BL lines without BANK1 hypermethylation, BANK1mRNA expressions were variable, ranging from <5% to 130% of GCB cells. To confirm that BANK1 hypermethylation is present in primary lymphoma cases, methylation status of 17 of the 37 CpGs were assessed in 23 cHL cases using en bloc formalin-fixed, paraffin-embedded materials and also laser-capture micro-issected Hodgkin/Reed-Sternberg (HRS) cells. There was evidence of BANK1 hypermethylation in the tumor cells in 9 of 23 cHL. Tumor cell specificity of BANK1 hypermethylation was further confirmed in 4 cHL cases using micro-dissected HRS cells. HRS cells were negative for BANK1 in 28 of 29 cHL cases examined by immunohistochemistry, suggesting that other mechanisms other than DNA methylation may be responsible for silencing BANK1expression. To investigate whether BANK1 has biological effects on B-cells related to lymphoma development, exogenous BANK1 was re-introduced to BC3, a PEL cell line showing marked BANK1 hypermethylation with absence of BANK1 expression. We established a stable doxycycline-inducible BC3 cell line expressing BANK1. Inhibition of cell growth was observed 2 to 3 days after doxycyline induction, and the number of viable cells with transfected BANK1 was only 25% compared to BC3 cells carry vehicle alone at day 6. An analysis of 5-bromo-2’ deoxyuridine (BrdU) incorporation after 48 hours of doxycline induction revealed that the fraction of cells in S-phase was reduced by 50% in the BANK1 transfectants, suggesting that BANK1has a negative effect on cell proliferation in these B cells. In summary, we have identified a novel IGH translocation partner and provide an example of an unusual consequence (gene inactivation) of IGH-associated translocation. We provide for the first time evidence of a potential role of BANK1 down-regulation in the development of B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Cerebrospinal Fluid Immunophenotyping in B-Cell Lymphomas: A Single Centre Experience on 99 Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2629-2629
Author(s):  
Anne Plonquet ◽  
Jehan Dupuis ◽  
Christo Christov ◽  
Christiane Copie-Bergman ◽  
Karim Belhadj ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
B Cells ◽  
B Cell ◽  
Lumbar Puncture ◽  
Cell Population ◽  
Cell Lymphoma ◽  
International Prognostic Index ◽  
B Cell Lymphoma ◽  
Neurological Symptoms ◽  
B Cell Lymphomas

Abstract Central nervous system involvement in B-cell lymphomas is almost always lethal. Recognized risk factors include Burkitt histology and high LDH level, multiple extranodal sites or an age-adjusted International Prognostic Index of > 1 in the case of diffuse large B-cell lymphoma (DLBCL) (van Besien et al. Blood 1998, Haioun, Ann Oncol 2000). Standard diagnostic approaches such as imaging (MRI, CT) and cytological study of the cerebrospinal fluid (CSF) are flawed with a large number of false negative results. Accordingly, currently used prophylactic approaches based on these uncertain criteria carry a risk of over- or undertreating patients. Lately, flow cytometry (FCM) has been shown to detect a high incidence of “occult” CNS disease in patients at risk (Hedge et al. Blood 2005), thus paving the way towards a more specific management of these cases. We performed an exploratory single-center study on 99 CSF samples from patients with B-cell lymphomas. FCM results did not influence the therapeutic strategy. CSF samples were processed as soon as possible following lumbar puncture (always within 3hrs). They were washed in saline buffer, counted in Trypan Blue (to evaluate cellularity & viability), and 1/3 was labelled with the following antibodies: CD45, CD3, CD14, CD19, CD5, and CD10. Two thirds of the remaining cells were either labelled with the same antibodies (in case paucicellularity did impede interpretation of the first labelling on one third of the sample), or with one of two complementary antibody combinations: CD20, CD22, CD19 or Kappa/Lambda/CD19 according to cellularity and clinical data. From July 2005 to April 2007, 99 samples from 87 consecutive patients were analyzed. Median age was 61 years and 64% patients had an IPI score of 3 or more. Forty six patients were devoid of neurological symptoms at the time of analysis. Forty had DLBCL, 6 Burkitt. Thirteen out of the 99 samples could not be analyzed because they were massively contaminated with blood, 7 because they showed non specific fluorescence, thus leaving 79 (80%) analyzable samples from 75 patients. Although some samples contained an excess of B-cells (22% with ≥ 5% and < 10% CD19+ cells, 22% with ≥10% CD19+ cells), none showed a clearly phenotypically abnormal B-cell population. On a clinical basis, all were treated with curative intent and 82% had meningeal prophylaxis. No patient developed CNS localization with a median follow-up of 15 months.Twenty-five samples were taken from 19 patients with neurological symptoms (cranial nerve/radicular palsies n=9, CNS tumors n=6, epiduritis n=4). Three cases showed a clearly phenotypically abnormal B-cell population (1 case of Burkitt lymphoma, 2 cases of lymphoplasmacytoid lymphoma), all being also positive by cytology.In conclusion, although preliminary, these results suggest that in our hand FCM appears not more sensitive that cytology in detecting CSF infiltration by neoplastic B-cells in aggressive B-cell lymphoma when performing a single exploratory lumbar puncture. A larger cohort of patients needs to be explored with standardization of FCM immunophenotyping technique in order to improve further diagnostic accuracy of CSF localization in lymphoma.

Deregulation of CCND1 or BCL2 in the Immature B-Cell Stage Determines the Resulting Lymphoma Histology

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 416-416
Author(s):  
Masaharu Tashima ◽  
Momoko Nishikori ◽  
Wataru Kishimoto ◽  
Ryo Yamamoto ◽  
Tomomi Sakai ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Population ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Cell Stage ◽  
Genetic Event

Abstract Abstract 416 In B-cell lymphomas, several chromosomal translocations are associated with specific histological subtypes. CCND1/IGH is detected in more than 90 % of the cases with mantle cell lymphoma (MCL), and BCL2/IGH is characteristically observed in follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center (GC) B-cell origin. Although these strong correlations are clinically recognized, their biological mechanism is not clearly explained so far. According to the results of translocation breakpoint mapping, both CCND1 and BCL2 translocations are considered to be generated by an error during physiological VDJ rearrangement of the IGH gene in the precursor-B cell stage. We hypothesized that the occurrence of these translocations in the immature B-cell stage, probably as an initial genetic event, should have a special impact on the determination of resulting lymphoma histology. We generated a mouse model mimicking human lymphoma with CCND1 or BCL2 translocation by lentivirally introducing these genes into Tp53+/− B6 mouse bone marrow cells and transplanting them to lethally irradiated wild-type B6 mice. In this model, CCND1 or BCL2 is expressed from immature to mature B cell stage under the control of the CD19 promoter, and subsequently mutations accumulate in the background of Tp53 haploinsufficiency. Both mice developed B-cell lymphomas several months after the transplantation, but their tumors showed some different features. CCND1-Tp53+/− mice developed B220lowCD5+CD23− tumors that expand into the B-cell area with sparing the GC, whereas BCL2-Tp53+/− mice preferentially developed B220highCD5−CD23+ tumors that tend to localize in the GC. Additionally, somatic hypermutation (SHM) analysis of the IGH gene of the tumor cells revealed obviously higher mutation frequency in the BCL2-Tp53+/− mice than in the CCND1-Tp53+/- mice (p<0.004). These results indicate that the primary gene deregulation of CCND1 and BCL2 determines the upcoming lymphoma of MCL-type and GC B-cell lymphoma-type, respectively. MCL has been postulated to be derived from naïve pre-GC B cells, with few SHM introduced in the immunoglobulin variable region. But the MCL-like tumor cells generated in CCND1-Tp53+/−mice seem to be originated from B-1a B cells, a distinct B cell population that does not enter GC and has few SHM in nature. There has been a debate whether B-1 cell population exists in humans, but it is recently proposed that the phenotype of human B-1 cells is CD20+CD27+CD43+CD70− by testing sort-purified B cell fractions for fundamental B-1 cell functions based on mouse studies (J Exp Med 2011;208:67–80). Interestingly, flow cytometric analysis of the human MCL tumor cells has shown that they mostly express this provisional B-1 cell phenotype, supporting the idea that human MCL is also derived from B-1 cells. Our mouse model precisely reproduces the link between CCND1 and BCL2 translocations and the resulting lymphoma subtypes in humans. It is assumed that these translocations trigger the cell expansion of different B-cell subgroups, which consequently leads to the development of lymphoma of distinct histology. Our findings provide new insights into the mechanism of lymphoma subtype determination. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TCL1: a shared tumor-associated antigen for immunotherapy against B-cell lymphomas

Blood ◽  
2012 ◽  
Vol 120 (8) ◽  
pp. 1613-1623 ◽  
Author(s):  
Jinsheng Weng ◽  
Seema Rawal ◽  
Fuliang Chu ◽  
Hyun Jun Park ◽  
Rakesh Sharma ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Patient Specific ◽  
Autologous Tumor ◽  
B Cell Lymphomas ◽  
B Cell Malignancies

Abstract Immunotherapy with therapeutic idiotype vaccines offers promise for treatment of B-cell malignancies. However, identification of novel immunogenic lymphoma-associated antigens that are universally expressed is necessary to overcome the barriers of patient-specific idiotype vaccines. Here, we determined whether T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene could be a target for immunotherapy of B-cell malignancies. We show that TCL1 mRNA and protein are selectively expressed in normal B cells but markedly hyperexpressed in multiple human B-cell lymphomas, including follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, and splenic marginal zone B-cell lymphoma. We demonstrated that TCL1-specific CD8+ T cells can be generated from HLA-A*0201 (HLA-A2)+ normal donors and identified TCL171-78 (LLPIMWQL) as the minimal epitope recognized by these T cells. More importantly, TCL171-78 peptide-specific T cells were present in the peripheral blood and tumor-infiltrating lymphocytes of lymphoma patients, could be expanded in vitro, and lysed autologous tumor cells but not normal B cells in an HLA-A2–restricted manner. Our results suggest that TCL1 is naturally processed and presented on the surface of lymphoma cells for recognition by cytotoxic T cells and can serve as a novel target for development of immunotherapeutic strategies against common B-cell lymphomas.

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