Background: Activating Gα_q signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT₂R) forms a rare alternative. The role of wild-type CysLT₂R in uveal melanoma remains unknown. Methods: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. Results: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. Conclusions: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT₂R an attractive therapeutic target in uveal melanoma.

The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy.

Increased angiogenesis is associated with a higher metastasis- and mortality rate in uveal melanoma (UM). Recently, it was demonstrated that genetic events, such as 8q-gain and BAP1-loss, influence the level of immune infiltrate. We aimed to determine whether genetic events, and specific cytokines, relate to angiogenesis in UM. Data from UM patients who underwent enucleation between 1999 and 2008 were analysed. Microvascular density (MVD) and the presence of infiltrating immune cells were determined with immunohistochemistry (IHC) and immunofluorescence in 43 cases. Chromosome status, BAP1 IHC and mRNA expression of angiogenesis-related genes were known in 54 cases. Tumours with monosomy 3/BAP1-loss showed a higher MVD compared to tumours with disomy 3/normal BAP1 expression (p = 0.008 and p = 0.004, respectively). Within BAP1-positive lesions (n = 20), 8q-gain did not relate to MVD (p = 0.51). A high MVD was associated with an increased expression of angiopoietin 2 (ANGPT2) (p = 0.041), Von Willebrand Factor (VWF) (p = 0.010), a decreased expression of vascular endothelial growth factor B (VEGF-B) (p = 0.024), and increased numbers of tumour-infiltrating macrophages (CD68+, p = 0.017; CD68+CD163+, p = 0.031) and lymphocytes (CD4+, p = 0.027). Concluding, vascular density of UM relates to its genetic profile: Monosomy 3 and BAP1-loss are associated with an increased MVD, while an early event (gain of 8q) is not independently related to MVD, but may initiate a preparation phase towards development of vessels. Interestingly, VEGF-B expression is decreased in UM with a high MVD.

A high HLA expression in uveal melanoma (UM) is part of the prognostically unfavorable inflammatory phenotype. We wondered whether the presence of soluble HLA (sHLA) in the aqueous humour is associated with clinical, histopathological or genetic tumour characteristics, and represents tumour HLA expression and intratumoural inflammation. Aqueous humour from 108 UM patients was analysed for the presence of sHLA, using a Luminex assay specific for HLA Class I. Clinical and genetic parameters were compared between sHLA-positive and negative eyes. A qPCR analysis was performed on tumour tissue using a Fluidigm assay. In 19/108 UM-containing eyes, the sHLA level in the aqueous was above the detection limit. Tumours in sHLA-positive eyes were significantly larger, more frequently involved the ciliary body, and more often showed monosomy 3, gain of chromosome 8q and loss of BAP1 staining. Melanoma-related survival was worse in patients with sHLA-positive aqueous humour. sHLA in the aqueous did not represent the tumour’s HLA expression and did not relate to immune cell infiltration in the tumour. We conclude that UM-containing eyes may contain sHLA in the aqueous humour, where it is a prognostically-unfavourable sign and may influence local immune responses.

Hypoxia-inducible factor 1-alpha (HIF1a) and its regulator von Hippel–Lindau protein (VHL) play an important role in tumour ischemia. Currently, drugs that target HIF1a are being developed to treat malignancies. Although HIF1a is known to be expressed in uveal melanoma (UM), it is as yet unknown which factors, such as tumour size or genetics, determine its expression. Therefore, we aimed to determine which tumour characteristics relate to HIF1a expression in UM. Data from 64 patients who were enucleated for UM were analysed. Messenger RNA (mRNA) expression was determined with the Illumina HT-12 v4 chip. In 54 cases, the status of chromosomes 3 and 8q, and BRCA1-associated protein 1 (BAP1) protein expression (immunohistochemistry) were determined. Findings were corroborated using data of 80 patients from the Cancer Genome Atlas (TCGA) study. A significantly increased expression of HIF1a, and a decreased expression of VHL were associated with monosomy 3/loss of BAP1 expression. The relationship between BAP1 loss and HIF1a expression was independent of chromosome 3. The largest basal diameter and tumour thickness showed no relationship with HIF1a. HIF1a expression related to an increased presence of infiltrating T cells and macrophages. From this study, we conclude that HIF1a is strongly related to tumour genetics in UM, especially to loss of BAP1 expression, and less to tumour size. Tumour ischemia is furthermore related to the presence of an inflammatory phenotype.

Inflammation, characterized by high numbers of infiltrating leukocytes and a high HLA Class I expression, is associated with a bad prognosis in uveal melanoma (UM). We wondered whether mutations in GNA11 or GNAQ differentially affect inflammation and HLA expression, and thereby progression of the disease. We analyzed data of 59 primarily enucleated UM eyes. The type of GNAQ/11 mutation was analyzed using dPCR; chromosome aberrations were determined by Fluorescence in Situ Hybridization (FISH), karyotyping, and single nucleotide polymorphism (SNP) analysis, and mRNA expression by Illumina PCR. Comparing tumors with a GNAQ mutation with those with a GNA11 mutation yielded no significant differences in histopathological characteristics, infiltrate, or HLA expression. When comparing the Q209L mutations with Q209P mutations in tumors with monosomy of chromosome 3, a higher mitotic count was found in the Q209P/M3 tumors (p = 0.007). The Kaplan-Meier (KM) curves between the patients of the different groups were not significantly different. We conclude that the type (Q209P/Q209L) or location of the mutation (GNA11/GNAQ) do not have a significant effect on the immunological characteristics of the tumors, such as infiltrate and HLA Class I expression. Chromosome 3 status was the main determinant in explaining the difference in infiltrate and HLA expression.