Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma.

BACKGROUND: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. METHODS: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays. RESULTS: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse. CONCLUSION: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Progesterone receptor B recruits a repressor complex to a half-PRE site of the estrogen receptor alpha gene promoter.

In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

The interleukin (IL)-31/IL-31R axis contributes to tumor growth in human follicular lymphoma.

Interleukin (IL)-31A binds to an heterodimer composed of IL-31 receptor A (IL-31RA) and Oncostatin M Receptor (OSMR). The IL-31/IL-31R complex is involved in the pathogenesis of various skin diseases, including cutaneous T-cell lymphoma. No information is available on the relations between the IL-31/IL-31R complex and B-cell lymphoma. Here we have addressed this issue in follicular lymphoma (FL), a prototypic germinal center(GC)-derived B-cell malignancy. IL-31 enhanced primary FL cell proliferation through IL-31R-driven signal transducer and activator of transcription factor 1/3 (STAT1/3), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation. In contrast, GC B cells did not signal to IL-31 in spite of IL-31R expression. GC B cells expressed predominantly the inhibitory short IL-31RA isoform, whereas FL cells expressed predominantly the long signaling isoform. Moreover, GC B cells lacked expression of other IL-31RA isoforms potentially involved in the signaling pathway. IL-31 protein expression was significantly higher in surface membrane than in cytosol of both FL and GC B cells. IL-31 was detected in plasma membrane microvesicles from both cell types but not released in soluble form in culture supernatants. IL-31 and IL-31RA expression was higher in lymph nodes from FL patients with grade IIIa compared with grade I/II, suggesting a paracrine and/or autocrine role of IL-31/IL-31RA complex in tumor progression through microvesicle shedding.

Promotion and inhibition of haemopoiesis by NK cells: a model for immune-mediated haemopoietic suppression.

Human NK cells, besides mediating cytotoxic functions, can exert a number of regulatory activities on the proliferation and maturation of hematopoietic stem cells through the release of soluble factors. A preliminary characterization of these factors demonstrates that NK cell-derived colony stimulating activities (CSA) are represented by GM-CSF and IL3, whereas the colony inhibiting activity (CIA) produced by NK cells is identifiable as TNF. CSA is mainly released by resting NK cells, while, following appropriate activation, the latter cells produce large amounts of CIA. NK cells also influence B cell activation by releasing a 50 kd BCGF and a 26 kd BCDF. The latter factor, which is active in vitro only in the presence of IL2, is identifiable as IL6. The production of regulatory cytokines by NK cells may have some bearing on the pathogenesis of a number of autoimmune disorders.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

Complementary IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicular and diffuse large B-cell lymphoma growth in vivo.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Incidental finding of peripheral B-cell non-Hodgkin lymphoma, lymphocytic/CLL type, of the gallbladder in a patient with chronic cholecystitis.

Although lymphoma involvement of the gallbladder, especially by MALT and large-cell types, is rare, this possibility should be considered in patients with symptoms of acute cholecystitis. A cholecystectomy was performed in a 79-year-old male patient with a clinical diagnosis of chronic cholecystitis. Histologically, the specimen showed an incidental finding of a small lymphocytic lymphoma (CLL) by morphologic and immunophenotyping studies, subsequently confirmed with flow cytometric analysis of blood. During follow-up, multiple lymph node enlargement was detected. An axillary node, excised and submitted to our department, was positive for lymphoma involvement. The bone marrow was negative.

A novel role of the CX3CR1/CX3CL1 system in the cross-talk between chronic lymphocytic leukemia cells and tumor microenvironment.

Several chemokines/chemokine receptors such as CCR7, CXCR4 and CXCR5 attract chronic lymphocytic leukemia (CLL) cells to specific microenvironments. Here we have investigated whether the CX(3)CR1/CX(3)CL1 axis is involved in the interaction of CLL with their microenvironment. CLL cells from 52 patients expressed surface CX(3)CR1 and CX(3)CL1 and released constitutively soluble CX(3)CL1. One third of these were attracted in vitro by soluble CX(3)CL1. CX(3)CL1-induced phosphorylation of PI3K, Erk1/2, p38, Akt and Src was involved in induction of CLL chemotaxis. Leukemic B cells upregulated CXCR4 upon incubation with CX(3)CL1 and this was paralleled by increased chemotaxis to CXCL12. Akt phosphorylation was involved in CX(3)CL1-induced upregulation of CXCR4 on CLL. In proliferation centers from CLL lymph node and bone marrow, CX(3)CL1 was expressed by CLL cells whereas CX(3)CR1 was detected in CLL and stromal cells. Nurselike cells (NLCs) generated from CLL patient blood co-expressed surface CX(3)CR1 and CX(3)CL1, but did not secrete soluble CX(3)CL1. Only half of NLC cell fractions were attracted in vitro by CX(3)CL1. In conclusion, the CX(3)CR1/CX(3)CL1 system may contribute to interactions between CLL cells and tumor microenvironment by increasing CXCL12-mediated attraction of leukemic cells to NLC and promoting directly adhesion of CLL cells to NLC.

Expression of CD5 and CD38 by human CD5- B cells: requirement for special stimuli.

In this study the mode of expression of CD5 by human tonsillar CD5- B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5+ and CD5- cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5- B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15-25% of the total CD5- B cells were induced to express CD5. Unlike CD5- B cells, CD5+ B cells proliferated vigorously in response to PMA as assessed by [3H]thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5- B cells was not related to the selective expansion of some CD5+ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5- B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [3H]thymidine. Furthermore, mitomycin C treatment of the CD5- B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5+ B cells but not CD5- B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5+ and CD5- B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5- B cells. In this respect, the CD5- B cells that converted into CD5+ cells (inducible CD5+ B cells) resembled the cells from the CD5+ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5- B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5- B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for differential responsiveness of human CD5+ and CD5- B cell subsets to T cell-independent mitogens.

Tonsillar resting B cells were separated into CD5+ and CD5- cell subsets and stimulated with the thymus-independent mitogens, Staphylococcus aureus Cowan strain I (SAC) or insolubilized anti-mu monoclonal antibodies (a mu Ab). CD5+ cells incorporated [3H]thymidine more efficiently than unfractionated cells when stimulated with SAC and their response was augmented by the addition of interleukin (IL) 2 to the cultures. CD5+ cells also proliferated in response to a mu Ab provided that IL 2 was present, SAC-, but not a mu Ab-stimulated CD5+ cells produced IgM and IgG molecules when IL 2 was added to the cultures and also secreted autoantibodies with rheumatoid factor activity and sometimes also with anti-single-stranded, but not double-stranded, DNA activity. The efficient response of CD5+ cells was not explained by the fact that they contained cells already activated in vivo. Thus, they did not express the CD23, CD69, CD71 and CD39 activation markers, failed to incorporated [3H]thymidine and to secrete Ig spontaneously or in response to IL 2 and were found to be in a quiescent state by cell cycle flow cytometric analysis. In contrast to CD5+ cells, CD5- cells displayed very little or no [3H]thymidine incorporation in response to SAC or to a mu Ab and their poor responsiveness was not altered by changing either the doses of the stimulants, the timing of the cultures, by co-culturing the cells together with CD5+ cells, or by adding IL 2 or IL 4. Immunofluorescence studies showed that freshly prepared CD5- cells did not have surface activation markers but that they expressed them following SAC stimulation. Thus, unlike that observed for CD5+ cells, SAC seems to be capable of activating CD5- cells but does not appear to be a sufficient stimulus for driving the cells into the subsequent phases of the cell cycle. The above findings, that demonstrate marked differences in the response to CD5+ and CD5- cells to thymus-independent stimuli, may bear relevance for the understanding of the normal clonal expansion of CD5+ cells as well as for the pathogenesis of autoimmune diseases.

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells.

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodide-stained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.

In vitro stimulation of human tonsillar subepithelial B cells: requirement for interaction with activated T cells.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.