Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.

Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igβ and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.

The current study aimed to identify differentially expressed B cell‑associated genes in peripheral blood mononuclear cells and observe the changes in B cell activation at different stages of coronary artery disease. Groups of patients with acute myocardial infarction (AMI) and stable angina (SA), as well as healthy volunteers, were recruited into the study (n=20 per group). Whole human genome microarray analysis was performed to examine the expression of B cell‑associated genes among these three groups. The mRNA expression levels of 60 genes associated with B cell activity and regulation were measured using reverse transcription‑quantitative polymerase chain reaction. The mRNA expression of the B cell antigen receptor (BCR)‑associated genes, CD45, NFAM, SYK and LYN, were significantly upregulated in patients with AMI; however, FCRL3, CD79B, CD19, CD81, FYN, BLK, CD22 and CD5 mRNA expression levels were significantly downregulated, compared with patients in the SA and control group. The mRNA levels of the T‑independent B cell‑associated genes, CD16, CD32, LILRA1 and TLR9, were significantly increased in AMI patients compared with SA and control patients. The mRNA expression of genes associated with T‑dependent B cells were also measured: EMR2 and CD97 were statistically upregulated, whereas SLAMF1, LY9, CD28, CD43, CD72, ICOSL, PD1, CD40 and CD20 mRNAs were significantly downregulated in AMI group patients compared with the two other groups. Additionally the gene expression levels of B cell regulatory genes were measured. In patients with AMI, CR1, LILRB2, LILRB3 and VAV1 mRNA expression levels were statistically increased, whereas, CS1 and IL4I1 mRNAs were significantly reduced compared with the SA and control groups. There was no statistically significant difference in B cell‑associated gene expression levels between patients with SA and the control group. The present study identified the downregulation of genes associated with BCRs, B2 cells and B cell regulators in patients with AMI, indicating a weakened T cell‑B cell interaction and reduced B2 cell activation during AMI. Thus, improving B2 cell‑mediated humoral immunity may be a potential target for medical intervention in patients with AMI.

Epstein-Barr virus (EBV)-associated diffuse large B-cell lymphoma (DLBCL) of the elderly constitutes a provisional clinicopathological entity in the current World Health Organization (WHO) classification and its genomic features remain sparsely characterized. We investigated a cohort of 26 cases of untreated de novo EBV-positive DLBCL of the elderly by high-resolution array-based comparative genomic profiling and fluorescence in situ hybridization (FISH). Moreover, we screened for activating mutations affecting nuclear factor (NF)-κB pathway signaling and chromatin remodeling (EZH2, CD79B, CARD11 and MYD88) due to their impact of gene expression signatures and postulated upcoming therapeutic targetability. We identified an overlap between genomic aberrations previously described to be exclusive features of plasmablastic lymphoma (PL), post-transplant lymphoproliferative disorder (PTLD) and DLBCL, respectively, indicating a close cytogenetic relationship between these entities. Few mutations affecting CD79B and CARD11 and no MYD88 mutations were detectable, hinting at EBV-mediated activation of NF-κB as an alternative to pathologically enforced B-cell receptor signaling in this rare entity.

Copy number alterations (CNAs) are thought to account for 85% of the variation in gene expression observed among breast tumours. The expression of cis-associated genes is impacted by CNAs occurring at proximal loci of these genes, whereas the expression of trans-associated genes is impacted by CNAs occurring at distal loci. While a majority of these CNA-driven genes responsible for breast tumourigenesis are cis-associated, trans-associated genes are thought to further abet the development of cancer and influence disease outcomes in patients. Here we present a network-based approach that integrates copy-number and expression profiles to identify putative cis- and trans-associated genes in breast cancer pathogenesis. We validate these cis- and trans-associated genes by employing them to subtype a large cohort of breast tumours obtained from the METABRIC consortium, and demonstrate that these genes accurately reconstruct the ten subtypes of breast cancer. We observe that individual breast cancer subtypes are driven by distinct sets of cis- and trans-associated genes. Among the cis-associated genes, we recover several known drivers of breast cancer (e.g. CCND1, ERRB2, MDM2 and ZNF703) and some novel putative drivers (e.g. BRF2 and SF3B3). siRNA-mediated knockdown of BRF2 across a panel of breast cancer cell lines showed significant reduction in cell viability for ER-/HER2+ (MDA-MB-453) cells, but not in normal (MCF10A) cells thereby indicating that BRF2 could be a viable therapeutic target for estrogen receptor-negative/HER2-enriched (ER-/HER2+) cancers. Among the trans-associated genes, we identify modules of immune response (CD2, CD19, CD38 and CD79B), mitotic/cell-cycle kinases (e.g. AURKB, MELK, PLK1 and TTK), and DNA-damage response genes (e.g. RFC4 and FEN1). siRNA-mediated knockdown of RFC4 significantly reduced cell proliferation in ER-negative normal breast and cancer lines, thereby indicating that RFC4 is essential for both normal and cancer cell survival but could be a useful biomarker for aggressive (ER-negative) breast tumours.

BACKGROUND

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The CD79 molecule, encoded by the CD79a and CD79b genes, is a signaling unit of the B-cell receptor complex, which transmits signals of B-cell activation, growth, and differentiation. They are B-cell-specific and expressed at most stages of B-cell development. Although plasma cells have been believed to lack these gene products, the regulation of CD79 expression in plasma cells is still controversial. In particular, the regulation of CD79b expression remains unclear. We sought to examine CD79b expression in normal and neoplastic plasma cells by immunohistochemical analysis. Out of the 23 clinical samples and 11 cell lines of plasma cell myeloma (PCM), none of the clinical samples and only 1 of 11 cell lines expressed CD79b immunohistologically, whereas non-neoplastic plasma cells in reactive hyperplastic lymph nodes exhibited loss of CD79b protein expression. This finding is quite different from our previous report on CD79a. Not only immunocytochemistry, but also RT-PCR and Western blot analysis of PCM cell lines gave identical results. Interestingly, we detected mRNA transcripts of CD79b in PCM cell lines, although protein translation was lacking. These findings suggest that expression of CD79b is downregulated in both plasma cells and plasma cell myeloma, and this process is possibly under post transcriptional regulation.

Primary CNS diffuse large B-cell lymphoma (PCNS-DLBCL) and systemic DLBCL harbor mutations in MYD88 and CD79B. DNA methyltransferase (MGMT) is methylated in some DLBCL. Our goal was to investigate the frequencies of these events, which have not been previously reported within the same series of patients with PCNS-DLBCL. Fifty-four cases of PCNS-DLBCL from two institutions were analyzed by Sanger sequencing for MYD88 and CD79B, and pyrosequencing for MGMT. MYD88 mutations were identified in 68.8% (35 of 51 cases), with L265P being the most frequent mutation. Mutations other than L265P were identified in 21.6% of cases, of which eight novel MYD88 mutations were identified. Of mutated cases, 17.6% had homozygous/hemizygous MYD88 mutations, which has not been previously reported in PCNS-DLBCL. CD79B mutations were found in six of 19 cases (31.6%), all in the Y196 mutation hotspot. MGMT methylation was observed in 37% (20 of 54 cases). There was no significant difference in median overall survival (OS) between the wild type and mutated MYD88 cases, or between methylated and unmethylated MGMT cases. However, a significant difference (P = 0.028) was noted in median OS between the wild type and mutated CD79B cases.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression, e.g. post-transplant lymphoproliferative disorders (PTLD) or HIV (AIDS-related PCNSL). These cases are poorly characterized, have dismal outcome and are typically Epstein-Barr virus (EBV)-tissue positive. We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. 47 were EBV-tissue negative: 45 EBV(-) HIV(-) PCNSL, 2 EBV(-) HIV(+) PCNSL; and 44 were EBV-tissue positive: 23 EBV(+) HIV(+) PCNSL, 21 EBV(+) HIV(-) PCNSL. As with prior studies, EBV(-) HIV(-) PCNSL had frequent MYD88, CD79B and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin (COO) sub-type. In contrast, these mutations were absent in all EBV-tissue positive cases and ABC frequency was low. Furthermore, copy number loss in HLA-class I/II and antigen presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV(+) HIV(-) PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-tissue positive PCNSL in the immunosuppressed is immunobiologically distinct from EBV(-) HIV(-) PCNSL, and despite expressing an immunogenic virus retains the ability to present EBV-antigens. Results provide a framework for targeted treatment.

The gut associated lymphoid tissues (GALT) of a juvenile bandicoot has been examined using histological and immunohistochemical techniques. The mesenteric lymph nodes were hyperfollicular and had well defined paracortical and medullary areas. Lymphocytes were densely packed throughout the cortex and paracortex and the mantles of the follicles. The GALT contained two distinct areas of tissue organisation. One consisted of large areas of aggregated follicles, whilst the other consisted of more linearly distributed follicles. The distribution of T and B cells in the tissue beds was documented using antibodies to surface markers CD3, CD5 and CD79b. T-cells were present in high numbers in the cortical region of the lymph node, whilst B-cells were predominant in the mantle of the follicles. Dispersed CD3 positive T-cells were abundant in the villi lacteals and present in high numbers in follicular areas of gut. CD79b positive B-cells were not observed in the lacteals but were abundant in the mantles of follicles. This is similar to that observed in other metatherians.

This paper describes the initial appearance and distribution of mature T and B cells in the developing immune tissues of the stripe-faced dunnart (Sminthopsis macroura) based on the use of species cross-reactive antibodies to the lymphocyte cell surface markers CD3, CD5 and CD79b. At birth no mature T or B cells were detected in the liver or bone marrow using anti-CD3, anti-CD5 or anti-CD79b antibodies. T cells were detected in the thymus with anti-CD3 by day 12 and anti-CD5 by day 50 postpartum, and T cells in the spleen were detected by day 43 and day 80 postpartum using anti-CD3 and anti-CD5, respectively. B cells were observed in the dunnart spleen by 43 days after birth. CD3- and CD79b-positive cells were detected in the lymph nodes by 50 days and CD5 by day 15 after birth, and in the gut-associated lymphoid tissues by day 50 and anti-CD5 by day 57 postpartum. The development and distribution of T and B cells in the immune tissues of dunnart pouch young is similar to that described in other marsupial species. Low numbers or absence of mature lymphocytes in immune tissues of early pouch young dunnarts further support the proposition that young marsupials are reliant on non-specific defence strategies and/or maternal strategies for a significant period of their time of development in the pouch.

The cellular response in the dermis of common wombats (Vombatus ursinus) with sarcoptic mange exhibited some typical aspects of an immune response to Sarcoptes scabiei. There was an induction phase for wombats experimentally infected with S. scabiei represented by absence of a dermal inflammatory infiltrate for at least 12 days after infection. T lymphocytes, plasma cells, mast cells, and neutrophils then entered the dermis, consistent with a type IV (delayed) hypersensitivity response. In free-living wombats with severe parakeratotic sarcoptic mange eosinophils were also present in the dermis suggesting that a type I (immediate) hypersensitivity response may develop after a type IV hypersensitivity response. Absence of plasma cells and B lymphocytes in free-living wombats with severe parakeratotic sarcoptic mange compared with their presence in wombats experimentally infected with S. scabiei suggested that some immune tolerance may develop with severe infections. A large proportion of cells in the dermal response were not identified but were possibly cells of connective tissue. The thickness of the epidermis increased within 4 days in response to S. scabiei infection. Some antibodies raised against human leucocyte antigens CD3, CD5, HLA-DP, DQ, DR, and CD79b cross-reacted with leucocyte antigens of common wombats and were used to identify cell types in inflammatory infiltrates using immunohistochemistry.

BACKGROUND

The classic immunophenotype for chronic lymphocytic leukemia (CLL) is CD19(+), restricted dim surface expression of kappa or lambda light chain, CD5(+), CD23(+), dim CD20(+), negative FMC7, and negative CD79b. However, the necessity of assaying for all 3 pan B-cell markers (CD20, FMC7, and CD79b) by flow cytometry has not been definitively documented for CLL.

METHODS

Qualitative patterns and semi-quantitative assessment of staining intensity for CD20, FMC7 and CD79b were performed in 70 cases with a current or prior diagnosis of CLL or CLL with increased prolymphocytes leukemia (CLL/PL). The concurrent morphology in 66 of 70 specimens was classified as typical CLL in 53 cases, CLL/PL in 10 cases, and large cell lymphoma in 3 cases.

RESULTS

Forty percent of the cases varied from the characteristic immunophenotype by having moderate or bright staining of CD20 (36%), FMC7 (7%), and/or CD79b (18%). Discrepant qualitative staining patterns were found between FMC7 and CD20 (21%), CD20 and CD79b (15%), and CD79b and FMC7 (10%). Semiquantitative measurement of staining intensity showed little correlation between CD79b and CD20 or FMC7. Moderate correlation was seen between CD20 and FMC7. No correlation was observed between morphology and intensity of marker expression.

CONCLUSIONS

Variable patterns and intensity of staining were seen for FMC7, CD20, and CD79b in this cohort of CLL samples. Dim or negative staining was most consistently seen for FMC7 (93% of specimens). Although FMC7 staining intensity was moderately correlated with CD20, CD79b intensity was poorly correlated with either CD20 or FMC7, and thus, may provide some independent information.

Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 μg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 μg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.

Mediastinal large B cell lymphomas are uncommon neoplasms that are thought to originate from thymic B cells. An unusual feature of these neoplasms is that they often lack surface immunoglobulin (Ig), a molecule ubiquitously expressed by most mature B cells. In the present study we have analyzed 12 cases of mediastinal large B cell lymphoma for the expression of the mb-1/CD79a polypeptide. This is a component, together with B29/CD79b, of a heterodimer that is associated with surface Ig on normal B cells. Our aim was to see whether loss of Ig in this type of lymphoma is associated with loss of the accompanying CD79a molecule. We have also evaluated 128 B cell lymphomas of other categories to see whether any of them show discordance between mb-1 and Ig expression and analyzed 30 T cell lymphomas as Ig-negative controls. We found that 5 of the 7 mediastinal large B cell lymphomas with interpretable staining results for both mb-1 and Ig, lack Ig but expressed CD79a (mb-1). This phenotype was very rare in other categories of B cell lymphoma, being found among 110 cases in only 5 cases that were all follicular lymphoma. The remaining 105 B cell lymphomas displayed mb-1+/Ig+ phenotype. All 30 T cell lymphomas were mb-1 negative. We conclude that discordant mb-1/Ig expression occurs commonly in mediastinal large B cell lymphomas. In addition, the finding that 11 of 12 of these neoplasms express a phenotype (CD10-, CD19+, CD20+, CD21-, CD22+, CD23-/+) that is very similar to that described for thymic medullary B cells reinforces the idea that most mediastinal large B cell lymphomas are of thymic B cell origin. The correlation between mb-1 and Ig staining patterns in B cell lymphomas of other categories reveals that in the majority (90%), expression of the antigen receptor complex parallels that of mature B cells. These data therefore confirm that the expression of the mb-1 protein provides independent strong evidence for the B lineage of lymphomas and may be used for their routine phenotypic characterization.

BACKGROUND

Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR) and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) patients.

METHODS

Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR), CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling.

RESULTS

Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p)-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB), p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB) was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells.

CONCLUSIONS

BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation-induced phosphorylation of signaling proteins in distinct cell populations can be used to identify aberrant signaling pathways.

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-kappaB, elongation factor 1alpha, CD79b, octamer binding factor 1, Ig H chain, stathmin, and gamma-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn->>PU.1->>GANP.

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igbeta, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing micro, kappa, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with mu or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Anaplastic diffuse large B-cell lymphoma (A-DLBCL) is a rare morphologic variant characterized by the presence of polygonal, bizarre-shaped tumor cells. However, the clinicopathologic and genetic features of this variant are largely unknown. In this study, we investigated 35 cases of A-DLBCL with regard to their clinical, pathologic, and genetic characteristics. The age of the patients ranged from 23 to 89 years (median age, 62 y) with a male to female ratio of 23:12. Twenty-two of 26 (85%) patients had Ann Arbor stage III or IV disease, and 17/26 (65%) patients had a high-intermediate or high International Prognostic Index score. For the 24 patients treated with aggressive chemotherapy regimens, the median overall survival (OS) was 16 months, and the 2-year OS rate was 36%. Immunophenotypically, 30/35 (86%) cases had a non-germinal center B-cell immunophenotype. CD30 expression was present in 18/35 (51%) cases, and the p53 protein stain was positive in 28/35 (80%) cases. Fifteen of 35 (43%) cases expressed both BCL2 and MYC (double expressor). Twenty-nine of 32 (91%) cases tested positive for RELA, RELB, or c-Rel in the nucleus, indicating activation of the NFκB signaling pathway. Cytogenetically, 11/27 (41%) cases had concurrent MYC and BCL2 and/or BCL6 abnormalities (translocation or extra copy), including 5 cases with triple abnormalities. TP53 mutation was found in 17/30 (57%) cases, whereas the MYD88 L265P, CD79B, and CARD11 mutations were found in 7/35, 4/30, and 5/30 cases, respectively. We compared the A-DLBCL group with 50 patients with DLBCL without anaplastic features (common DLBCL). The OS of patients with A-DLBCL was significantly worse than that of patients with DLBCL without anaplastic features (P=0.004). Cases of A-DLBCL more often had a high International Prognostic Index score and a non-germinal center B-cell immunophenotype, more frequently expressed CD30 and p53, and more often had mutations of TP53 and concurrent abnormalities of MYC and BCL2 and/or BCL6 (P<0.05). In conclusion, A-DLBCL displays clinicopathologic features that distinguish it from ordinary DLBCL. Most patients follow an aggressive clinical course and have a poor outcome. Cases of A-DLBCL have a high frequency of TP53 mutation and genetic abnormalities of MYC, BCL2, and BCL6.

Primary Central Nervous System Lymphoma (PCNSL) and Metastatic (or Secondary) Central Nervous System Lymphoma (MCNSL) are rare central nervous system (CNS) malignancies that exhibit aggressive clinical behavior and have a poor prognosis. The majority of CNS lymphomas are histologically classified as diffuse large-B cell lymphoma (DLBCL). DLBCL harbors a high frequency of mutations in MYD88 and CD79b. The MYD88 p.L265P mutation occurs at high frequency in CNS lymphoma and is extremely rare in non-hematologic malignancies. Currently, brain biopsy is considered the gold standard for CNS lymphoma diagnosis. However, brain biopsy is invasive, carries a risk of complications, and can delay initiation of systemic therapy. Circulating tumor DNA (ctDNA) in the cerebrospinal fluid (CSF) can be utilized to detect tumor-derived mutations. Testing of CSF-ctDNA is a minimally-invasive methodology that can be used to assess the genomic alterations present in CNS malignancies. We present a case of an 82-year-old man with a history of testicular lymphoma who presented with speech difficulty and a multifocal enhancing left inferior frontal mass. Analysis for both CSF-cytology and flow cytometry did not show evidence of neoplastic cells. A brain biopsy was performed and microscopic examination showed DLBCL. We isolated CSF-ctDNA and used droplet digital PCR (ddPCR) to detect the most common lymphoma-associated mutations in MYD88, L265P, and V217F. In conjunction, we evaluated the patient-matched CNS lymphoma tissue for MYD88 mutations. We detected the MYD88 p.L265P mutation in formalin fixed paraffin embedded (FFPE) tissue from the brain biopsy and the CSF-ctDNA. In contrast, both the tumor tissue and the CSF ctDNA were negative for the MYD88 p.V217F mutation. This study shows that testing CSF ctDNA for MYD88 mutations is a potentially minimally-invasive approach to diagnosing patients with suspected CNS lymphomas.

Blockage of B cell receptor signaling with ibrutinib presents a promising clinical approach for treatment of B-cell malignancies. However, many patients show primary resistance to the drug or develop secondary resistance. In the current study, cDNA microarray and Western blot analyses revealed CD79B upregulation in the activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) that display differential resistance to ibrutinib. CD79B overexpression was sufficient to induce resistance to ibrutinib and enhanced AKT and MAPK activation, indicative of an alternative mechanism underlying resistance. Conversely, depletion of CD79B sensitized primary refractory cells to ibrutinib and led to reduced phosphorylation of AKT or MAPK. Combination of the AKT inhibitor or the MAPK inhibitor with ibrutinib resulted in circumvention of both primary and acquired resistance in ABC-DLBCL. Our data collectively indicate that CD79B overexpression leading to activation of AKT/MAPK is a potential mechanism underlying primary ibrutinib resistance in ABC-DLBCL, and support its utility as an effective biomarker to predict therapeutic response to ibrutinib.

CD79a and CD79b proteins associate with Ig receptors as integral signaling components of the B cell Ag receptor complex. To study B cell development in zebrafish, we isolated orthologs of these genes and performed in situ hybridization, finding that their expression colocalized with IgH-μ in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to enhanced GFP to identify B cells, as demonstrated by PCR analysis of IgH-μ expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene (Rag)2:mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry+ cells and the timing of Ig H and L chain expression revealed simultaneous expression of both IgH-μ- and IgL-κ-chains, without progressing through the stage of IgH-μ-chain alone. Rag2:mCherry+ cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared with CD79a and CD79b:GFP+ B cells, which showed strongly reduced Rag mRNAs. Thus, B cell development in zebrafish does not go through a Raghi CD79+IgH-μ+ pre-B cell stage, different from mammals. After the generation of CD79:GFP+ B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-μ exon usage, similar to in mammals. This confirmed a conserved role for CD79 in B cell development and differentiation, without the requirement of a pre-B cell stage in zebrafish.

The surface expression of CD79b, using the monoclonal antibody (Mab) CB3-1, on B lymphocytes from normal individuals and patients with B cell chronic lymphocytic leukemia (CLL) has been analyzed using triple-staining cells for flow cytometry. In addition, the clinical significance of CD79b expression in CLL patients and its possible value for the evaluation of minimal residual disease (MRD) was explored. A total of 15 peripheral blood (PB) samples from healthy blood donors, five bone marrow (BM) samples from normal donors and 40 PB samples from CLL untreated patients were included in the study. In addition we studied the expression of CD79b in B lymphocytes from five CLL patients after fludarabine treatment in order to support our method. The expression of CD79b in B lymphocytes from PB was analyzed by flow cytometry, using simultaneous staining with the Mabs CD22, CD79b, CD19 and CD5, CD79b and CD19. Since normal immature bone marrow B cells are CD79b-/dim+ on their surface, in BM samples we used the combination CD45, CD79b and CD19 selecting mature B lymphocytes according to their bright CD45 intensity. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD19+ cells. For data analysis, the PAINT-A-GATE PRO software (Becton Dickinson) was used. Dilution experiments of CD79b- CLL cells and CD79bdim+ CLL cells with normal PB and BM cells were performed in order to assess the sensitivity level of the technique for detection of CD79b-/dim+residual CLL cells. All B lymphocytes from normal samples showed reactivity for the CD79b antigen. In contrast, CD79b was absent in 18/40 CLL patients (42.5%) and 20/40 CLL cases (50%) exhibited a low CD79b expression. Therefore, CD79b- B lymphocytes would be restricted to the CLL population and thus could be considered a 'tumor phenotype' for monitoring MRD in CLL patients. Dilution experiments indicate that the detection limit with this marker almost reaches the levels obtained by molecular biology methods as the PCR technique. All cases studied after fludarabine presented leukemic cells in their PB or BM samples detected by flow cytometry. Upon comparing the clinical and morphological characteristics of CD79b- and CD79b+ cases, all atypical CLL cases included in the present study were CD79b+ and advanced clinical stage (B and C Binet stage) was most frequently observed in CD79b+ cases than in CD79b- cases.

Constitutive nuclear factor-kappa B (NF-κB) activation has been reported in ocular adnexal lymphoma (OAL). TNFAIP3/A20 is a "global" inhibitor of NF-κB pathway. We have shown that OAL has preferential loss of the 6q23.3 region where TNFAIP3/A20 exist, which is suggested to involve in lymphomagenesis of OAL. The mechanisms causing NF-κB activity in OAL remain elusive. Recently, NF-κB canonical pathway genes including CARD11, CD79B and MYD88 were shown to be frequently mutated in diffuse large B-cell lymphomas. In this study, we analyzed the mutation status of these genes by direct sequencing in 24 OAL cases including 9 cases with loss of 6q23.3 previously identified by array comparative genomic hybridization. We showed that genetic alterations of these genes were not found in OAL, a finding differing from that of most B-cell lymphomas. Genetic or epigenetic alterations in other genes are likely to be relevant in pathogenesis of OAL case without A20 loss.