Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia

BCL-6 mutations are accumulated during B-cell transit through the germinal center (GC) and provide a histogenetic marker for B-cell tumors. On the basis of a comprehensive analysis of 308 B-cell neoplasms, we (1) expand the spectrum of tumors associated with BCL-6 mutations; (2) corroborate the notion that mutations cluster with GC and post-GC B-cell neoplasms; and (3) identify heterogeneous mutation frequency among B-lineage diffuse large cell lymphoma (B-DLCL) subsets. Mutations are virtually absent in acute lymphoblastic leukemia (P <.001) and mantle cell lymphoma (P <.05), whereas they occur frequently in GC or post-GC neoplasms, including lymphoplasmacytoid lymphoma, follicular lymphoma, MALT lymphomas, B-DLCL and Burkitt lymphoma. Among B-DLCL, mutations occur frequently in systemic nodal B-DLCL, primary extranodal B-DLCL, CD5(+) B-DLCL, CD30(+) B-DLCL, and primary splenic B-DLCL, suggesting a similar histogenesis of these B-DLCL subsets. Conversely, mutations are rare in primary mediastinal B-DLCL with sclerosis (10.0%; P <.01), supporting a distinct histogenesis for this lymphoma. Longitudinal follow-up of B-DLCL transformed from follicular lymphoma shows that they BCL-6 mutations may accumulate during histologic progression. Mutations also occur in some B-cell chronic lymphocytic leukemias, small lymphocytic lymphomas, and hairy cell leukemias, consistent with the hypothesis that a fraction of these lymphoproliferations are related to GC-like cells. Finally, the molecular pattern of 193 mutational events reinforces the hypothesis that mutations of BCL-6 and immunoglobulin genes are caused by similar mechanisms. (Blood. 2000;95:651-659)     

Biallelic DNA rearrangements and deletions within the BCL-6 gene in B-cell non-Hodgkin's lymphoma

Summary. Chromosomal translocations involving band 3q27 are recently described common specific cytogenetic abnormalities in B-cell neoplasms, and the BCL-6 gene, identified on 3q27, was shown to be disrupted and over-expressed in lymphoma cells having these chromosomal translocations. In the present study we found rearrangements within the BCL-6 gene in seven out of 3 5 cases with B-cell non-Hodgkin's lymphoma (NHL). Further analysis revealed that three of these patients with BCL-6 abnormality had multiple rearranged bands hybridized with probes from a single restriction fragment within the major translocation cluster (MTC). suggesting that independent DNA rearrangements would occur on both alleles. Additionally, Southern blot analysis indicated that three patients carry deletions encompassing the area containing the first exon of the BCL-6 gene. Our results suggest that biallelic DNA rearrangements and deletions would occasionally occur in NHL patients with BCL-6 abnormality.     

New chromosome abnormalities and lack of BCL-6 gene rearrangements in Argentinean diffuse large B-cell lymphomas

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphomas. Cytogenetic studies have revealed a broad spectrum of clonal genetic abnormalities and complex karyotypes. The purpose of this study was to contribute to the understanding of the genomic alterations associated with this group of lymphomas. METHODS: Cytogenetic, fluorescence in situ hybridization (FISH) and molecular analyses were performed in 30 cases with DLBCL: 20 de novo DLBCL (dn-DLBCL) and 10 DLBCL secondary to follicular lymphoma (S-DLBCL). RESULTS: A total of 37 different structural chromosomal rearrangements were found: 27% translocations, 54% deletions, and 19% other alterations. Chromosomes 8, 6, 2, and 9 were the most commonly affected. Interestingly, translocation t(3;14)(q27;q32) and/or BCL-6 gene rearrangements were not observed either by cytogenetic studies or by FISH analysis. Fifteen novel cytogenetic alterations were detected, among them translocations t(2;21)(p11;q22) and t(8;18)(q24;p11.3) appeared as sole structural abnormalities. Translocation t(14;18)(q32;q21) and/or BCL-2-IGH gene rearrangements were the genomic alterations most frequently observed: 50% of S-DLBCL and 30% of dn-DLBCL. Deletions del(4)(q21), del(6)(q27), del(8)(q11), and del(9)(q11) were recurrent. The most common gains involved chromosome regions at 12q13-q24, 7q10-q32, and 17q22-qter; 6q was the most frequently deleted region, followed by losses at 2q35-qter, 7q32-qter, and 9q13-qter. Four novel regions of loss were identified: 5q13-q21, 2q35-qter (both recurrent in our series), 4p11-p12, and 17q11-q12. CONCLUSIONS: These studies emphasize the value of combining conventional cytogenetics with FISH and molecular studies to allow a more accurate definition of the genomic aberrations involved in DLBCL.     

Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.The immune system uses immune receptors to sense and acquire antigens. Antigen binding induces a series of dynamic changes in the biophysical behaviors and biochemical features of the immune receptors, and these changes determine the fate of a cell (1–3). However, it has been difficult to accurately capture and thus comprehensively investigate these changes because they usually occur very quickly after immune recognition (1, 4). For example, recent live cell imaging studies showed that the B lymphocytes swiftly accumulate the surface-expressed B-cell receptors (BCRs) into the contact interface between the B cells and the antigen-presenting substrates to form a specialized membrane structure, the B-cell immunological synapse (IS) (1, 4). Moreover, both our studies and those of others showed that these accumulation events are sensitive to the biochemical and biophysical features of the antigens that B cells likely encounter in vivo (4, 5). These features include but are not limited to antigen density (6, 7), antigen affinity (6, 7), antigen valency (8–13), the mobility of the antigen (14–17), the stiffness of the substrates presenting the antigen (18, 19) and the mechanical forces delivered to the BCRs by the antigens (20, 21). These facts highlight a long-standing question in immunology: how can the initiation of B-cell activation process the information of antigen specificity, density, affinity, valency, mobility, substrate stiffness, and mechanical forces in such an efficient way? To attempt to address this intriguing question, a detailed understanding of the precise BCR sorting mechanisms within the B-cell IS during the initiation of B-cell activation is required. However, it is technically difficult to accurately capture these events due to their fast and dynamic nature. It is challenging to capture an entire molecular event from the same B-cell before and immediately after antigen recognition, and it is more difficult to capture multiple events in parallel from multiple cells in a synchronized manner. An attractive solution for this dilemma is to develop a precisely controllable trigger point for BCR and antigen recognition by using photoactivatable antigens, which are initially inactive but become immediately active on the illumination of UV light. Indeed, photoactivatable systems have been used to investigate the kinetics of second-messenger activity through caged calcium (22) and caged cAMP (23). Additionally, the T-cell receptor was studied using major histocompatibility complex (MHC) presenting photoactivatable peptide (24, 25).In this report, we dissected the dynamic responses during the initiation of B-cell activation by using a photoactivatable antigen based experimental system in combination with high-resolution high speed total internal reflection fluorescence microscopy (TIRFM) imaging techniques. We caged the widely used model antigen 4-hydroxy-3-nitrophenyl acetyl (caged-NP) that is only converted to its antigenic form after exposure to UV photons. The photoactivation of caged-NP in contact with NP-specific B1-8-BCR–expressing B cells provides a precisely controllable trigger point to perform high resolution temporal analyses of the formation of BCR microclusters and the B-cell IS in response to antigen stimulation. By combining the unique strengths of the caged-NP–based photoactivatable antigen system with TIRFM-based live cell and single molecule imaging techniques, we examined the basal response of a quiescent B cell exposed to coverslips presenting the caged-NP for 360 s and then examined the changes in the responses of the same B cell immediately after the recognition of the photoactivated-NP antigen for another 360 s. To our knowledge, this system represents the first temporally seamless imaging experimental procedure for the study of the molecular events during the initiation of B-cell activation. We illuminated the probing behaviors in quiescent B cells as defined by the unceasing extension of membrane pseudopods in random directions. We found that BCR and antigen recognition promptly terminated the probing responses. We also dissected the sophisticated BCR sorting mechanism within the B-cell IS during the initiation of B-cell activation.     

FADD expression and caspase activation in B-cell lymphomas resistant to Fas-mediated apoptosis

We have previously shown that malignant B cells from non-Hodgkin's lymphomas (NHL) are resistant to Fas-mediated apoptosis. To determine the mechanisms underlying this resistance, we analysed by Western blotting the expression of several apoptotic regulators, caspase 3, caspase 8, FADD and poly(ADP-ribose) polymerase (PARP) in fresh lymphoma cells, isolated from 16 B-NHL biopsy samples of different histological subtypes, and displaying variable levels of Fas expression. The profiles of expression of these apoptotic regulators were monitored in cell lysates at different times following Fas with or without CD40 stimulation. Expression of FADD and of the uncleaved forms of PARP, caspase 3 and caspase 8 were detected in all untreated NHL samples. Low levels of PARP cleavage were noted in three untreated samples. Fas stimulation alone induced neither significant apoptosis nor significant changes in the expression profiles of FADD, caspases 3 and 8 and PARP in the 16 samples, except for variations in FADD and caspase 8 expression levels in a minority of samples. Fas/CD40 co-stimulation induced apoptosis and cleavage of caspase 3, caspase 8 and PARP in the five NHLs tested; expression of FADD was not modified. Our results showed (1) that induction of apoptosis in B-NHLs by Fas/CD40 co-stimulation used the same caspase executioner machinery as the normal Fas pathway, and (2) that NHL cells which resisted Fas-mediated apoptosis displayed no defect in either expression or functionality of caspases 3 and 8, nor in FADD expression. The dysfunction underlying NHL resistance to apoptosis must therefore lie upstream of caspase 8, or could alternatively be influenced by anti-apoptotic regulators of the Bcl-2 family.     

B-cell activation during HIV-1 infection. III. Down-regulating effect of mitogens

Spontaneous in vitro production of HIV-1-specific antibodies, a hallmark of infected subjects, is often down-regulated by the addition of pokeweed mitogen. We observed that a decrease in such ongoing anti-HIV-1 antibody synthesis could also be induced in cultures from most patients by addition of phytohemagglutinin and Concanavalin A, but not by Epstein-Barr virus, a selective B-cell mitogen. In most cases, this down-regulatory effect of mitogens was evident within the first 24 h of culture. The observed mitogen-associated decrease in spontaneous antibody synthesis was prevented by treating peripheral blood mononuclear cells with agents inhibiting non-major histocompatibility complex-restricted cytotoxic activity or by adding third-party cells to the cultures. In most cases, the mitogen-induced effect was also counteracted by removal of T lymphocytes or CD8+ T-cell sub-population. These findings recall a similar phenomenon observed in normal subjects following intentional immunization, and indicate that mitogen-induced down-regulation of spontaneous in vitro anti-HIV-1-antibody production most probably occurs through a lectin-dependent cytotoxic effect on activated B cells.     

B-cell activation in human plasma cell dyscrasias

We studied the abnormal in vitro polyclonal B-cell activity observed in patients with multiple myeloma and Waldenström's macroglobulinaemia. Numbers of cells spontaneously secreting immunoglobulin (Ig) in freshly isolated suspensions of peripheral blood mononuclear cells and pokeweed mitogen (PWM) stimulated cultures of blood mononuclear cells were determined with a protein. A reverse haemolytic plaque assay. These data were correlated with both the clinical and laboratory parameters of disease. Furthermore, Ig secreted into supernatants of PWM-stimulated cultures was examined by a light chain radioimmunoassay for evidence of in vitro activation of malignant B-cells. The mean level of circulating immunoglobulin secreting cells (IgSC) in patients was elevated when compared to that of normal subjects. The highest values were observed in those patients with the highest levels of serum paraprotein. However, experiments with cycloheximide suggested that such increased circulating IgSC values were often caused by the detection of Ig passively adsorbed to blood mononuclear cells. The studies with PWM stimulation of blood mononuclear cells were particularly revealing. Cultures of patient blood mononuclear cells with PWM showed depressed IgSC responses as a group compared to cultures of normal blood mononuclear cells: nevertheless, approximately half the patients demonstrated a sizeable response to PWM. No evidence for preferential activation of Ig secretion by the malignant B-cell clone was observed. Impaired PWM induced responses were associated with advanced or progressive clinical disease.     

Coexpression of MYC and BCL-2 predicts prognosis in primary gastrointestinal diffuse large B-cell lymphoma

AIM:To investigate whether MYC and BCL-2 coexpression has prognostic significance in primary gastrointestinal diffuse large B-cell lymphoma(PGI-DLBCL)patients,and explore its associations with patients’clinical parameters.METHODS:Fresh and paraffin-embedded tumor tissue samples from 60 PGI-DLBCL patients who had undergone surgery at the Tianjin Medical University Cancer Institute and Hospital from January 2005 to May 2010 were obtained,and 30 lymphoid tissue samples from reactive lymph nodes of age-and sexmatched patients represented control samples.Staging and diagnostic procedures were conducted according to the Lugano staging system.All patients had been treated with three therapeutic modalities:surgery,chemotherapy,or radiotherapy.Expression of MYC and BCL-2 were detected at both protein and m RNA levels by immunohistochemistry and real-time RT-PCR.RESULTS:Positive expression levels of MYC and BCL-2proteins were detected in 35%and 45%of patients,respectively.MYC+/BCL-2+protein was present in30%of patients.MYC and BCL-2 protein levels were correlated with high MYC and BCL-2 m RNA expression,respectively(both P0.05).We found that advancedstage disease(atⅡE-Ⅳ)was associated with MYC and BCL-2 coexpression levels(P0.05).In addition,MYC+/BCL-2+patients had more difficulty in achieving complete remission than others(P0.05).Presenceof MYC protein expression only affected overall survivaland progression-free survival(PFS)when BCL-2 proteinwas coexpressed.The adverse prognostic impact ofMYC+/BCL-2+protein on PFS remained significant(P0.05)even after adjusting for age,Lugano stage,international prognostic index,and BCL-2 proteinexpression in a multivariable model.CONCLUSION:MYC+/BCL-2+patients have worsechemotherapy response and poorer prognosis thanpatients who only express one of the two proteins,suggesting that assessment of MYC and BCL-2 expressionby immunohistochemistry has clinical significance inpredicting clinical outcomes of PGI-DLBCL patients.     

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23.

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells induces some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line, including BL markers (cALLA and BLA), proliferation markers (transferrin receptor and BK19.9), and cell adhesion-related molecules (LFA-1 and LFA-3). Increased CD23 expression in cells expressing EBNA-2 was apparent from monoclonal anti-CD23 antibody binding to the cell surface, from immunoprecipitation of the 45-kDa and 90-kDa CD23 proteins with monoclonal antibody, and from RNA blots probed with labeled CD23 DNA. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation.