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Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.

Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and approximately half of those diagnosed will die of metastasis. This study investigates whether UM progression is driven by a subpopulation of stem-like cells, termed "cancer stem cells" (CSCs). Methods: Expression of postulated stem cell markers aldehyde dehydrogenase (ALDH), CD44, and CD133 was analyzed in UM cell lines and primary UM short-term cultures (STCs) established from tumor samples. Additionally, the notion of a "cellular hierarchy" within UM was investigated. Finally, the phenomenon of phenotypic plasticity in response to environmental factors was explored. Results: We demonstrate that expression of ALDH, CD44, and CD133 does not select for a subpopulation of stem-like cells in either UM cell lines or UM STCs. Furthermore, there is an absence of a cellular hierarchy in cell lines and all cells in culture are able to drive tumor progression. Last, we show that established UM cell lines and UM STCs are plastic in nature and switch their phenotype in response to environmental stimuli. Conclusions: We hypothesize that this capacity to undergo phenotypic plasticity may be a consequence of neural crest lineage and renders the exploration of the CSC hypothesis extremely challenging in UM.

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. (C) 2016 by the American Academy of Ophthalmology.

Invasion through stromal extracellular matrix (ECM) is part of the complex, multistep process of tumour cell invasion and metastasis. Our group has previously demonstrated that calcium and calmodulin are important in another step in the metastatic cascade - that of attachment of cells to ECM. Interestingly, the non-steroidal anti-oestrogen tamoxifen (which also has calmodulin antagonist activity), used in the treatment of breast cancer and now in metastatic cutaneous melanoma, can inhibit the attachment of normal and neoplastic cells to ECM. In this study, we investigated whether such drugs, known to inhibit cell attachment, could also subsequently reduce their invasion through a layer of human fibronectin. We examined the ability of the specific calmodulin antagonist J8, tamoxifen and its two major metabolites, N-desmethyltamoxifen (N-des) and 4-hydroxytamoxifen (4-OH), as well as the pure anti-oestrogen ICI 182,780 and 17beta-oestradiol to inhibit invasion of the human cutaneous melanoma cell line, A375-SM, uveal melanoma cells and uveal melanocytes. A375-SM cells and uveal melanoma cells showed a high level of invasion (15.2% and 33.7% respectively) compared with melanocytes (around 5%) under the experimental conditions used. Submicromolar concentrations of N-des, tamoxifen, J8 and 17beta-oestradiol significantly reduced the invasiveness of the A375-SM cell line. The uveal melanoma cells also showed similar inhibition, although at higher concentrations of these agents. 4-OH and ICI 182, 780 had little or no effect on invasion of A375-SM cells (these were not tested on uveal melanoma cells). All cells used in this study were found to be negative for type I nuclear oestrogen receptors, reinforcing the possibility that tamoxifen and 17beta-oestradiol can act via mechanisms unrelated to binding to classical oestrogen receptors to inhibit tumour cell invasion.