Analysis of immunoglobulin V genes suggests cutaneous marginal zone B-cell lymphomas recognise similar antigens

Extranodal marginal zone B-cell lymphomas (EMZL) are thought to develop from reactive infiltrates that represent immune responses to external or auto-antigens. Except for gastric EMZL, the antigenic triggers of EMZL development are mostly unknown, although a subset of cutaneous EMZL have been associated with Borrelia burgdorferi infections. To further evaluate whether a common antigen may be promoting the development of cutaneous EMZL, the immunoglobulin heavy chain variable (V(H)) genes from eight USA cases were sequenced and analysed. All used V(H)3 family gene segments, with 2/8 using the same V3-30 segment, 2/8 using the closely related V3-30.3 or V3-33 segments, 6/8 containing mutations and 2/7 showing evidence of ongoing mutation. Many of the complimentarity-determining region 3s (CDR3s) also showed similarities in length and displayed conserved amino acid motifs in the non-templated areas between the diversity and joining segments. The use of similar V(H) gene segments and conserved CDR3 amino acid motifs suggests that some of these cutaneous EMZL may bind the same or similar antigen via their surface immunoglobulin receptor. Analysis of the somatic mutations present in many of the V(H) genes was also consistent with antigen directly stimulating the growth of cutaneous EMZL.     

V(H) gene family utilization in different B-cell lymphoma subgroups

V(H) gene family specific polymerase chain reaction (PCR) amplification was performed in 87 B-cell lymphoma samples from 4 different subgroups. No apparent restriction in the VH gene usage was found in follicular lymphomas, lymphoplasmacytoid lymphomas or large B-cell lymphomas, whereas a biased VH1 utilization was shown in patients with chronic lymphocytic leukemia. Eleven of 18 chronic lymphocytic leukemia cases utilized the VH1 gene family, and nucleotide sequencing of the VH1 gene rearrangements revealed that a majority utilized the DP10 (51p1) germline gene, which has been reported to be strongly associated with autoimmune disease. No VH5 or VH6 rearrangements were amplified in the chronic lymphocytic leukemia subgroup, 2 gene families which previously have been found to be over-represented in these patients. In a high proportion (40%) of large B-cell lymphomas, VH gene family-specific PCR failed to amplify any rearrangement. Using primers hybridizing to the framework regions 2 and 3 and Southern blot analysis of the immunoglobulin heavy chain locus, clonal rearrangements were displayed in two-thirds of these PCR negative cases. However, the rearrangement status could not be elucidated in 5 of 35 patients with large B-cell lymphoma.     

Low rate of somatic hypermutations characterize progressive B-cell lymphomas

Abstract: Immunoglobulin heavy (IgH) chain gene rearrangements were characterized in 40 samples from 15 patients with B-cell lymphomas at different time points during tumour progression. Using polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) analysis of variable heavy (V_H) chain gene segments, we found that 6 cases displayed alterations in their IgH chain rearrangements at relapse. These alterations were mainly observed in follicular or transformed lymphomas, but no association to clinical features was found. Nucleotide sequence analysis revealed a low frequency of mutations in 3 cases, whereas 1 case displayed an extensive mutation rate in a compartment with transformed morphology at relapse. The mutations observed most probably resulted from somatic hypermutations. Further, the mutations were scattered randomly over the V₁₁ gene segment and no significant bias favouring amino acid substitutions was observed in 3 cases, suggesting that the tumour cells had not been subjected to antigen-driven selection. In 1 case, however, the mutation pattern indicated that the tumour cells had been affected by an antigen selection process. In the 2 remaining cases, the original V_HDJ_H rearrangement could no longer be detected by V_H gene family specific PCR at relapse, but using primers specific for the framework region 2 or 3 altered rearrangements were demonstrated, implying that mutations had been introduced in framework region 1. However, the majority of the tumour cell clones analysed were relatively stable during tumour progression, which make them eligible for analysis of minimal residual disease using the V_H gene regions as molecular markers.     

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.     

Lower expression of CD81 B-cell receptor in lymphoproliferative diseases associated with hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is frequently associated with type II mixed cryoglobulinaemia (MC), a benign lymphoproliferative disease (LPD). More recently, HCV has been implicated as a possible aetiologic factor of B-cell non-Hodgkin lymphoma (B-NHL). CD81, a B-cell surface receptor, has been proposed as a receptor for HCV binding and entry in circulating B cells. The stimulation of CD81 complex enables B cells to respond to lower concentrations of antigen and finally induces B-cell proliferation. We studied the phenotypic expression of CD81, CD19 and CD5 on circulating B cells in HCV patients LPD-positive or LPD-negative. Sixty-two patients were anti-HCV antibody positive. Among HCV positive patients, 44 were HCV RNA positive with an histologically proven chronic active hepatitis of whom 10 had a B-NHL, 14 an MC and 24 no extrahepatic manifestation. Eighteen patients were HCV RNA negative with evidence of resolved infection. A control group included 40 healthy subjects. Peripheral blood mononuclear cells (PBMC) were stained for surface expression of CD81, CD19 and CD5 using monoclonal antibodies, and were analyzed by flow cytometry. The percentage of PBMC expressing CD81, CD19 and CD5 receptors were compared between the groups by univariate analysis. Logistic regression model variables were then evaluated to correlate the presence of an LPD with HCV infection characteristics (i.e. age, gender, genotype, duration of infection, HCV RNA positivity, liver histological lesions), or phenotypic expression of CD81, CD19 and CD5 receptors on PBMC. HCV antibody-positive compared with HCV-negative subjects had a higher expression of CD19 receptor (23 +/- 13 vs 13 +/- 1%, P = 0.003). Among HCV RNA positive-patients, LPD+ compared with LPD- patients had a lower expression of CD81 (58 +/- 28 vs 82 +/- 18%, P = 0.001) and CD5 receptor (66 +/- 16 vs 74 +/- 13%, P = 0.04). In multivariate analysis, the expression of CD81 receptor was a negative (OR = 0.15, 95% CI = 0.04-0.64, P = 0.01) and CD19 receptor a positive (OR = 4.81, 95% CI =1.29-17.88, P = 0.02) predictive factor for an LPD. We found two negative predictive factors for HCV RNA positivity, i.e. age (OR = 0.23, 95% CI. = 0.08-0.62, P = 0.003) and the expression of CD81 receptor (OR = 0.34, 95% CI = 0.13-0.89, P = 0.02). In patients with a chronic active HCV infection, the presence of a lymphoproliferative disease, either MC or B-NHL, is associated with lower expression of CD81 and higher expression of CD19 receptor on peripheral B cells.     

Telomere length and correlation with histopathogenesis in B-cell leukemias/lymphomas

Telomere length was recently reported to correlate with cellular origin of B-cell malignancies in relation to the germinal center (GC). In this report, we measured telomere length by quantitative-PCR in 223 B-cell lymphomas/leukemias and correlated results with immunoglobulin (Ig) mutation status and immunostainings for GC/non-GC subtypes of diffuse large B-cell lymphoma (DLBCL). Shortest telomeres were found in Ig-unmutated chronic lymphocytic leukemia (CLL) [median telomere to single copy gene value (T/S) 0.33], differing significantly to Ig-mutated CLL (0.63). Contrary to this, mantle cell lymphomas (MCLs) exhibited similar telomere lengths regardless of Ig mutation status (0.47). Telomere length differed significantly between GC-like (0.73) and non-GC-like DLBCLs (0.43), and follicular lymphomas (FLs) had shorter telomeres (0.53) than GC-DLBCL. Hairy cell leukemias, which display Ig gene intraclonal heterogeneity, had longer telomeres (0.62) than FLs and non-GC-DLBCL, but shorter than GC-DLBCL. We conclude that although DLBCL and CLL subsets can be clearly distinguished, telomere length reflects many parameters and may not simply correlate with GC-related origin.     

ALK-positive large B-cell lymphomas with cryptic SEC31A-ALK and NPM1-ALK fusions

We report 2 ALK-positive large B-cell lymphoma cases showing granular cytoplasmic and cytoplasmic/nuclear ALK immunostaining in which cryptic ALK rearrangements were identified by fluorescent in situ hybridization and molecular analysis. In the first case, the ALK-involving t(2;3)(p23;q27) masked the cryptic SEC31A-ALK fusion generated by an insertion of the 5′ end of SEC31A (4q21) upstream of the 3′ end of ALK. This rearrangement was associated with loss of the 5′ end of ALK and duplication of SEC31A-ALK on der(20). In the second case with complex rearrangements of both chromosomes 2, a submicroscopic NPM1-ALK fusion created by insertion of the 3′ end of ALK into the NPM1 locus was evidenced. Further studies of SEC31A-ALK showed that this variant fusion transforms IL3-dependent Ba/F3 cells to growth factor independence, and that the ALK inhibitor TAE-684 reduces cell proliferation and kinase activity of SEC31A-ALK and its downstream effectors ERK1/2, AKT, STAT3 and STAT5.     

Critical role of the IgM Fc receptor in IgM homeostasis,B-cell survival,and humoral immune responses

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM–antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.     

Anaplastic large-cell lymphomas of B-cell phenotype are anaplastic lymphoma kinase (ALK) negative and belong to the spectrum of diffuse large B-cell lymphomas

There is controversy in the literature as to whether anaplastic large-cell lymphoma of B-cell phenotype is related to the t(2;5)-positive T- or 'null' cell lymphoma of the same morphology. We report a study of 24 lymphomas with morphological features of anaplastic large-cell lymphoma which expressed one or more B-cell markers and lacked T-lineage markers. Clinical features were more in keeping with large B-cell lymphoma than with classical t(2;5)-positive anaplastic large-cell lymphoma, and immunostaining for anaplastic lymphoma kinase (ALK) protein provided no evidence for the (2;5) translocation (or one of its variants). The staining patterns for CD20 and CD79 were typical of diffuse large B-cell lymphoma, CD30 expression was variable, and most cases (15/22) lacked epithelial membrane antigen (EMA). These findings support the view that 'B-cell anaplastic large-cell lymphoma' is unrelated to t(2;5)-positive (ALK-positive) lymphoma, and that it represents a morphological pattern occasionally encountered among diffuse large B-cell lymphomas. By the same reasoning, most tumours diagnosed as 'ALK-negative anaplastic large-cell lymphoma of T-cell or null phenotype' probably belong to the spectrum of peripheral T-cell lymphomas.     

Germ-line configuration of the T-cell receptor beta-chain gene in B-cell chronic lymphoproliferative disorders which co-express T-cell antigens

In 7 cases of chronic B-cell lymphoproliferative disorders-6 chronic lymphocytic leukaemias and 1 non-Hodgkin lymphoma in leukaemic phase--which co-expressed T-cell markers (CD3, CD2) the clonal origin was investigated at the DNA level. In accordance with the diagnosis, all cases showed a monoclonally rearranged configuration of the immunoglobulin genes. On the contrary, the T-cell receptor beta chain gene always retained a germ-line organization. These findings demonstrate that B-cell chronic lymphoproliferative disorders which co-express T-cell-related markers are truly composed of monoclonal B-cell elements.     

Heterogeneous intracellular expression of B-cell receptor components in B-cell chronic lymphocytic leukaemia (B-CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B-CLL-derived cell line

B-cell chronic lymphocytic leukaemia (B-CLL) cells display low amounts of surface immunoglobulins (sIg). To investigate the mechanisms underlying this phenomenon, we performed a thorough study of surface and intracellular expression of the B-cell receptor (BCR) components in B-CLL cells using flow cytometry. There was an heterogeneous pattern of expression. Overall, 20 of 22 samples showed reduced sIgM levels, compared with normal B cells. Among them, three (15%) had very low to undetectable intracellular IgM levels and variable amounts of CD79a and CD79b; nine (45%) had low intracellular CD79b levels but appreciable levels of IgM and CD79a; and eight (40%) had relatively normal intracellular levels of all BCR components. To investigate whether surface BCR levels could be controlled by the rate of CD79b synthesis, adenoviral vectors encoding CD79b were generated and used for gene transfer experiments. Delivery of CD79b to non-B cells transfected with IgM and CD79a lead to high-level expression of a functional BCR. Moreover, CD79b gene transfer in a B cell line derived from a B-CLL patient and characterised by low intracellular levels of endogenous CD79b consistently increased sIgM levels. These findings indicate that the phenotype of B-CLL cells in a subset of patients may depend primarily on poor CD79b expression, and suggest that upregulation of CD79b expression may correct the phenotype of these cells.     

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.     

Developments over the last 60 years in diffuse large B-cell lymphomas

At the time of the formation of the British Society of Haematology diffuse large B-cell lymphoma was not recognised as a specific entity and was included in the category of ‘large cell’ or ‘aggressive’ lymphomas. These were fatal in 95% of cases. Today the cure rate in adults entered into clinical trials is ~70% and a large number of British physicians have contributed to this progress.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Survival of human lymphoma cells requires B-cell receptor engagement by self-antigens

The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one V_H4-34⁺ ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of V_H4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.Theory and indirect evidence have supported the notion of antigenic stimulation in the pathogenesis of human B-cell lymphomas for the past half century (1). Human B-cell lymphomas selectively retain expression of the B-cell receptor (BCR) even in the face of frequent chromosomal translocations that disrupt the Ig heavy chain (IgH) locus, suggesting that the signaling pathways emanating from the BCR may sustain the survival of malignant B cells (2). Foreign antigens have been implicated in certain lymphomas, including the hepatitis C virus in splenic marginal zone lymphoma (3) and Helicobacter pylori in mucosa-associated lymphoid tissue-type lymphomas (4). However, no discernible foreign antigen is present in the majority of lymphoma cases, suggesting a possible role for self-antigens in lymphoma etiology.Examination of the Ig variable (V) regions has lent further support to the concept of antigen-dependent BCR signaling in lymphoid malignancies. In chronic lymphocytic leukemia, for example, a small subset of germ-line–encoded IgH variable gene (V_H) segments are rearranged recurrently (5). The same observation has been made in mantle cell lymphoma (MCL), although the recurrent V_H segments in these lymphomas are not fully concordant with those in chronic lymphocytic leukemia (CLL) (6). Nearly one-third of CLL cases use “stereotyped” BCR sequences in which malignant cells from different patients have almost identical IgH V sequences, including the third complementarity determining region (CDR3) that is diversified during V_H-D_H-J_H (VDJ) joining (5). This observation suggests that CLL BCRs may bind to an antigens because CDR regions typically dictate antibody reactivity. Indeed, CLL BCRs can react with many different self-antigens (7), including antigens released by apoptotic cells (8, 9). Additionally, BCRs derived from CLL patients can bind to a conserved epitope within the second framework region (FR2) of their own IgV_H (10). Because a large component of the germ-line IgV_H repertoire can form antibodies with self-reactivity (11), these findings might merely reflect the derivation of malignant B cells from self-reactive B cells. An alternative, nonmutually exclusive hypothesis is that a self-reactive BCR is essential to maintain the malignant phenotype in an ongoing fashion. This hypothesis has not yet been tested because of the absence of an appropriate model system.Chronic active BCR signaling drives NF-κB signaling in cell line models of the activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), which is essential for their viability (12). Unlike “tonic” BCR signaling (13), which is presumed to be antigen-independent, chronic active BCR signaling in ABC DLBCL has the hallmarks of antigen-dependent BCR signaling in normal B cells (14), including prominent clustering of the BCR on the cell surface (12). Moreover, ∼20% of DLBCL patients have gain-of-function mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signaling motifs of the BCR subunits CD79A and CD79B, providing genetic evidence that BCR signaling is integral to the pathogenesis of ABC DLBCL (12). Although these mutations increase the amplitude of BCR signaling, they cannot initiate BCR signaling de novo (12), leading us to consider a role for antigen in initiating and maintaining chronic active BCR signaling in ABC DLBCL.This possibility was supported by a clinical trial in relapsed/refractory DLBCL of ibrutinib, an inhibitor of Bruton’s tyrosine kinase, which links BCR activity to the NF-κB pathway (15). In ABC DLBCL, ibrutinib produced responses in 37% of patients, lengthening their survival. Although ABC DLBCL tumors with CD79A or CD79B mutations responded more frequently to ibrutinib, responses were also observed in 30% of cases without these mutations, suggesting that the BCR activity of these tumors may depend on a nongenetic process, such as self-antigen engagement of the BCR (15). Moreover, ibrutinib has also proved effective in other B-cell malignancies, such as CLL (16), in which no genetic mechanisms of BCR activity have been reported. In the present study, we sought to provide experimental evidence that the IgV_H regions of ABC DLBCL BCRs are required for their survival and to elucidate the role of self-antigens in this process.     

Expression of granulocyte colony-stimulating factor receptor on CD10-positive human B-cell precursors

We examined the expression of CD10 and G-CSF receptor (G-CSFR) on the lymphoid population of mononuclear cells obtained from bone marrow (BM) using two-colour analysis. In the BM of children with ALL in remission, the CD10⁺ population was significantly increased (20.6 ± 5.1% compared with that of controls (2.5 ± 0.5%). More than half (61.3 ± 2.9%) of the CD10⁺ cells coexpressed G-CSFR, but not CD13. These results indicate G-CSFR⁺ B-cell precursors are markedly increased in BM of ALL in remission, suggesting the probable involvement of G-CSF in the human early B-cell ontogeny.     

Rearrangement of the BCL6 gene in B-cell lymphoid neoplasms: comparison with lymphomas associated with BCL2 rearrangement

We report a series of B-cell neoplasms with regard to rearrangement of the BCL6 gene on chromosome band 3q27. Southern blot analysis using probes from the major translocation cluster (MTC) region of the BCL6 revealed rearrangement in 21/197 patients (10.7%) with B-cell neoplasms studied at presentation, and 11/25 patients (44%) first studied at relapse. In non-Hodgkin's lymphoma (NHL) studied at diagnosis, rearrangements of the BCL6 gene were not closely associated with a specific histopathologic subtype but distributed in subcategories in the Working Formulation. The incidence in follicular lymphoma was 12.1%, with significantly higher frequency in mixed and large cell subtypes, and that in diffuse aggressive lymphoma was 14.1%. Comigration analysis using probes from the immunoglobulin genes revealed association of the BCL6 gene with one of the three immunoglobulin loci in 9/25 cases analysed. A comparative study between NHL associated either with BCL2 or BCL6 rearrangement showed that advanced disease and bone marrow involvement were more frequent in BCL2(+) NHL. In contrast, extranodal involvement was more frequently observed in the BCL6(+) NHL. The survival curve of BCL6(+) NHL was characterized by a rapid decline followed by a plateau. Of the total of 32 BCL6(+) patients, six carried both BCL2 and BCL6 rearrangements; five of these six showed clinicopathological properties characteristic of follicular lymphoma, suggesting that the presence of the two genetic abnormalities does not necessarily have synergistic effects on malignant phenotypes. The high level of BCL6 expression in follicular lymphoma cell lines carrying a BCL2 rearrangement suggests that the deregulated BCL2 gene may have an effect on the development of genetic abnormalities of the BCL6 gene. The present study suggests that BCL6 gene rearrangement is primarily involved in large cell lymphoma irrespective of growth pattern of neoplastic cells, and that BCL6(+)BCL2(?) NHL could be curable with modern intensive chemotherapy.