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Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during antigen-driven B cell differentiation in the germinal center reaction. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Most B cell lymphomas require B cell antigen receptor expression, and in several instances chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Most B cell malignancies derive from germinal center B cells, most likely due to the high proliferative activity of these B cells and aberrant mutations caused by their naturally active mutagenic processes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis., (© 2024. The Author(s).)

Classic Hodgkin lymphoma (cHL) constitutes a B-cell neoplasm derived from germinal center lymphocytes. Despite high cure rates (80-90%) obtained with the current multiagent protocols, a significant proportion of cHL patients experience recurrences, characterized by a lower sensitivity to second-line treatments. The genomic background of chemorefractory cHL is still poorly understood, limiting personalized treatment strategies based on molecular features. In this study, using a targeted next-generation sequencing (NGS) panel specifically designed for cHL research, we compared chemosensitive and chemorefractory diagnostic tissue samples of cHL patients. Furthermore, we longitudinally examined paired diagnosis-relapsesamples of chemorefractory cHL in order to define patterns of dynamic evolution and clonal selection. Pathogenic variants in NOTCH1 and NOTCH2 genes frequently arise in cHL. Mutations in genes associated with epigenetic regulation (CREBBP and EP300) are particularly frequent in relapsed/refractory cHL. The appearance of novel clones characterized by mutations previously not identified at diagnosis is a common feature in cHL cases showing chemoresistance to frontline treatments. Our results expand current molecular and pathogenic knowledge of cHL and support the performance of molecular studies in cHL prior to the initiation of first-line therapies.

In eukaryotic cells, the genome is three dimensionally (3D) organized with DNA interaction dynamics and topology changes that regulate gene expression and drive cell fate. Upon antigen stimulation, naive B cells are activated and form germinal centers (GC) for the generation of memory B cells and plasma cells. Thereby, terminal B-cell differentiation and associated humoral immune response require massive but rigorous 3D DNA reorganization. Here, we review the dynamics of genome reorganization during GC formation and the impact of its alterations on lymphomagenesis from the nucleosome structure to the higher order chromosome organization. We particularly discuss the identified architects of 3D DNA in GC B cells and the role of their mutations in B-cell lymphomas., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lymphomas are cancers deriving from lymphocytes, arising preferentially in secondary lymphoid organs, and represent the 6th cancer worldwide and the most frequent blood cancer. The majority of B cell Non-Hodgkin lymphomas (B-NHL) develop from germinal center (GC) experienced mature B cells. GCs are transient structures that form in lymphoid organs in response to antigen exposure of naive B cells, and where B cell receptor (BCR) affinity maturation occurs to promote B cell differentiation into memory B and plasma cells producing high-affinity antibodies. Genomic instability associated with the somatic hypermutation (SHM) and class-switch recombination (CSR) processes during GC transit enhance susceptibility to malignant transformation. Most B cell differentiation steps in the GC are at the origin of frequent B cell malignant entities, namely Follicular Lymphoma (FL) and GCB diffuse large B cell lymphomas (GCB-DLBCL). Over the past decade, large sequencing efforts have provided a great boost in the identification of candidate oncogenes and tumor suppressors involved in FL and DLBCL oncogenesis. Mouse models have been instrumental to accurately mimic in vivo lymphoma-specific mutations and interrogate their normal function in the GC context and their oncogenic function leading to lymphoma onset. The limited access of biopsies during the initiating steps of the disease, the cellular and (epi)genetic heterogeneity of individual tumors across and within patients linked to perturbed dynamics of GC ecosystems make the development of genetically engineered mouse models crucial to decipher lymphomagenesis and disease progression and eventually to test the effects of novel targeted therapies. In this review, we provide an overview of some of the important genetically engineered mouse models that have been developed to recapitulate lymphoma-associated (epi)genetic alterations of two frequent GC-derived lymphoma entities: FL and GCB-DLCBL and describe how those mouse models have improved our knowledge of the molecular processes supporting GC B cell transformation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mossadegh-Keller, Brisou, Beyou, Nadel and Roulland.)

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells., Competing Interests: Declaration of Interests A.M.M. is consulting for Epizyme and Constellation Pharmaceuticals, and receives research funding from Janssen Pharmaceuticals; S.H.K. is consulting for Northrop Grumman; C.E.M. is a co-founder and equity stake holder for Onegevity Health and Biotia, Inc.; C.S. has performed consultancy for Seattle Genetics, Curis Inc., Roche, AbbVie, Juno Therapeutics, and Bayer, and has received research funding from Bristol-Myers Squibb and Trillium Therapeutics, Inc. There are no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Human B-cell lymphoma 6 (BCL6) gene, usually coding protein of 706 amino acids, is closely associated with large B cell lymphoma. Researches showed that protein mutation or change of expression levels usually happened in the mounting non-hodgkin lymphoma (NHL). Thus BCL6 is considered to be involved in germinal center (GC)-derived lymphoma., Results: The BCL61-350 gene codons were optimized for prokaryotic system. After expression of BCL61-350 in E. coli, the BCL61-350 protein was purified with Ni column. Then the BCL61-350 protein, mixing with QuickAntibody-Mouse5W adjuvant, was injected into Balb/c mice. After immunization and cell fusion, a stable cell line named 1E6A4, which can secrete anti-BCL6 antibody, was obtained. The isotype of 1E6A4 mAb was determined as IgG2a, and the affinity constant reached 5.12×1010 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the 1E6A4 mAb was able to detect BCL6 specifically and sensitively., Conclusions: BCL61-350 antigen has been successfully generated with an effective and feasible method, and a highly specific antibody named 1E6A4 against BCL6 has been screened and characterized in this study, which was valuable in clinical diagnosis., Competing Interests: We have the following interests: Qinghai Yang and Huiling Chen are employed by Fuzhou Maixin Biotech. Co., Ltd. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

CD74 is a type II transmembrane glycoprotein that functions as an MHC class II chaperone and displays diverse roles in immune responses. Recently, anti-CD74 immunotherapy has shown promise as an effective treatment strategy for lymphoid neoplasms in preclinical models. Using a human anti-CD74 antibody (SP7219), we defined the expression of CD74 protein in both normal and over 790 neoplastic hematolymphoid tissue samples. We found that CD74 is expressed broadly in normal B-cell compartments including primary and secondary lymphoid follicles and in the thymic medulla. The vast majority of lymphomas expressed CD74, including Hodgkin lymphomas (98%), B-cell lymphomas (96%), extranodal NK/T-cell lymphomas (88%), mature T-cell lymphomas (80%), and plasma cell myeloma (75%). Our findings confirm and expand previous observations regarding the expression of CD74 and suggest that CD74 expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and plasma cell malignancies., (© 2018 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Objectives: KLHL6 is a recently described BTB-Kelch protein with selective expression in lymphoid tissues and is most strongly expressed in germinal center B cells., Methods: Using gene expression profiling as well as immunohistochemistry with an anti-KLHL6 monoclonal antibody, we have characterized the expression of this molecule in normal and neoplastic tissues. Protein expression was evaluated in 1,058 hematopoietic neoplasms., Results: Consistent with its discovery as a germinal center marker, KLHL6 was positive mainly in B-cell neoplasms of germinal center derivation, including 95% of follicular lymphomas (106/112). B-cell lymphomas of non-germinal center derivation were generally negative (0/33 chronic lymphocytic leukemias/small lymphocytic lymphomas, 3/49 marginal zone lymphomas, and 2/66 mantle cell lymphomas)., Conclusions: In addition to other germinal center markers, including BCL6, CD10, HGAL, and LMO2, KLHL6 immunohistochemistry may prove a useful adjunct in the diagnosis and future classification of B-cell lymphomas., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Purpose of Review: There is mounting evidence that heterogeneity of the epigenome is a feature of many cancers, including B-cell lymphomas, and presents important clinical implications. The purpose of this review is to explain the biological and clinical relevance of this epigenetic phenomenon in B-cell neoplasms., Recent Findings: Here, we summarize new findings demonstrating that B-cell lymphomas display increased DNA methylation heterogeneity compared to their normal counterparts. This plasticity of cytosine methylation manifests both as intertumor and intratumor heterogeneity and is associated with worse prognosis and poor clinical outcome in lymphoma patients. Recent studies of different subtypes of B-cell lymphomas have revealed that epigenetic aberrations and heterogeneous cytosine methylation patterning are common features of all neoplasms derived from B-lymphocytes, irrespective of maturation stage. With regard to mechanisms driving this process, recent reports suggest that cytosine methylation heterogeneity arises through passive and active processes. One factor implicated in active generation of cytosine methylation heterogeneity is activation-induced cytidine deaminase, which mediates DNA methylation changes and introduces epigenetic heterogeneity in normal germinal center B cells, the cells of origin of mature B-cell neoplasms such as diffuse large B-cell lymphoma and follicular lymphoma., Summary: Understanding the scope and mechanism of epigenetic heterogeneity in cancer is of paramount importance to our understanding of clonal plasticity and treatment responses in B-cell lymphomas.

Methods for tracking B-cell repertoires and clonal history in normal and malignant B-cells based on immunoglobulin variable region (IGV) gene analysis have developed rapidly with the advent of massive parallel next-generation sequencing (mpNGS) protocols. mpNGS permits a depth of analysis of IGV genes not hitherto feasible, and presents challenges of bioinformatics analysis, which can be readily met by current pipelines. This strategy offers a potential resolution of B-cell usage at a depth that may capture fully the natural state, in a given biological setting. Conventional methods based on RT-PCR amplification and Sanger sequencing are also available where mpNGS is not accessible. Each method offers distinct advantages. Conventional methods for IGV gene sequencing are readily adaptable to most laboratories and provide an ease of analysis to capture salient features of B-cell use. This chapter describes two methods in detail for analysis of IGV genes, mpNGS and conventional RT-PCR with Sanger sequencing.

Most human B cell lymphomas originate from germinal center (GC) B cells. This is partly caused by the high proliferative activity of GC B cells and the remodeling processes acting at the immunoglobulin (Ig) loci of these cells, i.e., somatic hypermutation and class-switching. Mistargeting of these processes can cause chromosomal translocations, and the hypermutation machinery may also target non-Ig genes. As somatic hypermutation is exclusively active in GC B cells, the presence of somatic mutations in rearranged IgV genes is a standard criterium for a GC or post-GC B cell origin of lymphomas. Beyond this, ongoing somatic hypermutation during lymphoma clone expansion indicates that the lymphoma has an active GC B cell differentiation program. The proto-oncogene BCL6 is specifically expressed in GC B cells and also acquires somatic mutations as a physiological by-product of the somatic hypermutation process, albeit at a lower level than IgV genes. Thus, detection of BCL6 mutations is a further genetic trait of a GC experience of a B cell lymphoma. Typically, B cell lymphomas retain key features of their specific cells of origin, including a differentiation stage-specific gene expression pattern. This is at least partly due to genetic lesions, which "freeze" the lymphoma cells at the differentiation stage at which the transformation occurred. Therefore, identification of the normal B cell subset with the most similar gene expression pattern to a particular type of B cell lymphoma has been instrumental to deduce the precise cell of origin of lymphomas.We present here protocols to analyze human B cell lymphomas for a potential origin from GC B cells by determining the presence of mutations in rearranged IgV genes and the BCL6 gene, and by comparing the gene expression pattern of lymphoma cells with those of normal B cell subsets by genechip or RNA-sequencing analysis.

Thymocyte selection-associated high-mobility group box (TOX) is aberrantly expressed in cutaneous T-cell lymphomas. In a recent study, TOX expression was noted unexpectedly in the follicle center (germinal center) B-cells of reactive lymph nodes and tonsils, used as external controls. To evaluate whether TOX is also expressed by cutaneous B-cell lymphomas, TOX immunohistochemistry was performed on skin biopsies of 44 patients with primary and secondary cutaneous B-cell proliferations. TOX was expressed not only in the reactive follicle center cells of lymph nodes, tonsils, cutaneous lymphoid hyperplasia, and primary cutaneous marginal zone lymphomas, but also by the neoplastic follicle center cells of 16/17 patients with primary cutaneous follicle center lymphoma (PCFCL) and 7/7 patients with cutaneous manifestations of systemic follicular lymphoma (FL). Notably, TOX showed a very similar expression pattern as BCL6, a marker of germinal center B-cells. In 4/10 patients with a BCL6(+) primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL,LT) and in 2/2 patients with a secondary cutaneous BCL6(+) diffuse large B-cell lymphoma (DLBCL), TOX was expressed by more than 50 % of the neoplastic B-cells. In contrast, in 3/3 BCL6(-) PCDLBCL,LT, TOX was completely negative or weakly expressed by a minor proportion of the neoplastic B-cells. In conclusion, TOX is expressed not only by neoplastic T-cells, but also by both reactive and neoplastic follicle center (germinal center) B-cells and a proportion of BCL6(+) PCDLBCL,LT and secondary cutaneous BCL6(+) DLBCL. The functional significance of TOX expression in reactive and neoplastic B-cells remains to be elucidated.

Purpose of Review: Perturbation of the epigenome is emerging as a central driving force in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and follicular lymphoma. The purpose of this review is to explain how alteration of different layers of the epigenome contributes to the biology and clinical features of these tumors., Recent Findings: Key new findings implicate DNA methylation heterogeneity as a core feature of DLBCL. Epigenetic diversity is linked to unfavorable clinical outcomes, clonal selection at relapse, and is driven at least in part because of the actions of activation-induced cytosine deaminase, which is a unique feature of B-cell lymphomas. Somatic mutations in histone modifier genes drive lymphomagenesis through the establishment of aberrant gene-specific histone modification signatures. For example, EZH2 somatic mutations drive silencing of bivalent gene promoters through histone 3 lysine 27 trimethylation, whereas KMT2D (MLL2) mutations disrupt specific sets of enhancers through depletion of histone 3 lysine 4 mono and dimethylation (H3K4me1/me2)., Summary: Appreciation of the epigenome in determining lymphoma clonal heterogeneity and in driving lymphoma phenotypes through altered promoter and enhancer histone modification profiles is leading to a paradigm shift in how we understand and design therapies for DLBCL and follicular lymphoma.

GNA13 is the most frequently mutated gene in germinal center (GC)-derived B-cell lymphomas, including nearly a quarter of Burkitt lymphoma and GC-derived diffuse large B-cell lymphoma. These mutations occur in a pattern consistent with loss of function. We have modeled the GNA13-deficient state exclusively in GC B cells by crossing the Gna13 conditional knockout mouse strain with the GC-specific AID-Cre transgenic strain. AID-Cre(+) GNA13-deficient mice demonstrate disordered GC architecture and dark zone/light zone distribution in vivo, and demonstrate altered migration behavior, decreased levels of filamentous actin, and attenuated RhoA activity in vitro. We also found that GNA13-deficient mice have increased numbers of GC B cells that display impaired caspase-mediated cell death and increased frequency of somatic hypermutation in the immunoglobulin VH locus. Lastly, GNA13 deficiency, combined with conditional MYC transgene expression in mouse GC B cells, promotes lymphomagenesis. Thus, GNA13 loss is associated with GC B-cell persistence, in which impaired apoptosis and ongoing somatic hypermutation may lead to an increased risk of lymphoma development., (© 2016 by The American Society of Hematology.)

Aims: Large B cell lymphomas with MYC and BCL6/3q27 rearrangements, designated MYC/BCL6 DHL, are uncommon. Our aim was to better characterize this group of tumours., Methods and Results: We studied the clinicopathological features and outcome of 13 patients with MYC/BCL6 DHL and compared this group to a group of 83 MYC/BCL2 DHL patients. There were eight men and five women, with a median age of 63 years. Eleven tumours were classified as diffuse large B cell lymphomas (DLBCL) and two were B cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (BCLU). Immunohistochemical analysis showed that these tumours were positive for BCL6 (100%), BCL2 (eight of 10; 80%) and CD10 (eight of 10; 80%). Nine of 12 (75%) cases had a germinal centre B cell (GCB) immunophenotype; in one case data were incomplete. All patients were treated with chemotherapy. The clinicopathological features of MYC/BCL6 DHL were similar to MYC/BCL2 DHL, except that MYC/BCL6 DHL had a GCB immunophenotype less often. Patients with MYC/BCL6 DHL had a poor overall survival, similar to patients with MYC/BCL2 DHL (P = 0.32)., Conclusions: MYC/BCL6 DHL is an aggressive B cell lymphoma and patients often have an aggressive clinical course and poor prognosis, similar to patients with MYC/BCL2 DHL., (© 2015 John Wiley & Sons Ltd.)

Background: Activated B lymphocytes harbor programmed DNA double-strand breaks (DSBs) initiated by activation-induced deaminase (AID) and repaired by non-homologous end-joining (NHEJ). While it has been proposed that these DSBs during secondary antibody gene diversification are the primary source of chromosomal translocations in germinal center (GC)-derived B cell lymphomas, this point has not been directly addressed due to the lack of proper mouse models., Methods: In the current study, we establish a unique mouse model by specifically deleting a NHEJ gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in GC B cells, which results in the spontaneous development of B cell lymphomas that possess features of GC B cells., Results: We show that these NHEJ deficient lymphomas harbor translocations frequently targeting immunoglobulin (Ig) loci. Furthermore, we found that Ig translocations were associated with distinct mechanisms, probably caused by AID- or RAG-induced DSBs. Intriguingly, the AID-associated Ig loci translocations target either c-myc or Pvt-1 locus whereas the partners of RAG-associated Ig translocations scattered randomly in the genome. Lastly, these NHEJ deficient lymphomas harbor complicated genomes including segmental translocations and exhibit a high level of ongoing DNA damage and clonal heterogeneity., Conclusions: We propose that combined NHEJ and p53 defects may serve as an underlying mechanism for a high level of genomic complexity and clonal heterogeneity in cancers.

Purpose of Review: This review provides current knowledge on the role of microRNAs (miRNAs) in lymphoma with an emphasis on mature B-cell lymphoma., Recent Findings: Although miRNAs were previously used to stratify lymphoma classification, prognosis, or treatment response, recent publications portray this class of small noncoding RNAs as critical players in the lymphomagenesis process. Although functional studies provide ample evidence for their role as lymphoma drivers or suppressors, genetic studies providing the underlying mechanisms for these phenotypes are still lacking. As more whole-genome sequencing of lymphoma cases become available, this gap may be soon filled., Summary: Current findings highlight the potential role of miRNAs molecules as important factors in lymphomagenesis.

Ataxia telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin lymphomas, including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B cells causes cell autonomous, clonal mature B-cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly, naive B-cell-specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. Although EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increases the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas toward pre-GC-derived small lymphocytic neoplasms, sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naive B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.e. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-GC B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL.

Although there has been increased attention paid to the critical nature of nodal involvement in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), the B-cell compartment it is most closely related to and its relationship to the follicle remain uncertain. A clinicopathologic investigation of 60 extramedullary biopsies of LEF1+ CLL/SLL, including 29 cases with perifollicular/follicular (PF/F) growth, was therefore performed. A subset of PF/F cases demonstrated inner mantle zone preservation or intra-mantle zone growth. All PF/F and 16/31 other cases contained CD21+ follicular dendritic cells. No cytogenetic, IGHV mutational or gene usage differences were seen between PF/F and diffuse cases. PF/F cases were more often kappa positive (p<0.03) and had fewer involved nodal sites (p=0.0004). These findings suggest that at least a subset of bona fide CLL/SLL is related to the follicle, most likely the outer mantle zone, and that at least a subset of the diffuse cases may represent "later" disease.

Follicular lymphoma (FL) is a B-cell neoplasm resulting from the transformation of germinal center (GC) B cells. Although t(14;18) and ectopic B-cell lymphoma 2 (BCL2) expression constitute the genetic hallmark of FL, t(14;18)(pos) B cells bearing genotypic and phenotypic features of FL cells can be found in the blood of most healthy individuals. Nevertheless, the localization of these FL-like cells (FLLCs) in nonmalignant GC-rich tissues and the functional consequences of BCL2 overexpression have not been evaluated thus far. Among 85 reactive lymph node (RLN) samples, 14% were found to contain high levels of t(14;18) by quantitative polymerase chain reaction. In t(14;18)(hi) RLNs, CD20(pos)BCL2(pos)CD10(pos) FLLCs consistently accumulated within the GC, essentially as nonproliferative CXCR4(neg) centrocytes. Moreover, they displayed a reduced response to proliferative stimuli in vitro. Altogether, our findings provide new insights into in situ FLLC functional properties and suggest that these cells have not acquired the ultimate genetic events leading to FL transformation., (© 2014 by The American Society of Hematology.)

Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of hematological malignancies; however, the extent to which deregulated phosphorylation may be involved in B-cell non-Hodgkin lymphoma (B-NHL) pathogenesis is largely unknown. To identify phosphorylation events important in B-NHLs, we performed mass spectrometry-based, label-free, semiquantitative phosphoproteomic profiling of 11 cell lines derived from three B-NHL categories: Burkitt lymphoma, follicular lymphoma, and mantle-cell lymphoma. In all, 6579 unique phosphopeptides, corresponding to 1701 unique phosphorylated proteins, were identified and quantified. The data are available via ProteomeXchange with identifier PXD000658. Hierarchical clustering highlighted distinct phosphoproteomic signatures associated with each lymphoma subtype. Interestingly, germinal center-derived B-NHL cell lines were characterized by phosphorylation of proteins involved in the B-cell receptor signaling. Of these proteins, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) was identified with the most phosphorylated tyrosine peptides in Burkitt lymphoma and follicular lymphoma. PAG1 knockdown resulted in perturbation of the tyrosine phosphosignature of B-cell receptor signaling components. Significantly, PAG1 knockdown increased cell proliferation and response to antigen stimulation of these germinal center-derived B-NHLs. These data provide a detailed annotation of phosphorylated proteins in human lymphoid cancer. Overall, our study revealed the utility of unbiased phosphoproteome interrogation in characterizing signaling networks that may provide insights into pathogenesis mechanisms in B-cell lymphomas., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs). However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs) are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) and primary cutaneous follicle center lymphoma (PCFCL) are characterized by an activated B-cell (ABC)-genotype and a germinal center B-cell (GCB)-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.

MYC is a potent oncogene initially identified as the target of the t(8;14)(q24;q32) chromosome translocation in Burkitt lymphoma. MYC gene alterations have been identified in other mature B-cell neoplasms that are usually associated with an aggressive clinical behavior. Most of these tumors originate in cells that do not normally express MYC protein. The oncogenic events leading to MYC up-regulation seem to overcome the inhibitory effect of physiological repressors such as BCL6 or BLIMP1. Aggressive lymphomas frequently carry additional oncogenic alterations that cooperate with MYC dysregulation, likely counteracting its proapoptotic function. The development of FISH probes and new reliable antibodies have facilitated the study of MYC gene alterations and protein expression in large series of patients, providing new clinical and biological perspectives regarding MYC dysregulation in aggressive lymphomas. MYC gene alterations in large B-cell lymphomas are frequently associated with BCL2 or BCL6 translocations conferring a very aggressive behavior. Conversely, MYC protein up-regulation may occur in tumors without apparent gene alterations, and its association with BCL2 overexpression also confers a poor prognosis. In this review, we integrate all of this new information and discuss perspectives, challenges, and open questions for the diagnosis and management of patients with MYC-driven aggressive B-cell lymphomas.

Background: Programmed death-1 (PD-1/CD279) is a cell-surface protein expressed in activated T cells and a subset of T lymphocytes including follicular helper T cells (TFH ). The interaction between PD-1 and its ligands plays a role in immune response and evasion of malignancies. In nodal follicular lymphoma, the number of intratumoral PD-1-positive lymphocytes is associated with overall survival., Objectives: To investigate 28 cases of primary cutaneous B-cell lymphoma, including the subtypes PCFCL (n = 10), PCMZL (n = 10) and DLBCL-LT (n = 8) for the number and density of PD-1-positive cells., Methods: Immunohistochemical staining and a computerized morphometric analysis for evaluation were applied. The results were correlated with the clinical outcome. To distinguish between activated T cells and TFH we performed PD-1/bcl-6 double staining and compared these results with CXCL-13 staining. Double staining for PD-1 and PAX-5 was used to investigate whether tumour cells were positive for PD-1., Results: The PD-1-positive cells represented tumour-infiltrating T cells (TILs). Only a minor subset was represented by TFH . Patients with DLBCL-LT had a significantly lower number of PD-1-positive TILs than those with PCMZL (P = 0·012) and PCFCL (P = 0·002) or both (P = 0·001). The difference between PCMZL and PCFCL did not reach significance (P = 0·074). The tumour cells were negative for PD-1., Conclusions: A higher number of PD-1-expressing cells was found in indolent PCMZL and PCFCL than in high-malignant DLBCL-LT. The PD-1-positive cells represented not only TFH , but also other activated T cells as a part of the tumour microenvironment. The tumour cells in all investigated types of PCBCL did not show aberrant PD-1 expression., (© 2013 British Association of Dermatologists.)

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and fewer than half of patients are cured with standard first-line therapy. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL, one of the most common cancers in veterinary oncology, is morphologically similar to hDLBCL and is treated using similar chemotherapeutic protocols. With genomic technologies, it is now possible to molecularly evaluate dogs as a potential large-animal model for hDLBCL. We evaluated canine B-cell lymphomas (cBCL) using immunohistochemistry (IHC) and gene expression profiling. cBCL expression profiles were similar in many ways to hDLBCLs. For instance, a subset had increased expression of NF-κB pathway genes, mirroring human activated B-cell (ABC)-type DLBCL. Furthermore, immunoglobulin heavy chain ongoing mutation status, which is correlated with ABC/germinal center B-cell cell of origin in hDLBCL, separated cBCL into two groups with statistically different progression-free and overall survival times. In contrast with hDLBCL, cBCL rarely expressed BCL6 and MUM1/IRF4 by IHC. Collectively, these studies identify molecular similarities to hDLBCL that introduce pet dogs as a representative model of hDLBCL for future studies, including therapeutic clinical trials.

Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may be derived from tRNA, but the function of these molecules remains undefined. Here, we demonstrate that one such tRNA-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICER1-dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mRNA transcripts in a sequence-specific manner. Expression of CU1276 is abundant in normal germinal center B cells but absent in germinal center-derived lymphomas, suggesting a role in the pathogenesis of this disease. Furthermore, CU1276 represses endogenous RPA1, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can be derived from tRNA, thus defining a class of genetic entities with potentially important biological roles.

The human germinal centre-associated lymphoma gene is specifically expressed in germinal centre B-lymphocytes and germinal centre-derived B-cell lymphomas, but its function is largely unknown. Here we demonstrate that human germinal centre-associated lymphoma directly binds to Syk in B cells, increases its kinase activity on B-cell receptor stimulation and leads to enhanced activation of Syk downstream effectors. To further investigate these findings in vivo, human germinal centre-associated lymphoma transgenic mice were generated. Starting from 12 months of age these mice developed polyclonal B-cell lymphoid hyperplasia, hypergammaglobulinemia and systemic reactive amyloid A (AA) amyloidosis, leading to shortened survival. The lymphoid hyperplasia in the human germinal centre-associated lymphoma transgenic mice are likely attributable to enhanced B-cell receptor signalling as shown by increased Syk phosphorylation, ex vivo B-cell proliferation and increased RhoA activation. Overall, our study shows for the first time that the germinal centre protein human germinal centre-associated lymphoma regulates B-cell receptor signalling in B-lymphocytes which, without appropriate control, may lead to B-cell lymphoproliferation.

Germinal centers (GCs) are sites of B-cell clonal expansion, hypermutation, and selection. GCs are polarized into dark (DZ) and light zones (LZ), a distinction that is of key importance to GC selection. However, the difference between the B cells in each of these zones in humans remains unclear. We show that, as in mice, CXCR4 and CD83 can be used to distinguish human LZ and DZ cells. Using these markers, we show that LZ and DZ cells in mice and humans differ only in the expression of characteristic "activation" and "proliferation" programs, suggesting that these populations represent alternating states of a single-cell type rather than distinct differentiation stages. In addition, LZ/DZ transcriptional profiling shows that, with the exception of "molecular" Burkitt lymphomas, nearly all human B-cell malignancies closely resemble LZ cells, which has important implications for our understanding of the molecular programs of lymphomagenesis.

Fas-associated death domain protein is a key component of the extrinsic apoptotic pathway. In addition, in animal models, Fas-associated death domain protein phosphorylation at serine 194 has been shown to affect cell proliferation, especially in T lymphocytes. The importance of Fas-associated death domain protein phosphorylation at serine 194 for the proliferation of B lymphocytes, however, is uncertain. Here we show in reactive lymph nodes that serine 194 phosphorylated Fas-associated death domain protein is expressed predominantly in the dark (proliferative) zone of germinal centers. In B-cell non-Hodgkin lymphoma cell lines, serine 194 phosphorylated Fas-associated death domain protein levels are substantially higher in highly proliferating cells and lower in serum-starved cells. We also used immunohistochemical analysis to assess Fas-associated death domain protein phosphorylation at serine 194 expression in 122 B-cell non-Hodgkin-type lymphomas. The mean percentage of serine 194 phosphorylated Fas-associated death domain protein positive tumor cells was 81% in Burkitt lymphoma, 41% in diffuse large B-cell lymphoma, 18% in follicular lymphoma, 18% in plasma cell myeloma, 12% in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 11% in mantle cell lymphoma, and 2% in chronic lymphocytic leukemia/small lymphocytic lymphoma (P < .0001, Kruskal-Wallis test). Furthermore, in chronic lymphocytic leukemia/small lymphocytic lymphoma, serine 194 phosphorylated Fas-associated death domain protein was detected predominantly in proliferation centers. In the entire study group, the percentage of cells positive for serine 194 phosphorylated Fas-associated death domain protein correlated significantly with the proliferation index Ki-67 (Spearman R = 0.9, P < .0001). These data provide evidence that serine 194 phosphorylated Fas-associated death domain protein is involved in the proliferation of normal and neoplastic B cells and has features of a novel proliferation marker., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Follicular lymphoma (FL) is a germinal centre-derived indolent B-cell lymphoma representing the second most common Non Hodgkin lymphoma in the Western world. This chapter focuses on the pathology of FL and summarizes the current knowledge about genetic and molecular features that are relevant for the pathogenesis of this neoplasm. The translocation t(14;18) is present in approximately 90% of FL leading to the upregulation of the anti-apoptotic protein BCL2, that may constitute a promising molecular target for therapeutic approaches. FL lacking the t(14;18) also exist, and B-cells carrying the t(14;18) can be detected in a subset of healthy individuals. In addition to the t(14;18), secondary genetic alterations are present in most FL and, more recently, deeper insights into the methylation and microRNA expression patterns in the tumour cells have been gained. The tumour microenvironment appears to be particularly important for the biology and the clinical course of FL., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Context: Cap-mediated messenger RNA translation controlled by the eukaryotic initiation factor 4F (eIF-4F) complex plays a key role in human cancer. eIF-4F activity is controlled by a repressor binding protein (4E-BP1), which promotes translation when phosphorylated., Objective: To examine the level of expression and phosphorylation of 4E-BP1 in various subtypes of B-cell lymphoma and reactive lymphoid tissues., Design: Archival formalin-fixed, paraffin-embedded B-cell lymphoma samples and reactive lymphoid tissues were immunostained and examined for expression of 4E-BP1 and phosphorylated 4E-BP1. Expression of components of the eIF-4F complex and unphosphorylated and phosphorylated 4E-BP1 was confirmed using Western immunoblotting on lysates of frozen lymphoma samples and reactive tissues., Results: Immunohistochemical analysis demonstrated weak to undetectable 4E-BP1 staining within benign, reactive germinal centers (N = 10). In contrast, 4E-BP1 was consistently expressed (moderate to strong staining) in 98% of various subtypes of mature B-cell lymphoma (N = 50). 4E-BP1 expression was also demonstrable in all 4 lymph nodes with in situ or partial involvement by follicular lymphoma and in all 12 cases of BCL2-negative lymphoma. The level of phosphorylation of 4E-BP1 in lymphomas, evaluated by immunohistochemistry, was heterogeneous., Conclusions: The immunohistochemical expression pattern of 4E-BP1 exhibits regional and cellular specificity in reactive lymphoid tissues and may offer a diagnostic tool for distinguishing reactive follicles from neoplastic B-cell proliferations.

The aims of this study were to analyze the incidence and morphology of cyclin D1+ DLBCL and cases of Richter transformation (RT), and to elucidate possible molecular mechanisms of cyclin D1 overexpression. Seventy-two cases of de novo DLBCL and 12 cases of RT were included in this study. Cyclin D1 positivity was found in 10/66 (15%) cases of unselected de novo DLBCL and in 2/11 (18%) cases of RT. Seven independently identified cases of cyclin D1+ DLBCL, including one RT, were added to the study. Centroblastic morphology was found in 17/19 (89%) cases of cyclin D1+, most with a post-germinal center phenotype (CD10-, BCL6+, MUM1+). No alterations in the CCND1 gene indicative for a translocation t(11;14) were identified by FISH. Analysis of the MYC locus yielded gene copy alterations in five cases and no disruption of the gene locus in any case, suggesting an alternative mechanism of cyclin D1 deregulation.

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic "hit" in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways.

Background: During B-cell development, precursor B cells transiently express the pre-B-cell receptor composed of μ heavy chain complexed with VpreB and λ5 surrogate light chain polypeptides. Recent profiling studies unexpectedly revealed abundant transcripts of one member of the VpreB family, VpreB3, in a subset of mature B cells and Burkitt lymphoma., Design and Methods: Here we used a novel antibody to investigate the normal expression pattern of VpreB3 protein in human hematopoietic and lymphoid tissues, and to determine whether VpreB3 could serve as a useful diagnostic biomarker for select B-cell lymphomas., Results: We found that VpreB3 protein is normally expressed by precursor B cells in bone marrow and by a subset of normal germinal center B cells in secondary lymphoid organs. Among lymphoid malignancies, we found an association between VpreB3 expression and B-cell tumors with c-MYC abnormalities. VpreB3 was highly expressed in all cases of Burkitt lymphoma, whether of endemic or sporadic origin (44/44 cases, 100%), all cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (5/5 cases, 100%), and the majority of diffuse large B-cell lymphomas harboring a c-MYC translocation (15/18 cases, 83%). The expression of VpreB3 in diffuse large B-cell lymphomas without a c-MYC translocation was associated with c-MYC polysomy in 25/75 cases (33%) but only rarely observed in diffuse large B-cell lymphomas lacking a c-MYC abnormality (9/98 cases, 9%)., Conclusions: We conclude that for B-cell tumors with features suggesting a possible c-MYC translocation, such as intermediate to large cell size and high proliferation rate, the presence of VpreB3 should prompt subsequent confirmatory genetic testing, whereas the absence of VpreB3 is virtually always associated with wild-type c-MYC alleles.

In the germinal center (GC), B cells proliferate dramatically and diversify their immunoglobulin genes, which increases the risk of malignant transformation. The GC B-cell reaction relies on crosstalk with follicular dendritic cells (FDCs), to which the costimulatory receptor CD137 on FDCs and its ligand on GC B cells potentially contribute. We report that mice deficient for CD137 ligand (CD137L) are predisposed to develop B-cell lymphoma, with an incidence of approximately 60% at 12 months of age. Lymphoma membrane markers were characteristic of GC B cells. Longitudinal histologic analysis identified the GC as site of oncogenic transformation and classified 85% of the malignancies found in approximately 200 mice as GC-derived B-cell lymphoma. To delineate the mechanism underlying lymphomagenesis, gene expression profiles of wild-type and CD137L-deficient GC B cells were compared. CD137L deficiency was associated with enhanced expression of a limited gene set that included Bcl-10 and the GC response regulators Bcl-6, Spi-B, Elf-1, Bach2, and activation-induced cytidine deaminase. Among these are proto-oncogenes that mediate GC B-cell lymphoma development in humans. We conclude that CD137L ordinarily regulates the GC B-cell response and thereby acts as a tumor suppressor.

X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4(+)/Bcl-6(-)/Pax-5(-) B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas.

B lymphocytes provide the cellular basis of the humoral immune response. All stages of this process, from B-cell activation to formation of germinal centers and differentiation into memory B cells or plasma cells, are influenced by extrinsic signals and controlled by transcriptional regulation. Compared to naïve B cells, memory B cells display a distinct expression profile, which allows for their rapid secondary responses. Indisputably, many B-cell malignancies result from aberrations in the circuitry controlling B-cell function, particularly during the germinal centre (GC) reaction. Here, we review new insights into memory B-cell subtypes, recent literature on transcription factors regulating human B-cell differentiation and further evidence for B-cell lymphomagenesis emanating from errors during GC cell reactions.

Follicular lymphoma (FL) is a morphologically and genetically well-characterized B-cell non-Hodgkin lymphoma that can show predominantly follicular, combined follicular and diffuse, or predominantly diffuse growth patterns. Although approximately 85% of FLs harbor the translocation t(14;18)(q32;q21) and consistently display a follicular growth pattern, predominantly diffuse FLs are less well characterized on the phenotypical, molecular, and clinical level. We studied 35 predominantly diffuse FL by immunohistochemistry, classical chromosome banding analysis, fluorescence in situ hybridization (FISH), and gene expression profiling. A total of 28 of 29 analyzable cases lacked t(14;18), and 27 of 29 cases revealed a unifying chromosomal aberration, a deletion in 1p36. Morphologically, 12 FLs were grade 1 and 23 were grade 2, and the immunophenotype with frequent expression of CD10, BCL6, and CD23 was in line with a germinal center B-cell phenotype. The gene expression profiles of 4 predominantly diffuse FLs fell into the spectrum of typical FL, with a unique enrichment of specific gene signatures. Remarkably, patients with diffuse FL frequently presented with low clinical stage and large but localized inguinal tumors. These results suggest that predominantly diffuse FL represent a distinct subtype of t(14;18)-negative nodal FL with a unifying genetic alteration (deletion of 1p36) and characteristic clinical features.

To assess the role of interferon regulatory factor (IRF) 8 in B-cell development and lymphomagenesis, we studied its expression in reactive lymphoid tissues, its relationship to other B-cell transcription factors, and its expression in a series of 232 B-cell tumors and 30 cell lines representing a variety of B-cell developmental stages. We found that although IRF8 was detectable in most reactive B-cells, its expression levels differed with developmental stage. Germinal center B cells contained the highest levels of IRF8, with lower levels seen in mantle and marginal zone B cells and none in plasma cells. IRF8 was coexpressed with PAX-5, Pu.1, and B-cell lymphoma (BCL)-6, and similar to BCL-6, was absent from the small population of IRF4-positive germinal center B cells thought to be committed to postgerminal center developmental programs. Similarly, IRF8 was most strongly expressed in lymphomas of germinal center origin with lower levels present in mantle cell lymphomas, chronic lymphocytic leukemia, and marginal zone lymphomas, and no expression observed in plasmacytic/plasmablastic neoplasms. The reciprocal expression pattern with IRF4 in reactive tissues was generally maintained in lymphomas with some exceptions. These results suggest an important role for IRF8 during germinal center B-cell development and in related lymphomas, and provide a new diagnostic marker helpful in distinguishing B-cell non-Hodgkin lymphoma subtypes.

Centerin (SERPINA9/GCET1) is a protease inhibitor with expression restricted to germinal center B cells and lymphoid malignancies with germinal center B-cell maturation. Expression of the centerin gene transcript, along with bcl-6 and GCET2/HGAL, constitutes a molecular signature associated with a good prognosis in diffuse large B-cell lymphomas. A monoclonal antibody to centerin was generated and used for Western blotting, immunohistochemistry, and immunofluorescence. Centerin expression was demonstrated in Burkitt lymphoma Raji cells. An immunohistochemical survey of normal tissues showed centerin expression in germinal center B cells in lymphoid follicles in tonsil, lymph node, and lymphoid tissue in the gastrointestinal tract. Centerin was strongly expressed in most follicular lymphomas. In addition, 14 (47%) of 30 diffuse large B-cell lymphomas were positive for centerin, which correlated most closely with CD10 expression. Immunohistochemical expression of centerin further defines the germinal center cell origin of a subgroup of lymphomas.

Most human B cell non-Hodgkin's lymphomas (B-NHLs) derive from germinal centers (GCs), the structure in which B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR) before being selected for high-affinity antibody production. The pathogenesis of B-NHL is associated with distinct genetic lesions, including chromosomal translocations and aberrant SHM, which arise from mistakes occurring during CSR and SHM. A direct link between these DNA remodeling events and GC lymphoma development, however, has not been demonstrated. Here we have crossed three mouse models of B cell lymphoma driven by oncogenes (Myc, Bcl6 and Myc/Bcl6; refs. 5,6) with mice lacking activation-induced cytidine deaminase (AID), the enzyme required for both CSR and SHM. We show that AID deficiency prevents Bcl6-dependent, GC-derived B-NHL, but has no impact on Myc-driven, pre-GC lymphomas. Accordingly, abrogation of AID is associated with the disappearance of CSR- and SHM-mediated structural alterations. These results show that AID is required for GC-derived lymphomagenesis, supporting the notion that errors in AID-mediated antigen-receptor gene modification processes are principal contributors to the pathogenesis of human B-NHL.

Spectrins are a family of cytoskeletal proteins that organize and link membranes to subcellular motors and filaments. Although traditionally divided into erythroid and non-erythroid forms, the discovery of new spectrin isoforms in various tissues indicates that their distribution is not yet fully characterized. To our knowledge, there is no comprehensive analysis of spectrins in lymphoid malignancies. Using tumor microarrays of paraffin blocks, we immunohistochemically studied 10 lymph nodes with reactive lymphoid hyperplasia and 94 lymph nodes involved by B-cell malignant lymphoma. Expression of spectrins alphaI, alphaII, betaI, betaII, and betaIII was scored using a 20% cutoff for positive immunoperoxidase staining. All spectrin isoforms, except erythroid-specific alphaI spectrin, were detected in lymph nodes with reactive lymphoid hyperplasia. In contrast, various spectrins were lost in particular B-cell malignant lymphomas. Based on the absence of staining for one or more spectrin isoforms in at least 50% of cases, we identified three patterns: (1) loss of alphaII and betaII in follicular lymphoma, grades 2/3 and 3/3; nodular lymphocyte predominance Hodgkin's lymphoma; nodular sclerosis Hodgkin's lymphoma; (2) loss of betaI only in Burkitt lymphoma; and (3) loss of alphaII and betaI in mixed cellularity Hodgkin's lymphoma. In contrast, follicular lymphoma, grade 1/3 and diffuse large B-cell lymphoma retained spectrin in 67-100% of cases. The other lymphoma subtypes retained spectrin in greater than 50% of cases. We identified the loss of particular spectrin isoforms in B-cell malignant lymphomas that have a nodular growth pattern and/or are believed to arise from germinal center B-cells, that is follicular lymphoma, grades 2/3 and 3/3; Burkitt lymphoma; nodular sclerosis Hodgkin's lymphoma; mixed cellularity Hodgkin's lymphoma; and nodular lymphocyte predominance Hodgkin's lymphoma. The absence of particular spectrin isoforms may correlate with transformation or aggressive biologic behavior for some lymphoma subtypes.

Metastasis-associated protein 3 (MTA3) is a recently described cell-type specific component of the Mi-2-NURD transcriptional co-repressor complex that is expressed in breast epithelia and germinal centre B cells. In model B cell lines, MTA3 physically interacts with BCL6 and appears to be instrumental in maintenance of the germinal centre B cell transcriptional programme that precludes premature plasmacytic differentiation. Here, we report selective, in situ cell-type specific expression of MTA3 among lymphoid cells largely confined to the germinal centre B cell compartment. Centroblasts display greater expression than smaller, less proliferative centrocytes, with undetectable expression in quiescent plasma cells. Among B cell neoplasms, germinal centre B cell-like lymphomas likewise exhibit selective expression that generally escalates with increasing proliferative capacity. MTA3 protein expression was, in accord, highly predictive of the germinal centre B cell-like gene expression profile for diffuse large B cell lymphomas. Lastly, relative repression of a subset of known BCL6 targets, including BLIMP1 and p27kip1, was highest in diffuse large B cell lymphomas that co-expressed both MTA3 and BCL6 protein. Together, these novel data suggest a role for MTA3 in BCL6-mediated lymphomagenesis in germinal centre B cell-like neoplasms.

Bcl-6 is expressed in germinal centre derived B-cell non-Hodgkin lymphomas including diffuse large B-cell lymphoma (DLBCL) and is likely to play a major role in driving proliferation of a subset of DLBCLs, especially those of germinal centre B-cell subtype, but the role of c-Myc, which is important for proliferation in various lineages is not known. We used the highly standardised staining conditions of a tissue microarray to characterise co-expression of c-Myc and Bcl-6 in DLBCL. We carried out immunohistochemistry of 73 arrayed cases. The majority (62/73) did not express c-Myc, but 11 cases (15%) showed nuclear staining. 5/53 (9%) of Bcl-6 expressing cases co-expressed c-Myc, whereas a much higher proportion, 6/20 (30%), of Bcl-6 negative cases were positive for c-Myc. Overall survival of c-Myc expressing cases was the same as those that had absent expression. There was no significant correlation between c-Myc expression and DLBCL subtype (germinal centre or non-germinal centre subtypes).

Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place in response to stimulation by various antigens. To select a proper B-cell clone for antibody-mediated immunity, multiple apoptotic signals synchronize in the GC, both in negative and positive selection pathways. At the same time, GC have been known to be a major source of B-cell lymphomas including follicular and Burkitt's, and also some diffuse large B-cell lymphomas. Therefore, uncovering the biological characteristics of GC would greatly contribute to understanding lymphomagenesis, or progression of B-cell lymphomas of GC origin. Herein the authors briefly explain the expression and pathophysiological significance of apoptosis regulators in GC, focusing particularly on Bcl-2, Fas (CD95) and a transcription factor, nuclear factor of activated T cells, which seems to play a critical role in regulating cellular dynamics of GC B cells via B-cell antigen receptor. The expression of these molecules is then compared with that of the neoplastic counterpart B-cell lymphomas in order to consider lymphomagenesis of GC origin. In conclusion, follicular lymphoma closely reflected characteristics of GC among these B-cell lymphomas, although it acquires strong expression of apoptosis-resistant gene, bcl-2.