Comparing the Prognostic Value of BAP1 Mutation Pattern, Chromosome 3 Status, and BAP1 Immunohistochemistry in Uveal Melanoma.

Uveal melanoma (UM), a tumor of the eye, can be divided into 2 major classes correlating with patients' prognosis. Gene expression profiles and chromosome 3 status are correlated with tumor classification and prognosis. Somatic BAP1 mutations are another feature largely restricted to metastatic UM. Here we performed thorough BAP1 mutation analysis including sequencing and gene dosage analysis of all BAP1 coding exons as well as methylation analysis of the promoter CpG island in a set of 66 UMs. The results were compared with the BAP1 protein expression as determined by immunohistochemistry and the tumor-related survival of the patients. BAP1 sequencing and gene dosage analysis of BAP1 exons by multiplex ligation-dependent probe amplification revealed a mutation in 33 (89%) of 37 tumors with monosomy 3 (M3) or isodisomy 3. BAP1 mutations were not detected in any of the 28 tumors with disomy 3 or partial monosomy 3 (partM3). Most of the sequence mutations (21 of 28) were frame-shift, splice-site, or nonsense mutations leading to a premature termination codon. BAP1 protein as determined by immunohistochemistry was absent in all samples with a BAP1 mutation irrespective of the functional type of mutation. Kaplan-Meier analysis revealed a highly significant association between BAP1 protein staining and patients' survival (P=0.0004). The association between BAP1 mutation status and tumor-related survival was less pronounced but still significant (P=0.0023). We conclude that BAP1 protein staining is favorable over BAP1 mutation screening by Sanger sequencing for prognostic testing of UM patients.

The Pediatric Choroidal and Ciliary Body Melanoma Study: A Survey by the European Ophthalmic Oncology Group.

PURPOSE: To collect comprehensive data on choroidal and ciliary body melanoma (CCBM) in children and to validate hypotheses regarding pediatric CCBM: children younger than 18 years, males, and those without ciliary body involvement (CBI) have more favorable survival prognosis than young adults 18 to 24 years of age, females, and those with CBI. DESIGN: Retrospective, multicenter observational study. PARTICIPANTS: Two hundred ninety-nine patients from 24 ocular oncology centers, of whom 114 were children (median age, 15.1 years; range, 2.7-17.9 years) and 185 were young adults. METHODS: Data were entered through a secure website and were reviewed centrally. Survival was analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. MAIN OUTCOME MEASURES: Proportion of females, tumor-node-metastasis (TNM) stage, cell type, and melanoma-related mortality. RESULTS: Cumulative frequency of having CCBM diagnosed increased steadily by 0.8% per year of age between 5 and 10 years of age and, after a 6-year transition period, by 8.8% per year from age 17 years onward. Of children and young adults, 57% and 63% were female, respectively, which exceeded the expected 51% among young adults. Cell type, known for 35% of tumors, and TNM stage (I in 22% and 21%, II in 49% and 52%, III in 30% and 28%, respectively) were comparable for children and young adults. Melanoma-related survival was 97% and 90% at 5 years and 92% and 80% at 10 years for children compared with young adults, respectively (P = 0.013). Males tended to have a more favorable survival than females among children (100% vs. 85% at 10 years; P = 0.058). Increasing TNM stage was associated with poorer survival (stages I, II, and III: 100% vs. 86% vs. 76%, respectively; P = 0.0011). By multivariate analysis, being a young adult (adjusted hazard rate [HR], 2.57), a higher TNM stage (HR, 2.88 and 8.38 for stages II and III, respectively), and female gender (HR, 2.38) independently predicted less favorable survival. Ciliary body involvement and cell type were not associated with survival. CONCLUSIONS: This study confirms that children with CCBM have a more favorable survival than young adults 18 to 25 years of age, adjusting for TNM stage and gender. The association between gender and survival varies between age groups.

Suspected vitreous seeding of uveal melanoma: relevance of diagnostic vitrectomy.

AIM: To review all cases of suspected vitreous seeding of treated or untreated uveal melanoma at our clinic and to compare clinical, cytological and histological findings with patients' survival. METHODS: Retrospective non-randomised study of 23 patients with consecutive uveal melanoma who underwent diagnostic vitrectomy in our clinic between January 2000 and November 2013. Reason for vitrectomy was suspected dissemination of tumour cells inside the eye. Treated as well as treatment-naïve primary uveal melanomas were included in this study. Follow-up data of all patients were collected. RESULTS: The study included 23 patients with a mean age of 66 years. Four patients presented pigmented vitreous debris at initial presentation prior to treatment of the uveal melanoma. All but one of these four patients has been enucleated as a consequence of cytology-proven vitreous spreading of vital melanoma cells. The remaining 19 patients presented pigmented vitreous debris at a mean of 60 months following local tumour treatment. Thirteen of these patients had been treated with a ruthenium plaque (mean scleral dose 1295 Gy, mean apex dose 152 Gy), three with binuclid plaque (mean scleral dose 1005 Gy, mean apex dose 70 Gy) and three with proton beam radiation. Of the 19 patients, 10 showed only melanophages in the vitreous specimen, while the remaining 9 patients had vital tumour cells in vitreous cytology. Four out of these nine patients have been enucleated in the course of follow-up. During follow-up of our cohort of 23 patients, 4 patients died, but only 1 of them due to metastatic disease. CONCLUSION: The outcome of this small cohort study shows that obtaining a vitreous specimen helps to distinguish melanophages from vital tumour cells. We could not observe an increased risk of metastasis in patients who showed melanoma cell dissemination inside the eye, compared with those patients only showing melanophages. We therefore suggest to carefully re-evaluate the necessity of enucleation in every patient.

Zinc supplementation induces regulatory T cells by inhibition of Sirt-1 deacetylase in mixed lymphocyte cultures.

SCOPE: Zinc is an essential trace element, regulating immune function. Its deficiency results in immune dysfunction and transplant rejection. In here, a benefit of zinc supplementation for the induction of tolerance was investigated, focusing on the TH 1-dominated allogeneic immune reaction. METHODS AND RESULTS: Allogeneic immune reaction was modeled by mixed lymphocyte culture (MLC). The effect of zinc supplementation was monitored via expression of cytokines and surface lineage markers using ELISA and flow cytometry. Epigenetic analyses were performed to investigate mechanisms underlying zinc-induced changes in regulatory T cell (Treg) activation. Results reveal that Tregs are induced when MLCs are treated with 50 µM zinc causing a decrease in IFNγ production. IL-2 and IL-10 expression were not affected. The teleology of this effect includes the inhibition of histone deacetylase Sirt-1-mediated Foxp3 deacetylation, resulting in its decreased degradation. CONCLUSION: In conclusion, zinc should be considered to prevent graft-versus-host disease (GVHD) as it is capable of stabilizing iTregs, resulting in increased numbers of this cell type while not suppressing the immune system.

T-helper type 1 cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells.

OBJECTIVE: We examined the influence of zinc on T-helper type 1 (Th1)/T-helper type 2 (Th2) balance in human lymphocytes. METHODS: Human peripheral blood mononuclear cells or diluted whole blood were cultured for 8 d in the presence of zinc (30 or 60 microM) or 1 microM of N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (a zinc-specific chelator). Phytohemagglutinin-induced cytokine release was measured by enzyme-linked immunosorbent assay, and expression of CD56/CD69, CCR4/CD3, and CCR5/CD3 and intracellular labile zinc were detected by flow cytometry. RESULTS: We found that our in vitro supplementation resulted in an increase of intracellular labile zinc comparable to that of a 7-wk administration of 10 mg of zinc per day in vivo. Zinc triggered interferon-gamma release and impaired interleukin-10 release. Phenotypically, a Th2/Th1 shift could not be confirmed after detecting the Th1-specific chemokine receptor CCR5 or CCR4 for Th2 cells. Surprisingly, we detected a larger amount of CD56+ cells after zinc stimulation, leading us to the conclusion that the amount of interferon-gamma release after zinc supplementation might be attributed to the upregulation of natural killer cells after in vitro zinc supplementation rather than to a Th2/Th1 shift. CONCLUSION: We suggest that a nutritional intake of 10 mg of zinc increases the quantity of interferon-gamma-producing natural killer cells and strengthens the immune system against neoplasms and viral infections.