Upregulated Expression of Activation-Induced Cytidine Deaminase in Ocular Adnexal Marginal Zone Lymphoma with IgG4-Positive Cells.

Immunoglobulin G4-related disease (IgG4-RD) is a systemic disorder characterized by tissue fibrosis and intense lymphoplasmacytic infiltration, causing progressive organ dysfunction. Activation-induced cytidine deaminase (AID), a deaminase normally expressed in activated B-cells in germinal centers, edits ribonucleotides to induce somatic hypermutation and class switching of immunoglobulin. While AID expression is strictly controlled under physiological conditions, chronic inflammation has been noted to induce its upregulation to propel oncogenesis. We examined AID expression in IgG4-related ophthalmic disease (IgG4-ROD; n = 16), marginal zone lymphoma with IgG4-positive cells (IgG4+ MZL; n = 11), and marginal zone lymphoma without IgG4-positive cells (IgG4- MZL; n = 12) of ocular adnexa using immunohistochemical staining. Immunohistochemistry revealed significantly higher AID-intensity index in IgG4-ROD and IgG4+ MZL than IgG4- MZL (p < 0.001 and = 0.001, respectively). The present results suggest that IgG4-RD has several specific causes of AID up-regulation in addition to inflammation, and AID may be a driver of oncogenesis in IgG4-ROD to IgG4+ MZL.

An open-label phase 2 trial of entospletinib in indolent non-Hodgkin lymphoma and mantle cell lymphoma.

Spleen tyrosine kinase (Syk) mediates B-cell receptor signalling in normal and malignant B cells. Entospletinib is an oral, selective Syk inhibitor. Entospletinib monotherapy was evaluated in a multicentre, phase 2 study of patients with relapsed or refractory indolent non-Hodgkin lymphoma or mantle cell lymphoma (MCL). Subjects received 800 mg entospletinib twice daily. Forty-one follicular lymphoma (FL), 17 lymphoplasmacytoid lymphoma/Waldenström macroglobulinaemia (LPL/WM), 17 marginal zone lymphoma (MZL) and 39 MCL patients were evaluated. The primary endpoint was a progression-free survival (PFS) rate (defined as not experiencing progression or death) at 16 weeks for patients with MCL and at 24 weeks for patients with FL, LPL/WM and MZL. The most common treatment-emergent adverse events were fatigue, nausea, diarrhoea, vomiting, headache and cough. Common laboratory abnormalities were anaemia, neutropenia and thrombocytopenia; aspartate transaminase, alanine transaminase, total bilirubin and serum creatinine were all increased. PFS at 16 weeks in the MCL cohort was 63·9% [95% confidence interval (CI) 45-77·8%]; PFS at 24 weeks in the FL, LPL/WM, MCL and MZL cohorts was 51·5% (95% CI 32·8-67·4%), 69·8% (95% CI 31·8-89·4%), 56·6% (95% CI 37·5-71·8%) and 46·2% (95% CI 18·5-70·2%), respectively. Entospletinib had limited single-agent activity with manageable toxicity in these patient populations.

Protease activity of MALT1: a mystery unravelled.

Constitutive NF-κB (nuclear factor κB) activation in B-cell lymphomas relies greatly on the CARMA1 [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase) 1]-Bcl10-MALT1 (mucosa-associated lymphoid tissue translocation gene 1) signalling complex. Within this protein complex, MALT1 possesses a rather unique enzymatic activity, which allows it to cleave Bcl10, RelB and CYLD, among other substrates. The catalytic activity of MALT1 promotes activation of canonical and non-canonical NF-κB as well as other signalling pathways. However, even after a decade of intense research on MALT1, many mechanistic aspects of its enzymatic activity remain elusive. A recent article by Hachmann, Snipas, van Raam, Cancino, Houlihan, Poreba, Kasperkiewicz, Drag and Salvesen [(2012) Biochem. J. 443, 287-295] provides novel insight into the activation mechanism and the substrate specificity of MALT1. These intriguing findings convincingly demonstrate the importance of MALT1 dimerization for its catalytic activity and pave the way for novel therapeutic approaches that target this crucial regulator of lymphoma survival and proliferation.

Is elevated gastric tissue NOX2 associated with lymphoma of mucosa-associated lymphoid tissue?

Abstract Helicobacter pylori infection plays a crucial role in the pathogenesis of gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT). However, the host response to this infection is also important in the development of the disease. In particular, NADPH oxidases (NOXs) which generate reactive oxygen species are known to induce cell damage possibly leading to carcinogenesis. We analyze for the first time NOX expression in a series of well characterized gastric MALT lymphoma (GML) patients in comparison with controls. Our observation leads to the hypothesis that NOX2 expression is significantly associated with GML.

Ectopic lymphoid neogenesis and lymphoid chemokines in Sjogren's syndrome: at the interplay between chronic inflammation, autoimmunity and lymphomagenesis.

It has long been demonstrated that a subset of patients with Sjogren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the salivary glands (SG). These structures are characterised by periductal clusters of T and B lymphocytes, development of high endothelial venules and differentiation of follicular dendritic cells (FDC) networks. Evidence in patients with and animal models of SS demonstrated that the formation and maintenance of ELS in the SG is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. Several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing some of these factors during chronic inflammation in SS. In this review we focus on the cellular and molecular mechanisms regulating the formation of ELS in SS SG, with particular emphasis on the role of lymphoid chemokines. In addition, we summarise accumulating data in support of the notion that ELS in SS represent functional niches whereby autoreactive B cells undergo affinity maturation, clonal selection and differentiation into autoantibody producing cells, thus contributing to autoimmunity over and above secondary lymphoid organs. Furthermore, we review the emerging role of ELS and lymphoid chemokines in driving extranodal B cell lymphomagenesis in SS and we focus on recent evidence suggesting that ELS identify subsets of SS patients at increased risk of developing systemic manifestations and lymphoma.

MALT1 protease: a new therapeutic target in B lymphoma and beyond?

The identification of mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) as a gene that is perturbed in the B-cell neoplasm MALT lymphoma, already more than a decade ago, was the starting point for an intense area of research. The fascination with MALT1 was fueled further by the observation that it contains a domain homologous to the catalytic domain of caspases and thus, potentially, could function as a protease. Discoveries since then initially revealed that MALT1 is a key adaptor molecule in antigen receptor signaling to the transcription factor NF-κB, which is crucial for lymphocyte function. However, recent discoveries show that this function of MALT1 is not restricted to lymphocytes, witnessed by the ever-increasing list of receptors from cells within and outside of the immune system that require MALT1 for NF-κB activation. Yet, a role for MALT1 protease activity was shown only recently in immune signaling, and its importance was then further strengthened by the dependency of NF-κB-addicted B-cell lymphomas on this proteolytic activity. Therapeutic targeting of MALT1 protease activity might, therefore, become a useful approach for the treatment of these lymphomas and, additionally, an effective strategy for treating other neoplastic and inflammatory disorders associated with deregulated NF-κB signaling.

Effect of human cell malignancy on activity of DNA polymerase iota.

An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by cell division blocking that occurs in all normal cells except in testicles and in malignant cells.

Overexpression of B cell-activating factor of TNF family (BAFF) is associated with Helicobacter pylori-independent growth of gastric diffuse large B-cell lymphoma with histologic evidence of MALT lymphoma.

We have recently demonstrated that nuclear expression of BCL10 predicts Helicobacter pylori (HP) independence of early-stage gastric diffuse large B-cell lymphoma (DLBCL) with histologic evidence of mucosa-associated lymphoid tissue (MALT). In this study, we examined the role of B cell-activating factor of TNF family (BAFF) in mediating BCL10 nuclear translocation and HP independence of gastric DLBCL (MALT). We used immunohistochemistry and immunoblotting to measure the expression of BAFF, pAKT, BCL3, BCL10, and NF-kappaB. Transactivity of NF-kappaB was measured by electromobility shift assay. In lymphoma samples from 26 patients with gastric DLBCL (MALT), we detected aberrant expression of BAFF in 7 of 10 (70%) HP-independent and in 3 of 16 (18.8%) HP-dependent cases (P = .015). BAFF overexpression was associated with pAKT expression (P = .032), and nuclear expression of BCL3 (P = .014), BCL10 (P = .015), and NF-kappaB (P = .004). In B-cell lymphoma Pfeiffer cells, BAFF activated NF-kappaB and AKT; the activated NF-kappaB up-regulated BCL10, and the activated AKT caused formation of BCL10/BCL3 complexes that translocated to the nucleus. Inhibition of AKT by LY294002 (a PI3K inhibitor) blocked BCL10 nuclear translocation, NF-kappaB transactivity, and BAFF expression. Our results indicate that autocrine BAFF signal transduction pathways may contribute to HP-independent growth of gastric DLBCL (MALT).

Microcirculatory alteration in low-grade gastric mucosa-associated lymphoma by Helicobacter heilmannii infection: its relation to vascular endothelial growth factor and cyclooxygenase-2.

BACKGROUND: There are clinical reports that Helicobacter heilmannii, as well as Helicobacter pylori, has been clinically reported to cause gastric low-grade mucosa-associated lymphoid tissue-type (MALT) lymphoma, although its precise mechanism remains to be clarified. Thus, the present study was undertaken to elucidate the alteration of the microcirculatory structure and the relation to angiogenetic factors in mice infected with H. heilmannii for 3 and 6 months. METHODS: Immunohistochemical studies have been performed by FITC-dextran intra-aortic infusion or CD31, vascular endothelial growth factor-A, cyclooxygenase 2 antibodies using our recently established model of gastric mucosa-associated lymphoid tissue-type gastric B-cell lymphoma in C57BL/6 mice. RESULTS: Increased microcirculatory network was recognized surrounding the MALT lymphoma tissues by both the FITC-dextran infusion method and CD31 immunoreactivity. Vascular endothelial growth factor-A immunoreactivity was recognized within the lymphoma tissues as well as in the marginal area, while cyclooxygenase-2 immunoreactivity was localized in the area surrounding the MALT lymphoma tissues. CONCLUSION: Increased microvascular network as well as enhanced VEGF-A immunoreactivity was shown to be related to expansion of the MALT lymphoma formed by Helicobacter heilmannii infection.

TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphoma.

The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma.

Evaluation of JAK2 in B and T cell neoplasms: identification of JAK2(V617F) mutation of undetermined significance (JMUS) in the bone marrow of three individuals.

BACKGROUND/AIMS: The JAK2(V617F) mutation, which has been found in patients with myeloproliferative disorders (MPD), has not yet been evaluated in lymphoproliferative disorders by any adequately sensitive techniques. METHODS: We investigated whether low levels of JAK2(V617F) are present in lymphoid neoplasms using a highly sensitive and highly specific amplification refractory mutation system PCR (ARMS-PCR) assay. RESULTS: While 234 of 237 cases did not carry the JAK2(V617F) allele, it was identified in the bone marrow of 3 B cell lymphoma patients. The mutation was found to be neither associated with the lymphomas per se, nor with any signs, symptoms or laboratory findings of MPD. Moreover, JAK2(V617F) appeared subsequently in the peripheral blood of 2 of the 3 patients. CONCLUSION: These findings suggest that JAK2(V617F) arises in the bone marrow of individuals before clinical manifestation of any myeloid disorders. Presence of JAK2(V617F) in bone marrow might therefore increase the risk of future MPD development, just as monoclonal gammopathy of undetermined significance (MGUS) increases the risk of multiple myeloma. We term this phenomenon 'JAK2(V617F) of undetermined significance' (JMUS). Its clinical significance remains to be determined. To our knowledge, these findings represent the first identification of JAK2(V617F) in the bone marrow of patients without myeloid malignancies.

Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjögren's syndrome.

Demonstration of ectopic germinal center-like structures (GC-LSs) in chronically inflamed tissues in patients with autoimmune disorders is a relatively common finding. However, to what extent ectopic lymphoid structures behave as true GC and are able to support class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes is still debated. In addition, no information is available on whether CSR and SHM can take place in the absence of GCs at extrafollicular sites in an ectopic lymphoid tissue. In this study, we show that in salivary glands (SGs) of Sjögren's syndrome (SS) activation-induced cytidine deaminase (AID), the enzyme responsible for CSR and SHM is invariably expressed within follicular dendritic cell (FDC) networks but is not detectable in SGs in the absence of ectopic GC-LSs, suggesting that FDC networks play an essential role in sustaining the Ag-driven B cell proliferation within SS-SGs. We also show that the recently described population of interfollicular large B cells selectively expresses AID outside ectopic GC in the T cell-rich areas of periductal aggregates. Finally, we report that AID retains its exclusive association with numerous, residual GCs in parotid SS-MALT lymphomas, whereas neoplastic marginal zone-like B cells are consistently AID negative. These results strongly support the notion that ectopic lymphoid structures in SS-SGs express the molecular machinery to support local autoantibody production and B cell expansion and may play a crucial role toward lymphomagenesis.

cIAP2 is a ubiquitin protein ligase for BCL10 and is dysregulated in mucosa-associated lymphoid tissue lymphomas.

The pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas is associated with independent chromosomal translocations that lead to the upregulation of either BCL10 or MALT1 or the generation of a fusion protein, cIAP2-MALT1. While both BCL10 and MALT1 are critically involved in antigen receptor-mediated NF-kappaB activation, the role of cIAP2 is not clear. Here we show that cIAP2 is a ubiquitin ligase (E3) of BCL10 and targets it for degradation, inhibiting antigen receptor-mediated cytokine production. cIAP2-MALT1 lacks E3 activity, and concomitantly, the BCL10 protein is stabilized in MALT lymphomas harboring this fusion. Furthermore, BCL10 and cIAP2-MALT1 synergistically activate NF-kappaB. These results reveal cIAP2 as an inhibitor of antigenic signaling and implicate its dysfunction in MALT lymphomas.

Effect of interleukin-1beta and glutathione S-transferase genotypes on the development of gastric mucosa-associated lymphoid tissue lymphoma.

e tested whether polymorphic variations in glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and interleukin-1 (IL-1beta and IL-1RN) genes confer susceptibility to mucosa-associated lymphoid tissue lymphomas (MALT) in a Chinese population. The rates of GSTM1, GSTP1, IL-1beta and IL-1RN genotypes did not differ between patients and controls. However, GSTT1 null genotypes were significantly more common in patients with MALT lymphomas (43/75 vs. 138/321, p=0.029; OR=1.8, 95% CI: 1.1-3.0) than in controls. Our results suggest that a glutathione S-transferase defect plays a role in MALT lymphoma.

Cdc25A and cdc25B expression in malignant lymphoma of the thyroid: correlation with histological subtypes and cell proliferation.

Cdc25B and cdc25A phosphatases are representative stimulators of cell cycle progression, and recent studies have also indicated their oncogenic roles. In this study, we investigated the expression of these phosphatases in malignant lymphoma of the thyroid by immunohistochemistry. These phosphatases were not expressed in follicular cells in normal follicles, but were heterogeneously or diffusely expressed in the follicles in chronic thyroiditis and malignant lymphoma. In infiltrating lymphocytes in chronic thyroiditis, they were only occasionally expressed. Of the 47 cases of lymphoma, 30 (63.8%) were classified as high group for cdc25B because it was expressed in more than 25% of lymphoma cells. Cdc25B expression level was inversely associated with MIB-1 labeling index (p=0.0008), and aberrant p53 expression (p=0.0077). Furthermore, cases of marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MZBL) were more frequently classified as high group (p=0.0318) than those of diffuse large B-cell lymphoma (DLBL). On the other hand, 22 cases (46.8%) were regarded as high group for cdc25A, but its expression level was not linked to those parameters. These findings suggest that i) cdc25B plays a role in the early phase of thyroid lymphoma possibly including the malignant transformation from chronic thyroiditis, and ii) cdc25A may contribute to the progression of lymphoma.

Expression of activation-induced cytidine deaminase in human B-cell non-Hodgkin lymphomas.

Activation-induced cytidine deaminase (AID) induces somatic hypermutation (SHM), class switch recombination (CSR), and immunoglobulin gene conversion in B-lymphocytes. Here we report for the first time the expression of AID in healthy human B-lymphocytes and in B-cell non-Hodgkin lymphomas (B-NHL). AID mRNA expression in humans is restricted to the CD19(+)CD38(+)IgD(-) germinal center cells, namely the CD19(+)CD38(+)CD44(-) centroblasts. After in vitro stimulation of naive human B cells by CD40-L and IL-4, AID mRNA is strongly induced for only 48 hours. In a survey of human B-NHL AID was found to be constitutively expressed in follicular lymphoma and in diffuse large B-cell lymphoma but to be absent in B-precursor lymphoblastic leukemia, in mantle cell lymphoma, and in plasma cell myeloma. In B-cell chronic lymphatic leukemia, in immunocytoma, and in extranodal marginal zone B-cell lymphoma of MALT, AID mRNA was expressed only in some samples. In follicular lymphoma and diffuse large B-cell lymphoma, the expression of AID mRNA was coincident with the presence of SHM in the variable region exons of the immunoglobulin heavy-chain gene. In human B-NHL, the AID mRNA is spliced into 4 different variants but does not contain point mutations. Thus AID, which is highly regulated during healthy B-cell development, is constitutively expressed in human germinal center B-NHL and in subsets of nongerminal center B-NHL. This constitutive expression of AID may promote illegitimate DNA recombinations and somatic mutations in B-NHL.

Loss of Fas (CD95/APO-1) regulatory function is an important step in early MALT-type lymphoma development.

Fas (CD95, APO-1) mutations were found in autoimmune diseases and some lymphomas, suggesting impairment of Fas-mediated cell death signaling that may cause tumor development. Because mucosa-associated lymphoid tissue (MALT)-type lymphoma B cells recognize autoantigens and proliferate in response to antigen and T cell-mediated signals, it is suggestive that autoreactive B cell lymphoma precursor cells may have escaped the Fas-mediated checkpoint that normally operates in healthy individuals. Using different biochemical, molecular, and functional approaches, we analyzed the Fas signaling in malignant B cells from seven MALT-type lymphomas that were additionally characterized for the t(11;18)(q21;q21) and four gastric diffuse large B cell lymphomas (DLBL). All DLBLs and three of seven MALT-type lymphomas were resistant to Fas-mediated apoptosis in vitro. Moreover, four of five MALT-type lymphomas analyzed and one of three DLBLs analyzed showed mutations in Fas mRNA transcripts but no loss of heterozygosity in the Fas promotor region. Alternative mechanisms of resistance to apoptosis, such as decreased expression of Fas or production of soluble Fas were not operative. Therefore, it is suggestive that a subgroup of MALT-type lymphoma B cells, irrespective of t(11;18)(q21;q21), escape the censoring Fas pathway by mutating and inactivating Fas. This identifies a key regulatory step in early MALT-type lymphomagenesis.

Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.

Caspases are cysteine proteases essential to apoptosis. We have identified two families of caspase-like proteins, Paracaspases (found in metazoans and Dictyostelium) and metacaspases (found in plants, fungi, and protozoa). Metazoan paracaspase prodomains contain a death domain and immunoglobulin domains. Several plant metacaspase prodomains contain zinc finger motifs resembling those in the plant hypersensitive response/cell death protein Isd-1. The human paracaspase prodomain binds Bcl10, a protein involved in the t(1;14)(p22;q32) translocation of mucosa-associated lymphoid tissue (MALT) lymphoma. Another MALT lymphoma translocation, t(11;18)(q21;q21), fuses the IAP-2 gene to the MLT1/MALT1 locus, which encodes the human paracaspase. We find that this fusion activates NF-kappaB and that the caspase domain is required for this function, since mutation of the conserved catalytic cysteine attenuates NF-kappaB activation.