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.

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.

Expression of NTB-A in Normal Tissue, Non-Hodgkin Lymphomas and Investigation of Its Potential Therapeutic Impact for Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4741-4741
Author(s):  
Xiaoxian Zhao ◽  
Wouter Korver ◽  
Nichole Prescott ◽  
Arie Abo ◽  
Eric Hsi
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Cell Lymphoma ◽  
White Blood Cells ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Normal Tissues

Abstract Introduction: Expressed on T, B, NK cells and neutrophils, NTB-A belongs to the signaling lymphocytic activation molecule (SLAM) family of immune receptors. It was reported recently that cross-linking of NTB-A induces T cell activation and Ly108 (the presumed mouse orthologue of NTB-A) controls the production of reactive oxygen species in neutrophils. To date, little is known about its expression at the protein level in normal tissues, or in hematologic malignancies. Methods: We have generated monoclonal antibodies (mAbs) to NTB-A and used tissue microarrays (TMAs) to screen normal tissues and lymphomas. Western blotting and flow cytometry were used for confirmation of selected entities. Complement dependent cytotoxicity (CDC) assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). Results: NTB-A was not detected in normal tissues including heart, liver, breast, kidney, brain, lung, uterus, small intestine, skin, prostate, pancreas, ovary, bladder, testis and stomach (5/5 negative cases for all above tissues) by IHC. Expression was observed in lymphocytes of normal tonsil and spleen. In lymphomas, we found NTB-A expression in diffuse large B-cell lymphoma (DLBL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma and Burkitt lymphoma. Western blotting and flow cytometry of B-cell lymphoma cell lines DOHH2 (DLBL) and Raji (Burkitt) confirmed NTB-A expression in these lines. Myeloma cell lines were negative for expression. Flow cytometry of normal blood cells showed expression of NTB-A in B-cells, T-cells and NK cells (95.5 ± 6.7%, 62.6 ± 29.5% and 72.7 ± 26.5%, respectively) but not in CD66+ granulocytes. Furthermore, CD34+ progenitor cells from bone marrow were negative. Because of the expression in SLL, we focused on chronic lymphocytic leykemia (CLL) cells. All (15/15) patient samples were confirmed positive by flow cytometry. No significant differences in expression levels were observed comparing CLL B-cells vs normal B-cells (n = 10). However, anti-NTB-A antibodies were capable of inducing CDC in white blood cells from CLL patients (n=5) but not in normal white blood cells (n=5), in addition to CDC activity against Raji cells. Preliminary data also suggests that the CDC effect is greater in purified CLL B-cells than purified normal B-cells. Conclusions: These results show that NTB-A is expressed in normal lymphocytes but not in other solid tissues or in CD34+ progenitor cells. B cell malignancies, including Non-Hodgkin lymphoma cells appear to express NTB-A. In particular CLL patient cells express NTB-A, and an anti-NTB-A mouse mAb is capable of inducing CDC, suggesting NTB-A may be a potential immunotherapeutic target. Further studies examining the expression patterns in hematologic malignancies are ongoing.

A Germinal Center- Derived B Cell Lymphoma Model

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2008-2008
Author(s):  
Ryan T Phan ◽  
Khang Nguyen ◽  
Sonia Romero ◽  
Alice Nicolson ◽  
Phillipp Nham ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Dna Breaks ◽  
Dna Rearrangements ◽  
Human B Cell

Abstract Abstract 2008 Most human B-cell lymphomas represent mature phenotypes of germinal center (GC) or post-GC origin and are frequently associated with chromosomal translocations, often involving the rearrangement of immunoglobulin (Ig) loci to various cellular oncogenes, leading to oncogenic activation. The mechanisms underlying these processes, however, are not well understood. Several studies suggest that these genetic lesions arise from errors of physiologic DNA rearrangements in GC B cells, namely class switch recombination (CSR) and somatic hypermutation (SHM). Here we report the generation of a mouse model in which DNA breaks are physiologically instituted in mature B cells, yet inefficiently repaired via specific deletion of DNA repair gene XRCC4 in GC B cells, thus effectively creating an in vivo environment for errors in DNA rearrangements. These activated B cells exhibit significant increased chromosomal IgH locus breaks and reduced CSR. In p53-deficient background, these mice develop B-cell lymphoma from 5.5 to 16 months. These clonally developed tumors characteristically harbor chromosomal translocations and phenotypically resemble mature phenotypes. Many of these tumors bear mutated V genes, suggesting that those cells have transited through GC. Thus, this mouse model mimics human B-cell lymphoma and might be useful for the development of therapeutic interventions in B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Preferential Acquision of N-Glycosylation Sites in the VDJ Region in Germinal Center B-Cell-Like Difusse Large B-Cell Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1589-1589 ◽  
Author(s):  
Miguel Alcoceba ◽  
Elena Sebastián ◽  
Ana Balanzategui ◽  
Luis Marín ◽  
Santiago Montes-Moreno ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Marginal Zone Lymphoma ◽  
Large B Cell Lymphoma ◽  
Glycosylation Sites ◽  
Large B Cell

Abstract Abstract 1589 Introduction: Acquired potentially N-glycosylation sites are produced by somatic hypermutation (SHM) in the immunoglobulin (Ig) variable region. This phenomenon is produced in ∼9% of normal B-cells and seems to be related to certain B-cell lymphoproliferative disorders (B-LPDs) such as follicular lymphoma (FL, 79%), endemic Burkitt lymphoma (BL, 82%) and diffuse large B-cell lymphoma (DLBCL, 41%). These data suggest that new potential N-glycosylation sites could be related to germinal center B (GCB)-LPDs. By contrast, in other B-LPDs, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), MALT lymphoma, Waldenström macroglobulinemia (WM) or multiple myeloma (MM), these modifications have not been analyzed in deep. Aims: To evaluate the acquisition of potential N-glycosylation sites in B-LPDs, including immunohystochemical DLBCL subtypes (GCB and non-GCB) and specific non-GCB-LPDs, such as hairy cell leukemia (HCL), splenic marginal-zone lymphoma (SMZL), CLL, MCL, ocular extranodal marginal zone lymphoma (OAEMZL), MM and WM. Patients: A total of 953 sequences (203 from our group and 750 previously published sequences) of B-LPDs were included. Diagnosis distribution was as follows: DLBCL (n=235), MCL (n=235), CLL (n=166), MM (n=96), OAEMZL (n=82), SMZL (n=68), WM (n=38) and HCL (n=33). Methods: Acquired N-glycosylation sites were counted according to the sequence Asn-X-Ser/Thr, where X could be any amino acid except Pro. Natural motifs in germline sequences of IGHV1–08, IGHV4–34 e IGHV-5a were not considered. Fisher test was used to perform comparisons between groups. To distinguish DLBCL biological subtypes (GCB and non-GCB DLBCL), Hans' algorithm was used. Results: A total of 83 out of the 235 DLBCL cases acquired at least a new N-glycosylation site, a higher value than in normal B-cells (35% vs. 9%, p<0.0001). Higher incidence of these motifs in the group of GCB as compared to non-GCB DLBCL were observed (52% vs. 20%, p<0.0001). Those cases diagnosed of HCL, CLL, MCL, MM, WM, OAEMZL and SMZL presented a reduced number of new N-glycosylation sites, showing similar values than normal B-cells (range 3–18%, p=ns). Conclusions: We described for the first time the pattern of N-glycosylation in HCL, SMZL, OAEMZL and in the immunohystochemical DLBCL subtypes, where the GCB-DLBCL showed a higher number of new N-glycosylation sites with respect to non-GCB DLBCL and other non-GCB-LPDs. The presence of novel N-glycosylation sites in FL, BL and in GCB-DLBCL strongly suggests that these motifs are characteristic of the germinal center B-LPDs. 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 A Signaling Pathway Mediating Downregulation of BCL6 in Germinal Center B Cells Is Blocked by BCL6 Gene Alterations in B Cell Lymphoma

Cancer Cell ◽  
2007 ◽  
Vol 12 (3) ◽  
pp. 280-292 ◽  
Author(s):  
Masumichi Saito ◽  
Jie Gao ◽  
Katia Basso ◽  
Yukiko Kitagawa ◽  
Paula M. Smith ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Signaling Pathway ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Gene Alterations ◽  
Germinal Center B Cells

Intraocular Lymphoma: Diagnostic Approach and Immunophenotypic Findings in Vitrectomy Specimens

2009 ◽  
Vol 133 (8) ◽  
pp. 1233-1237 ◽  
Author(s):  
Kirtee Raparia ◽  
Chung-Che(Jeff) Chang ◽  
Patricia Chévez-Barrios
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Who Classification ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Intraocular Lymphoma ◽  
B Cell Lymphomas ◽  
Not Otherwise Specified ◽  
Large B Cell Lymphoma ◽  
Large B Cell

AbstractContext.—Diagnosis and classification of primary intraocular lymphoma can be challenging because of the sparse cellularity of the vitreous specimens.Objective.—To classify and clinically correlate intraocular lymphoma according to the World Health Organization (WHO) classification by using vitrectomy specimens.Design.—Clinical history, cytologic preparations, flow cytometry reports, and outcome of 16 patients diagnosed with intraocular lymphoma were reviewed.Results.—The study group included 10 women and 6 men. The mean age of the patients was 63 years (range, 19–79 years). Eleven patients had central nervous system involvement and 6 patients had systemic involvement. All cases were adequately diagnosed and classified according to the WHO classification by using combination of cytologic preparations and 4-color flow cytometry with a limited panel of antibodies to CD19, CD20, CD5, CD10, and κ and λ light chains. The cases included 9 primary diffuse large B-cell lymphomas of the CNS type; 2 diffuse large B-cell lymphomas, not otherwise specified; 1 extranodal, low-grade, marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT); 1 precursor B-lymphoblastic lymphoma; and 3 peripheral T-cell lymphomas, not otherwise specified. Of note, all 11 cases of diffuse large B-cell lymphoma were CD10−. All the patients received systemic chemotherapy and radiation therapy. Only 4 patients were free of disease at last follow-up (range, 18 months to 8 years), with severe visual loss.Conclusions.—Intraocular lymphoma cases can be adequately classified according to the WHO classification. Diffuse large B-cell lymphoma, CD10− and most likely of non–germinal center B-cell–like subgroup, is the most common subtype of non-Hodgkin lymphoma in this site, in contrast to ocular adnexal lymphoma for which MALT lymphoma is the most common subtype.

Constitutively Activated STAT3 Promotes Cell Proliferation and Survival in the Activated B Cell Subtype of Diffuse Large B Cell Lymphomas.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1621-1621
Author(s):  
Bihui Hilda Ye ◽  
Beibei Belinda Ding ◽  
Jian Jessica Yu ◽  
Raymond Y.-L. Yu ◽  
Lourdes M. Mendez ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
B Cell Lymphoma ◽  
Cell Development ◽  
B Cell Development ◽  
Reciprocal Relationship ◽  
B Cell Lymphomas ◽  
Large B Cell

Abstract During B cell development, cell proliferation and survival are regulated by stage-specific transcription factors. Accordingly, distinct oncogenic pathways are employed by B cell lymphomas representing different stages of B cell development. Diffuse large B cell lymphoma (DLBCL) contains at least two main phenotypic subtypes, i.e. the germinal center B cell-like (GCB-DLBCL) and the activated B cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated NF-kappaB and tends to be refractory to chemotherapy. In this study, we investigated the relationship between BCL6 and STAT3 expression/activation in DLBCL and normal GC B cells. Our results demonstrate that BCL6 directly inhibits transcription of the STAT3 gene by binding to two BCL6 sites in its 5′ regulatory region. As a result, high level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Specifically, in tonsillar GCs, STAT3 expression and activation is restricted to a previously uncharacterized subset of BCL6−Blimp-1− B cells in the apical light zone. The location and phenotype of these cells suggest that they are in the process of exiting the BCL6-directed GC program and transitioning to a plasma cell differentiation process governed by Blimp-1. The reciprocal relationship between BCL6 and STAT3 is also conserved in DLBCL such that STAT3 expression and activation is preferentially associated with the BCL6-low, ABC subtype. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB and Mcl-1, and increased expression of the cell cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define STAT3 activation as a second oncogenic pathway operating in ABC-DLBCL and suggest that blocking STAT3 may be potentially therapeutic in treatment of these aggressive lymphomas.

Activation Induced Cytidine Deaminase-Associated DNA Repair Pathways Influence Germinal Center B Cell Lymphomagenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1247-1247
Author(s):  
Xiwen Gu ◽  
Carmen J. Booth ◽  
Zongzhi Liu ◽  
Matthew P. Strout
Keyword(s):  
Dna Repair ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
Cytidine Deaminase ◽  
B Cell Lymphoma ◽  
Chromosome Abnormalities ◽  
B Cell Lymphomas ◽  
Activation Induced Cytidine Deaminase ◽  
Repair Pathways

Abstract Somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation induced cytidine deaminase (AID). Resulting uracil-guanine (U-G) mismatches are processed by UNG-dependent base-excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with B cell lymphomas, the role of downstream AID-associated DNA repair pathways in lymphomagenesis is not defined. Through deregulated expression of BCL6, IµHABcl6 mice develop an AID-dependent GC-derived lymphoma that resembles human diffuse large B cell lymphoma (DLBCL). We have previously demonstrated that IµHABcl6 Ung-/-Msh2-/- mice have a similar incidence (35% vs 27%) but a 2.5-fold shorter median time to development of B220+ IgM+ PNAhi CD138- DLBCL compared with IµHABcl6 mice (6.5 months vs. 16.2 months; P = 0.0003). This suggests that AID-associated DNA repair pathways serve to protect the GC B cell and delay BCL6-driven lymphomagenesis. To investigate the individual contribution of BER and MMR in the pathogenesis of GC-derived lymphoma, we have now generated IµHABcl6 Ung-/- and IµHABcl6 Msh2-/- single-deficient mice. The majority of IµHABcl6 Ung-/- mice remained healthy beyond 20 months with only 3 of 22 (13.6%) mice becoming sick starting at ∼16 months. Sick mice were found to have splenic lymphomas comprised of mature B220+ IgM+ PNAlow CD138- B cells. Histological examination revealed expanded follicles with a population of small lymphocytes, consistent with a follicular B cell lymphoma which has been shown to arise in Ung-/- mice. In contrast, 18 of 22 (81.8%) IµHABcl6 Msh2-/- mice rapidly succumbed to malignancy starting at ∼3 months and had a median survival of 6 months. Of 15 tumors available for analysis, there was 1 histiocytic sarcoma, 1 squamous cell carcinoma, 4 T cell lymphomas, and 9 B220+ IgM- PNA- CD138- pre-B cell lymphomas (determined by histology, immunophenotyping and gene expression profiling). None of the IµHABcl6 Ung-/- or IµHABcl6 Msh2-/- mice developed DLBCL. Since lack of UNG is strongly protective when MSH2 is present, we conclude that in the setting of deregulated BCL6, UNG promotes the development of DLBCL. In contrast, MSH2 is protective against the development of tumors in general and does not facilitate DLBCL in the absence of UNG. Combined with the observation that IµHABcl6 Ung-/-Msh2-/- mice develop DLBCL with a significantly shorter latency than IµHABcl6 mice, this data indicates that a complex interplay between AID-associated BER and MMR produces a net protective effect against lymphomagenesis. In the absence of UNG and MSH2, AID-generated U-G mismatches are not processed into strand lesions and are simply replicated, yielding C/G to T/A transition mutations. Thus, to assess how combined lack of UNG and MSH2 might promote the accelerated development of BCL6-driven lymphoma, we carried out spectral karyotyping and sequence analysis of AID target genes (IgJH4, cMyc, Pim1, RhoH, Cd79a, CD79b, H2afx, Pax5, and Cd83) in lymphomas from the different genotypes. IµHABcl6 DLBCLs (3/3) harbored various complex chromosome abnormalities, consistent with previous findings. Numerous clonal and sub-clonal chromosome abnormalities including translocations, duplications, deletions, and aneuploidies were also detected in IµHABcl6 Ung-/-Msh2-/- (4/4) and IµHABcl6 Ung-/- (2/2) lymphomas. Pre-B cell tumors from IµHABcl6 Msh2-/- mice could not be stimulated to produce metaphase chromosomes. Clonal and non-clonal mutations of the IgJH4 intronic region were identified in lymphomas from IµHABcl6 (2/3), IµHABcl6 Ung-/-Msh2-/- (4/4), and IµHABcl6 Ung-/- (2/3) mice, consistent with ongoing AID activity. No mutations were detected in 3 pre-B cell lymphomas, consistent with their pre-GC origin. Six clonal mutations within AID hotspots (all C/G to T/A) were identified in Pim1, RhoH, and Pax5 in 2 of 4 IµHABcl6 Ung-/-Msh2-/- DLBCLs. None of the other genotypes carried any clonal mutations of non-Ig genes. Thus, chromosome abnormalities in GC B cell lymphomas can arise through mechanisms independent of BER and MMR but may be due to off-target effects of AID on other genes that regulate cell cycle, apoptosis, or genomic stability. Disclosures: No relevant conflicts of interest to declare.

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