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Purpose: A lack of representative human disease models has limited the translation of new and more effective treatments in uveal melanoma (UM), the most common primary adult intraocular malignancy. To fill this critical need, we developed and characterized a multicenter biobank of UM patient-derived organoids (PDOs)., Methods: UM patients requiring enucleation from 2019 to 2024 donated tumor tissue for PDO generation. PDOs were cultured in Cultrex and compared to donor primary tumor using exome sequencing, RNA sequencing, and immunohistochemistry. The ability of PDOs to maintain the transformed phenotype was evaluated in an orthotopic xenograft model and monitored with fundus imaging. ATAC sequencing and drug response assays were done in a subset of PDOs to explore the feasibility of their use for mechanistic and translational studies., Results: PDOs were successfully established in 40 of 44 cases (91%), retained clinically relevant mutations and molecular markers from the primary tumor, and displayed similar gene expression profiles and well-validated clinical prognostic markers of the disease. PDOs retained tumorigenic capacity in an in vivo model resembling human disease progression. Finally, we demonstrated that PDOs were a feasible platform to identify and evaluate novel therapeutic targets and investigate differential, personalized drug response., Conclusions: PDO models offer a new platform with improved representation of human UM to aid in translational research for this dismal condition.

Purpose: Unfortunately, treatment of patients with uveal melanoma (UM) with metastatic disease is limited. Twenty percent of patients with UM harbor a mutation in the splicing factor gene SF3B1, suggesting that aberrant spliceosome function plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome-compromised leukemic cells to these compounds., Methods: We studied the effect of the splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1- and BAP1-mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24 hours to different concentrations of E7107. Tumor slices were stained with hematoxylin and eosin (H&E) and incubated with BAP1, MelanA, MIB-1, and caspase-3 antisera., Results: The E7107-exposed UM cell lines exhibited decreased cell viability and increased apoptosis, with the greatest effect on SF3B1-mutated UM cells. A similar effect on UM tumor slices was observed upon exposure to E7107. Additionally, RNA was isolated for differential isoform expression analysis. No significant difference in isoform usage was found genome-wide. However, specific genes were differentially expressed after E7107 treatment in the SF3B1-mutated samples. Moreover, E7107 had the greatest effect on intron retention., Conclusions: This study indicates/suggests that mutated SF3B1 UM cells are more sensitive to the splicing inhibitor E7107 than wild-type SF3B1 UM cells.

Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy with a high probability of metastatic disease. Although excellent treatment options for primary UM are available, therapy for metastatic disease remain limited. Drug discovery studies using mouse models have thus far failed to provide therapeutic solutions, highlighting the need for novel models. Here, we optimize zebrafish xenografts as a potential model for drug discovery by showcasing the behavior of multiple cell lines and novel findings on mutation-dependent compound synergism/antagonism using Z-Tada; an algorithm to objectively characterize output measurements., Methods: Prognostic relevant primary (N = 4) and metastatic UM (N = 1) cell lines or healthy melanocytes (N = 2) were inoculated at three distinct inoculation sites. Standardized quantifications independent of inoculation site were obtained using Z-Tada; an algorithm to measure tumor burden and the number, size, and distance of disseminated tumor cells. Sequentially, we utilized this model to validate combinatorial synergism or antagonism seen in vitro., Results: Detailed analysis of 691 zebrafish xenografts demonstrated perivitelline space inoculation provided robust data with high probability of cell dissemination. Cell lines with more invasive behavior (SF3B1mut and BAP1mut) behaved most aggressive in this model. Combinatorial drug treatment illustrated synergism or antagonism is mutation-dependent, which were confirmed in vivo. Combinatorial treatment differed per xenograft-model, as it either inhibited overall tumor burden or cell dissemination., Conclusions: Perivitelline space inoculation provides robust zebrafish xenografts with the ability for high-throughput drug screening and robust data acquisition using Z-Tada. This model demonstrates that drug discovery for uveal melanoma must take mutational subclasses into account, especially in combinatorial treatment discoveries.

Purpose: Ocular melanoma is a common primary malignant ocular tumor in adults with limited effective treatments. Epigenetic regulation plays an important role in tumor development. The switching/sucrose nonfermentation (SWI/SNF) chromatin remodeling complex and bromodomain and extraterminal domain family proteins are epigenetic regulators involved in several cancers. We aimed to screen a candidate small molecule inhibitor targeting these regulators and investigate its effect and mechanism in ocular melanoma., Methods: We observed phenotypes caused by knockdown of the corresponding gene and synergistic effects with BRD inhibitor treatment and SWI/SNF complex knockdown. The effect of JQ-1 on ocular melanoma cell cycle and apoptosis was analyzed with flow cytometry. Via RNA sequencing, we also explored the mechanism of BRD4., Results: The best tumor inhibitory effect was observed for the BRD4 inhibitor (JQ-1), although there were no statistically obvious changes in the shBRD4 and shBRD9 groups. Interestingly, the inhibitory effect of JQ-1 was decrease in the shBRD4 group. JQ-1 inhibits the growth of melanoma in various cell lines and in tumor-bearing mice. We found 17 of these 28 common differentially expressed genes were downregulated after MEL270 and MEL290 cells treated with JQ-1. Four of these 17 genes, TP53I11, SH2D5, SEMA5A, and MDGA1, were positively correlated with BRD4. In TCGA database, low expression of TP53I11, SH2D5, SEMA5A, and MDGA1 improved the overall survival rate of patients. Furthermore, the disease-free survival rate was increased in the groups with low expression of TP53I11, SH2D5, and SEMA5A., Conclusions: JQ-1 may act downstream of BRD4 and suppress ocular melanoma growth by inducing G1 cell cycle arrest.

Purpose: To investigate the correlation between apparent diffusion coefficient (ADC) histograms and high-risk clinicopathologic features related to uveal melanoma (UM) prognosis., Methods: This retrospective study included 53 patients with UM who underwent diffusion-weighted imaging (DWI) between August 2015 and March 2024. Axial DWI was performed with a single-shot spin-echo echo-planar imaging sequence. ADC histogram parameters of ADCmean, ADC50%, interquartile range (IQR), skewness, kurtosis, and entropy were obtained from DWI. The relationships between histogram parameters and high-risk clinicopathological characteristics including tumor size, preoperative retinal detachment, histological subtypes, Ki-67 index, and chromosome status, were analyzed by Spearman correlation analysis, Mann-Whitney U test, or Kruskal-Wallis test., Results: A total of 53 patients (mean ± SD age, 55 ± 15 years; 22 men) were evaluated. The largest basal diameter (LBD) was correlated with kurtosis (r = 0.311, P = 0.024). Tumor prominence (TP) was correlated with entropy (r = 0.581, P < 0.001) and kurtosis (r = 0.273, P = 0.048). Additionally, significant correlations were identified between the Ki-67 index and ADCmean (r = -0.444, P = 0.005), ADC50% (r = -0.487, P = 0.002), and skewness (r = 0.394, P = 0.014). Finally, entropy was correlated with monosomy 3 (r = 0.541, P = 0.017)., Conclusions: The ADC histograms provided valuable insights into high-risk clinicopathologic features of UM and hold promise in the early prediction of UM prognosis.

Purpose: Cell lines are being used in preclinical uveal melanoma (UM) research. Because not all cell lines harbor typical GNAQ or GNA11 hotspot mutations, we aimed at better classifying them and determining whether we could find genetic causes to explain the protein and mRNA expression profiles of the cell lines., Methods: We studied protein and mRNA expression of 14 UM cell lines and determined the presence of single nucleotide variants and small insertions and deletions with next-generation sequencing and copy number alterations with a single nucleotide polymorphism array. The lists of differentially expressed proteins and genes were merged, and shared lists were created, keeping only terms with concordant mRNA and protein expression. Enrichment analyses were performed on the shared lists., Results: Cell lines Mel285 and Mel290 are separate from GNA-mutated cell lines and show downregulation of melanosome-related markers. Both lack typical UM mutations but each harbors four putatively deleterious variants in CTNNB1, PPP1R10, LIMCH1, and APC in Mel285 and ARID1A, PPP1R10, SPG11, and RNF43 in Mel290. The upregulated terms in Mel285 and Mel290 did not point to a convincing alternative origin. Mel285 shows loss of chromosomes 1p, 3p, partial 3q, 6, and partial 8p, whereas Mel290 shows loss of 1p and 6. Expression in the other 12 cell lines was related to BAP1 expression., Conclusions: Although Mel285 and Mel290 have copy number alterations that fit UM, multi-omics analyses show that they belong to a separate group compared to the other analyzed UM cell lines. Therefore, they may not be representative models to test potential therapeutic targets for UM.

Purpose: The purpose of this study was to analyze the extent of DNA breaks in primary uveal melanoma (UM) with regard to radiotherapy dose delivery (single-dose versus fractionated) and monosomy 3 status., Methods: A total of 54 patients with UM were included. Stereotactic radiotherapy (SRT) was performed in 23 patients, with 8 undergoing single-dose SRT (sdSRT) treatment and 15 receiving fractionated SRT (fSRT). DNA breaks in the enucleated or endoresected tumors were visualized by a TUNEL assay and quantified by measuring the TUNEL-positive area. Protein expression was analyzed by immunohistochemistry. Co-detection of chromosome 3 with proteins was performed by immuno-fluorescent in situ hybridization., Results: The amount of DNA breaks in the total irradiated group was increased by 2.7-fold (P < 0.001) compared to non-irradiated tissue. Tumors treated with fSRT were affected more severely, showing 2.1-fold more DNA damage (P = 0.007) compared to the cases after single (high) dose irradiation (sdSRT). Monosomy 3 tumors showed less DNA breaks compared to disomy 3 samples (P = 0.004). The presence of metastases after radiotherapy correlated with monosomy 3 and less DNA breaks compared to patients with non-metastatic cancer in the combined group with fSRT and sdSRT (P < 0.05)., Conclusions: Fractionated irradiation led to more DNA damage than single-dose treatment in primary UM. As tumors with monosomy 3 showed less DNA breaks than those with disomy 3, this may indicate that they are less radiosensitive, which may influence the efficacy of irradiation.

Purpose: To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group., Methods: Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates., Results: The mean age at diagnosis was 17 years (range 5.0-24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018)., Conclusions: We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).

Purpose: Uveal melanoma (UM) is a rare disease with a high mortality, and new therapeutic options are being investigated. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, expressed in the testis, but also in cancers, including uveal melanoma. PRAME is considered a target for immune therapy in several cancers, and PRAME-specific T cell clones have been shown to kill UM cells., Methods: We studied the literature on PRAME expression in hematological and solid malignancies, including UM, and its role as a target for immunotherapy. The distribution of tumor features was compared between PRAME-high and PRAME-low UM in a 64-patient cohort from the Leiden University Medical Center (LUMC) and in the Cancer Genome Atlas (TCGA) cohort of 80 cases and differential gene expression analysis was performed in the LUMC cohort., Results: PRAME is expressed in many malignancies, it is frequently associated with a negative prognosis, and can be the target of T cell receptor (TCR)-transduced T cells, a promising treatment option with high avidity and safety. In UM, PRAME is expressed in 26% to 45% of cases and is correlated with a worse prognosis. In the LUMC and the TCGA cohorts, high PRAME expression was associated with larger diameter, higher Tumor-Node-Metastasis (TNM) stage, more frequent gain of chromosome 8q, and an inflammatory phenotype., Conclusions: We confirm that PRAME is associated with poor prognosis in UM and has a strong connection with extra copies of 8q. We show that PRAME-specific immunotherapy in an adjuvant setting is promising in treatment of malignancies, including UM.

Purpose: The purpose of this study was to profile protein expression liquid vitreous biopsies from patients with uveal melanoma (UM) using mass spectrometry to identify prognostic biomarkers, signaling pathways, and therapeutic targets., Methods: Vitreous biopsies were collected from two cohorts in a pilot study: comparative control eyes with epiretinal membranes (ERM; n = 3) and test eyes with UM (n = 8). Samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identified proteins were compared to data from a targeted multiplex ELISA proteomics platform., Results: A total of 69 significantly elevated proteins were detected in the UM vitreous, including LYVE-1. LC-MS/MS identified 62 significantly upregulated proteins in UM vitreous that were not previously identified by ELISA. Analysis of differential protein expression by tumor molecular classification (gene expression profiling [GEP] and preferentially expressed antigen in melanoma [PRAME]) further identified proteins that correlated with these classifications. Patients with high-risk GEP tumors displayed elevated vitreous expression of HGFR (fold-change [FC] = 2.66E + 03, P value = 0.003) and PYGL (FC = 1.02E + 04, P = 1.72E-08). Patients with PRAME positive tumors displayed elevated vitreous expression of ENPP-2 (FC = 3.21, P = 0.04), NEO1 (FC = 2.65E + 03, P = 0.002), and LRP1 (FC = 5.59E + 02, P value = 0.01). IGF regulatory effectors were highly represented (P value = 1.74E-16). Cross-platform analysis validated seven proteins identified by ELISA and LC-MS/MS., Conclusions: Proteomic analysis of liquid biopsies may provide prognostic information supporting gene expression of tumor biopsies. The use of multiple protein detection platforms in the same patient samples increases the sensitivity of candidate biomarker detection and allows for precise characterization of the vitreous proteome.

Purpose: The purpose of this study was to investigate the clinical features, tumor characteristics, including histopathology and cytogenetic analysis, and management of patients with uveal ring melanoma in New Zealand., Methods: A retrospective review was conducted on all uveal melanoma cases treated in a single national oncology center in New Zealand. The study period was from January 1, 2013 to December 31, 2022 (10 years). Written consent was obtained from all patients included in this case series., Results: Uveal ring melanoma of ciliary body origin (n = 4) comprised 0.7% of all uveal melanomas (n = 571). Ethnicity distribution was three patients of New Zealand European ancestry and one patient of Chinese/Pasifika ancestry. Three patients (75%) were symptomatic at presentation (spontaneous hyphema, glaucoma, and cataract), whereas one was asymptomatic but subsequently developed painful refractory glaucoma. All eyes underwent enucleation. Three eyes had primary iris biopsies with subsequent enucleation for refractory glaucoma and pain and one eye underwent primary enucleation. All cases demonstrated malignant tumor characteristics including diffuse 270 to 360-degree ciliary body ring growth pattern, epithelioid-cell type and presence of either BAP-1 expression loss or gain of MYC gene. Two cases (50%) developed distant organ metastasis - liver, parotid gland, and breast. Of those, one patient was deceased at the time of follow-up whereas one had completed treatment for metastases., Conclusions: Uveal ring melanoma is a rare tumor that is highly invasive and malignant. When detected, prompt definitive treatment should be advocated, and patient counselling should be given regarding the high risk of developing a painful, blind eye with increased risk of metastasis in the absence of treatment.

Purpose: Uveal melanoma (UM) is a tumor of the eye that metastasizes in approximately half of cases. Prognostic testing requires accessibility to tumor tissue, which is usually not available with eye-preserving therapies. Noninvasive approaches to prognostic testing that provide valuable information for patient care are therefore needed. The aim of this study was to evaluate the use of circulating cell-free plasma DNA analysis in UM patients undergoing brachytherapy., Methods: The study recruited 26 uveal melanoma patients referred to the department between February and October 2020. Blood samples were collected at various time points before, during, and after treatment, and deep amplicon sequencing was used to identify oncogenic variant alleles of the GNAQ and GNA11 genes, which serve as indicators for the presence of circulating tumor DNA (ctDNA)., Results: The results showed that all patients were ctDNA negative before brachytherapy. In 31% of patients, ctDNA was detected during therapy. The variant allele fraction of GNAQ or GNA11 alleles in ctDNA positive samples ranged from 0.24% to 2% and correlates with the largest basal diameter and thickness of the tumor., Conclusions: The findings suggest that brachytherapy increases the presence of tumor DNA in the plasma of UM patients. Thus ctDNA analysis may offer a noninvasive approach for prognostic testing. However, efforts are still required to lower the limit of detection for tumor-specific genetic alterations.

Purpose: The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity., Methods: We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC50) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq., Results: Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm2) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines., Conclusions: AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC50 and higher CRT exposure.

Purpose: Choroidal melanoma (CM) and ciliary body melanoma (CBM) are the two most common subtypes of uveal melanoma. Starting from the observation that CBM tends to have a higher metastatic potential than CM, we hypothesized that specific cytogenetic abnormalities could be associated with tumor location - reflecting distinct genetic signatures that would drive the risk of distant spread., Methods: Chromosomal alterations were investigated by molecular cytogenetic techniques in 217 and 97 patients with CM and CBM, respectively. Cox proportional hazards regression analysis was used to identify the independent predictors of distant metastasis., Results: Patients with CBM had larger tumor sizes (P < 0.001), higher disease stages (P < 0.001), and more frequently showed distant metastasis (P = 0.002) than those with CM. On analyzing the entire study cohort, we found that specific chromosomal alterations - including chromosome 8p loss (P < 0.001), 1p loss (P < 0.001), and monosomy 3 (P < 0.005) - were independent predictors of distant metastasis. Based on a decision-tree learning algorithm, we identified three specific subgroups of patients with uveal melanoma at high risk of distant spread. Monosomy 3 occurred significantly more frequently in patients with T3 CBM tumors., Conclusions: Specific cytogenetic abnormalities - including chromosome 8p loss, 1p loss, and monosomy 3 - are independent risk factors for distant metastasis in uveal melanoma. Larger tumor size at presentation and monosomy 3 contribute to a higher metastatic risk in patients with CBM.

Purpose: To investigate the presence of plasma circulating tumor DNA (ctDNA) in patients with uveal melanoma during and after primary tumor treatment., Methods: Detectability and variant allele frequency of ctDNA were assessed using a 129-oncogene panel using next-generation deep sequencing and hybridization capture in 69 patients with uveal melanoma undergoing primary treatment with enucleation (n = 8, during surgery) or plaque brachytherapy (n = 61; postoperative day 0, 1, 2, or 3). Follow-up assessments were performed in 39 patients over a median of 21 months (range, 3.2-31.9 months) of follow-up. Correlations between genomic data and disease parameters were performed., Results: Overall, ctDNA was detectable in 20 of 69 patients with uveal melanoma (28.9%) during the perioperative period. On the day of enucleation, ctDNA was detected in two of eight patients (25%). In patients undergoing brachytherapy, ctDNA was significantly more detectable on postoperative days 2 or 3 compared with postoperative day 0 or 1 (32.4% vs 0.0%; P = 0.0015). Patients with follow-up ctDNA that became detectable or had an increased variant allele frequency were significantly more likely to develop metastasis compared with patients with follow-up ctDNA that became undetectable or decreased variant allele frequency (P = 0.04). In patients with detectable vs. undetectable ctDNA, there was no significant difference in tumor size, stage or location., Conclusions: ctDNA is significantly more detectable at 48 to 72 hours after plaque brachytherapy compared with less than 48 hours. ctDNA can be detected during enucleation. Relative increases in ctDNA levels may herald the development of clinically apparent metastases.

Purpose: Over 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma., Methods: A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot., Results: AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells., Conclusions: Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

Purpose: The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is related to the tumor's genetic background., Methods: Characteristics of 144 patients with enucleated UM were analyzed together with mRNA expression levels of YAP/TAZ-related genes (80 patients from the The Cancer Genome Atlas [TCGA] project and 64 patients from Leiden, The Netherlands). VP was administered to cell lines 92.1, OMM1, Mel270, XMP46, and MM28 (UM), CRMM1 and CRMM2 (conjunctival melanoma), and OCM3 (cutaneous melanoma). Viability, growth speed, and expression of YAP1-related proteins were assessed., Results: In TCGA data, high expression of YAP1 and WWTR1 correlated with the presence of monosomy 3 (P = 0.009 and P < 0.001, respectively) and BAP1-loss (P = 0.003 and P = 0.001, respectively) in the primary UM; metastasis development correlated with higher expression of YAP1 (P = 0.05) and WWTR1 (P = 0.003). In Leiden data, downstream transcription factor TEAD4 was increased in cases with M3/BAP1-loss (P = 0.002 and P = 0.006) and related to metastasis (P = 0.004). UM cell lines 92.1, OMM1, and Mel270 (GNAQ/11-mutation, BAP1-positive) and the fast-growing cell line OCM3 (BRAF-mutation) showed decreased proliferation after exposure to VP. Two slow-growing UM cell lines XMP46 and MM28 (GNAQ/11-mutation, BAP1-negative) were not sensitive to VP, and neither were the two conjunctival melanoma cell lines (BRAF/NRAS-mutation)., Conclusions: High risk UM showed an increased expression of YAP/TAZ-related genes. Although most UM cell lines responded in vitro to VP, BAP1-negative and conjunctival melanoma cell lines did not. Not only the mutational background, but also cell growth rate is an important predictor of response to YAP/TAZ inhibition by VP.

Purpose: Adiponectin is an insulin-sensitizing and anticarcinogenic hormone that is encoded by a gene on chromosome 3. Here, we analyzed the expression of adiponectin and its receptor Adipor1 in primary uveal melanoma (UM) with regard to the monosomy-3 status and clinical factors, as well as the physiological response of UM cells to adiponectin., Methods: Immunohistochemistry was performed on the primary UM of 34 patients. Circulating melanoma cells (CMC) were isolated by immunomagnetic enrichment. Monosomy-3 was evaluated by Immuno-FISH. Gene expression was analyzed using the RNAseq data of The Cancer Genome Atlas study. Cultures of choroidal melanocytes and UM were established from the samples of two patients. The proliferative potential of the UM cell lines Mel-270 and OMM-2.5 was determined by immunocytochemistry, immunoblotting, cell cycle analysis, nucleolar staining, and adenosine triphosphate (ATP) levels., Results: UM with monosomy-3 exhibited a lower immunoreactivity for adiponectin and Adipor1, which was associated with monosomy-3-positive CMC and the development of extraocular growth or metastases. Both proteins were more abundant in the irradiated tumors and present in the cultured cells. Gene expression profile indicated the impairment of adiponectin-mediated signaling in the monosomy-3 tumors. Adiponectin induced a significant decline in the ATP levels, Ki-67 expression, cells in the G2/M phase, and nucleolar integrity in UM cultures., Conclusions: Adiponectin deficiency appears to enhance the metastatic potential of the UM cells with monosomy-3 and the termination of tumor dormancy. Counteracting insulin resistance and improving the serum adiponectin levels might therefore be a valuable approach to prevent or delay the UM metastases.

Purpose: A subgroup of uveal melanoma (UM) gives rise to metastases at a late stage. Our objective was to identify patient and tumor characteristics that are associated with UM-related death in patients who survived 5 years following enucleation., Methods: A retrospective analysis was performed in 583 primary UM cases, enucleated at the Leiden University Medical Center between 1983 and 2013. Univariable and multivariable Cox regression analyses were performed in the total cohort and separately in those surviving more than 5 years (n = 297)., Results: In the total cohort, the median age was 62.6 years, and the median tumor diameter was 12.0 mm. Monosomy 3 was detected in 53% of cases and gain of 8q in 47%. In the cohort surviving 5 years, the median age was 59.5 years, and the median tumor diameter was 11.0 mm. Monosomy 3 and gain of 8q were detected in 33% and 31% of cases, respectively. In the total cohort, male gender (P = 0.03), tumor diameter (P < 0.001), mitotic count (P < 0.001), extravascular matrix loops (P = 0.03), extraocular growth (P < 0.001), and gain of 8q (P < 0.001) were independently associated with UM-related death. In patients surviving 5 years after enucleation, univariable analysis revealed that age (P = 0.03), tumor diameter (P < 0.001), monosomy 3 (P = 0.04), and 8q gain (P = 0.003) were associated with subsequent UM-related death. Using a multivariable analysis, only male gender (P = 0.03) and gain of 8q (P = 0.01) remained significant., Conclusions: Predictors of UM-related death change over time. Among UM patients who survived the initial 5 years following enucleation, male gender and chromosome 8q status were the remaining factors related to UM-related death later on.

Purpose: MicroRNA-34a (miR-34a) has been implicated in many biological processes. It is downregulated in uveal melanoma, and introduction of miR-34a inhibits the proliferation and migration of uveal melanoma cells. Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) is a novel target of miR-34a identified first in retinal pigment epithelial cells. In this study, we sought to evaluate the interaction of miR-34a and LGR4 in uveal melanoma and its downstream mechanisms., Methods: The expression of LGR4, epithelial-mesenchymal transition (EMT)-associated factors, and matrix metalloproteinase 2 (MMP2) in uveal melanoma cells was assessed by immunoblotting and immunofluorescence analysis. MicroRNA-34a mimic molecules, LGR4 small interfering RNA (siRNA), or MMP2-specific siRNA were transiently transfected into uveal melanoma cells. In vitro scratch and Transwell assays were used to evaluate the migratory and invasive potential of the resultant uveal melanoma cells., Results: LGR4 is upregulated in uveal melanoma cells. Introduction of miR-34a significantly decreased the expression level of LGR4. Transfection with miR-34a or knockdown of LGR4 attenuated the aggressiveness of uveal melanoma cells. In addition, there was a decrease in the expression of mesenchymal markers N-cadherin, vimentin, and Snail following miR-34a introduction or knockdown of LGR4. Finally, MMP2 was found to be a downstream effector for miR-34a and LGR4 that regulates the migration and invasion of uveal melanoma cells., Conclusions: MicroRNA-34a negatively controls LGR4, thereby inhibiting the migration and invasion of uveal melanoma cells. Ultimately, both miR-34a and LGR4 impact the aggressiveness of uveal melanoma with alterations in the markers of the EMT. MMP2 is a downstream effector that influences the metastasis seen with uveal melanoma cells.

Purpose: Uveal melanoma (UM) is characterized by multiple chromosomal rearrangements and recurrent mutated genes. The aim of this study was to investigate if copy number variations (CNV) alone and in combination with other genetic and clinico-histopathological variables can be used to stratify for disease-free survival (DFS) in enucleated patients with UM., Methods: We analyzed single nucleotide polymorphisms (SNP) array data of primary tumors and other clinical variables of 214 UM patients from the Rotterdam Ocular Melanoma Study (ROMS) cohort. Nonweighted hierarchical clustering of SNP array data was used to identify molecular subclasses with distinct CNV patterns. The subclasses associate with mutational status of BAP1, SF3B1, or EIF1AX. Cox proportional hazard models were then used to study the predictive performance of SNP array cluster-, mutation-, and clinico-histopathological data, and their combination for study endpoint risk., Results: Five clusters with distinct CNV patterns and concomitant mutations in BAP1, SF3B1, or EIF1AX were identified. The sample's cluster allocation contributed significantly to mutational status of samples in predicting the incidence of metastasis during a median of 45.6 (interquartile range [IQR]: 24.7-81.8) months of follow-up (P < 0.05) and vice versa. Furthermore, incorporating all data sources in one model yielded a 0.797 C-score during 100 months of follow-up., Conclusions: UM has distinct CNV patterns that correspond to different mutated driver genes. Incorporating clinico-histopathological, cluster and mutation data in the analysis results in good performance for UM-related DFS prediction.

Purpose: The activation of the mitogen-activated protein kinase (MAPK) pathway has been suggested as the major downstream target when GNAQ and GNA11 (GNAQ/11) are mutated in uveal melanoma (UM). However, clinical trials with single agent MEK inhibitor showed no clinical significance in altering the overall outcome of the disease in UM; therefore, we investigated the correlation between naturally occurring mutations in GNAQ/11 and activation of MAPK pathway in vivo in primary UM., Methods: Screening for activating mutations in codons 183 and 209 of GNAQ/11 was carried out by sequencing and restriction fragment length polymorphism (RFLP) in a cohort of 42 primary UM. Activation of the MAPK pathway and other potential downstream signals was assessed by immunohistochemistry and/or Western blot analysis. Potential downstream signaling of mutant and wild type GNAQ/11 was studied by transient transfection assay in nonmutant cell lines., Results: Somatic mutations in GNAQ/11 were observed in 35/42 (83.3%) of primary UM. Tumors with GNAQ/11 mutations showed variations in the activation of ERK1/2 with significant tumor heterogeneity. Weak and undetectable ERK1/2 activation was observed in 4/35 (11.4%) and 8/35 (22.9%) of the GNAQ/11 mutant UM, respectively. Tumor heterogeneity of GNAQ/11 mutations was also observed in a subset of tumors., Conclusions: Our results indicate that there is marked variation in MAPK activation in UM with GNAQ/11 mutations. Thus, GNAQ/11 mutational status is not a sufficient biomarker to adequately predict UM patient responses to single-agent selective MEK inhibitor therapy.

Purpose: Cancer stem cells (CSCs) are a subpopulation of cells with the capacity to drive tumor growth. While there is evidence of the existence of CSCs in uveal melanoma (UM), there is no consensus on their defining markers. In this study, we examined putative CSC markers in UM cell lines, primary UM (PUM), and normal choroidal melanocytes (NCM)., Methods: Nonadherent sphere assays were used to assess the tumorigenic potential of 15 PUMs, 8 high (M3) and 7 low (D3) metastatic risk. Flow cytometry was used to compare the expression of CSC markers between 10 PUMs and 4 NCMs, as well as in 8 UM cell lines grown under adherent and nonadherent conditions. Based on the data generated and from TCGA analyses, CD166 was investigated in detail, including its effect on cell migration using a tumor transendothelial migration assay., Results: M3 PUM had a greater melanosphere-forming efficiency than D3 PUM. CD166 and Nestin expression was upregulated in PUM compared to NCM by flow cytometry. UM cell lines resistant to anoikis had increased levels of CD271, Nestin, and CD166 compared with adherent cells. TCGA analysis showed that patients with higher CD166 expression had a poorer prognosis: this was supported by a Mel270 CD166high subpopulation that had enhanced migratory capabilities compared with CD166low cells. IHC showed that CD166 is expressed in the cytoplasm and cell membrane of PUM cells., Conclusions: UM contain a population of cells with characteristics of CSCs. In particular, CD166high UM cells appear to represent a subpopulation with enhanced migratory capacity.

Purpose: Uveal melanoma (UM) is uniformly refractory to all available systemic chemotherapies, thus creating an urgent need for novel therapeutics. In this study, we investigated the sensitivity of UM cells to ICG-001, a small molecule reported to suppress the Wnt/β-catenin-mediated transcriptional program., Methods: We used a panel of UM cell lines to examine the effects of ICG-001 on cellular proliferation, migration, and gene expression. In vivo efficacy of ICG-001 was evaluated in a UM xenograft model., Results: ICG-001 exerted strong antiproliferative activity against UM cells, leading to cell cycle arrest, apoptosis, and inhibition of migration. Global gene expression profiling revealed strong suppression of genes associated with cell cycle proliferation, DNA replication, and G1/S transition. Gene set enrichment analysis revealed that ICG-001 suppressed Wnt, mTOR, and MAPK signaling. Strikingly, ICG-001 suppressed the expression of genes associated with UM aggressiveness, including CDH1, CITED1, EMP1, EMP3, SDCBP, and SPARC. Notably, the transcriptomic footprint of ICG-001, when applied to a UM patient dataset, was associated with better clinical outcome. Lastly, ICG-001 exerted anticancer activity against a UM tumor xenograft in mice., Conclusions: Using in vitro and in vivo experiments, we demonstrate that ICG-001 has strong anticancer activity against UM cells and suppresses transcriptional programs critical for the cancer cell. Our results suggest that ICG-001 holds promise and should be examined further as a novel therapeutic agent for UM.

Purpose: Adenocarcinomas or adenomas derived from pigmented ciliary epithelium (APCE) are exceptionally rare ocular tumors. These tumors have pigmented and epithelioid features, and some APCEs are negative for keratin markers and positive for melanocytic markers. It is especially difficult to distinguish APCEs from uveal melanoma (UM). Accordingly, we examined protein expression and genetic mutations associated with APCE to facilitate diagnosis., Methods: Five APCE and 11 UM samples were obtained from patients during surgical resection at our institute. APCE and UM ocular structures were compared comprehensively. Protein expression and genetic alterations involved in malignant melanoma were evaluated., Results: SOX10 was expressed diffusely in all 11 UMs and in surrounding uveal or choroidal melanocytes, but not in the APCEs or nontumorous pigmented epithelia. Additionally, the expression patterns of cytokeratins and melanocytic markers differed between UMs and APCEs. We identified BRAF V600E mutations in four of five APCE samples, but not in the 11 UM samples. Moreover, GNAQ or GNA11 mutations were found in 10 of the 11 UM samples, but not in APCE samples. NRAS mutations were not observed in either tumor group examined., Conclusions: APCE is a separate entity distinguished from UM by the absence of SOX10 expression and presence of the BRAF V600E mutation. These results have implications for diagnosis, providing a means to distinguish between UM and APCE.

Purpose: The American Joint Committee on Cancer (AJCC) staging system has been validated for use as a prognostic parameter in uveal melanoma (UM). We studied whether adding information regarding chromosome 3 and 8q status further enhances the prognostic value of this staging system., Methods: We retrospectively studied a cohort of 522 patients who had been treated for UM in two different centers between 1999 and 2015. The mean follow-up time was 47.7 months. Cumulative incidence curves were generated and regression analyses were performed for different combinations of AJCC staging and chromosome status. Death due to UM metastases was the primary endpoint., Results: In AJCC stage I cases, only patients with monosomy 3 as well as chromosome 8q gain died due to UM metastases (P < 0.001). Among patients with stage II and III tumors, those with monosomy 3 plus gain of chromosome 8q had the worst prognosis, whereas the clinical outcome of those with only one of these aberrations was intermediate (P < 0.001). Patients without monosomy 3 and 8q gain showed favorable prognosis, independent of their tumor's AJCC stage. In cases with monosomy 3, 8q gain, or both, adding AJCC stage improved the predictive value. Multivariable regression analyses demonstrated that AJCC staging and chromosome 3 and 8q status contain independent information about survival status., Conclusions: Combining information on AJCC staging and chromosome 3 and 8q status allows a more accurate prognostication in UM. We conclude that the prognostic value of the AJCC staging system can be improved by adding information regarding chromosome 3 and 8q status.

Purpose: Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors., Methods: Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice., Results: Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice., Conclusions: Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.

Purpose: Most of the uvea melanoma (UM) display a near-diploid (normal, -2N) karyotype with only a few chromosomal changes. In contrast to these simple aberrations 18% of the UM samples show a polyploid character (>2N) and this was associated with an unfavorable prognosis. This study attempts to gain insight in the prognostic value of polyploidy in UM., Methods: In 202 patients the ploidy status of the UM was determined using cytogenetic analysis, fluorescence-in-situ-hybridization (FISH), multiplex ligation dependent probe amplification (MLPA), and/or single nucleotide polymorphism (SNP) array analysis. Immunohistochemistry was used to determine the BAP1 expression and mutation analyses of BAP1 (coding regions) and the mutation hotspots for the SF3B1, EIF1AX, GNAQ, and GNA11 genes was carried out using Sanger sequencing or whole-exome sequencing., Results: Twenty-three patients had a polyploid UM karyotype (11.4%). Patients with a polyploid tumor had larger tumors (15.61 vs. 13.13 mm, P = 0.004), and more often loss of heterozygosity of chromosome 3 (P = 0.003). No difference in occurrence of mutations between polyploid and diploid tumors was observed for BAP1, SF3B1, EIF1AX, GNAQ, and GNA11. Polyploidy did not affect survival (P = 0.143). BAP1 deficiency was the only significant independent prognostic predictor for patients with polyploid tumors, with a 16-fold increased hazard ratio (HR 15.90, P = 0.009)., Conclusions: The prevalence of mutations in the UM related genes is not different in polyploid UM compared with diploid UM. Moreover, similar to patients with diploid UM, BAP1 mutation is the most significant prognostic predictor of metastasis in patients with polyploid UM.

Purpose: The purpose of this study is to study the genomic evolution of primary uveal melanoma., Methods: Primary uveal melanoma genomic DNA was assayed on the Illumina Human660W-Quad v1.0 DNA Analysis BeadChip. Raw signal intensity data were quantile normalized to estimate copy number aberration with the Genome Alteration Print algorithm. Distance between samples was calculated as the Manhattan distance between the copy number profiles of the tumors. From the distance matrix, a phylogenetic network (evolutionary relationship inference) was estimated using SplitsTree4., Results: Of the 57 tumors, one (1.8%) was discarded because of a failed assay, and seven (12.3%) were revealed to be mixtures of several cell populations that could not be resolved. Three clades of tumor were identified (A [59.2%], B [32.7%], and C [6.1%]), each following a distinct evolutionary path and each associated with metastatic status (P = 0.01). One tumor (2.0%) did not fit into any clade. From a normal diploid melanocyte, a few tumors (clade C) lose a large portion of chromosome 6q, but do not develop any mutations on 8q. In an alternate path, the vast majority of tumors (clade A and clade B [91.9%]) gain a copy of the telomeric half of 8q. A majority of these tumors (clade A) subsequently lose a copy of chromosome 3, as well as gain the centromeric half of 8q. The other tumors (clade B) gain copies of 6p, as well as regions on 11p and 22q., Conclusions: Our data suggest that there is little overlap in the subtypes of uveal melanoma after divergence (identified as clades A and B) and that these distinct subtypes are not likely to crossover or transform from one major clade to another.

Purpose: To compare the status of chromosomes 3 and 8 in 25-gauge transvitreal retinochoroidal (TVRC) biopsy specimens and enucleated eyes in order to evaluate for genetic heterogeneity and the utility of TVRC biopsy to obtain an adequate sampling of the tumor., Methods: Genetic heterogeneity was evaluated in 27 patients treated at Rigshospitalet between 2009 and 2013. The TVRC biopsy was performed to confirm diagnosis prior to enucleation and was subsequently analyzed using two techniques for chromosomes 1p, 3, 6, and 8: Fluorescence in situ hybridization (FISH) in all patients, and multiplex ligation-dependent probe amplification (MLPA) in 16 patients. Biopsies were compared with histological sections from matched enucleated eyes, which were microdissected following a hexagonal grid and analyzed with MLPA., Results: Twenty-four tumors were available for analysis. The TVRC biopsy identified chromosome 3 aberrations with MLPA in all cases (sensitivity = 100%), while FISH missed two cases (sensitivity = 89%). Conversely, FISH analysis demonstrated polyploidy of chromosome 3 in three additional cases missed by MLPA. Chromosome 8 aberrations were detected in 75% of cases with MLPA and 68% of cases with FISH. Heterogeneity of chromosomes 3 and 8 was shown in 3 (13%) and 11 tumors (46%), respectively, with an increased frequency of genetic aberrations toward the base of the tumor (P = 0.049). The study showed no difference in tumor size between heterogeneous and homogenous melanomas (P = 0.82)., Conclusions: Regardless of genetic heterogeneity, the TVRC biopsy identified all patients with a high risk of developing metastatic disease when a combination of chromosome 3 and 8 status was assessed.

Purpose: The purpose of this study was to determine whether radiation treatment induces chromosomal aberrations in uveal melanoma (UM) and to evaluate which tumor features determine success of karyotyping and FISH., Methods: Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH for chromosome 3 was performed in 248 samples. Thirty-six UMs had previously undergone irradiation. Karyotypes were analyzed, and the success rate of karyotyping/FISH was evaluated and compared with clinicopathologic tumor characteristics and prior irradiation., Results: Aberrations were observed in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16, and Y being altered in at least 15% of the tumors. Aberrations were more common and more complex in previously irradiated tumors (significant for chromosomes 5 [P = 0.004] and 13 [P = 0.04]). Karyotyping and FISH failed significantly more often in irradiated tumors (both P < 0.001). In nonirradiated cases, successful karyotyping was related to a large tumor prominence (P = 0.004) and a high mitotic count (P = 0.007). The success of FISH in these tumors was not associated with any of the studied parameters. In irradiated tumors, karyotyping succeeded more frequently in cases with a high mitotic count (P = 0.03), whereas FISH was more often successful in tumors with a high mitotic count (P = 0.001), a large diameter (P = 0.009) and large prominence (P = 0.008)., Conclusions: Karyotyping and FISH are more often successful in UMs with features characteristic of high tumor aggressiveness, whereas prior irradiation leads to multiple chromosome aberrations and to unsuccessful tests. It will be interesting to determine whether other techniques can provide reliable information on the chromosome status of previously irradiated UMs.

Purpose: Malignant melanomas of the ocular surface (conjunctival melanoma [CM]) and within the eye (uveal melanoma [UM]) show different types of metastatic behavior. While CM has a propensity to spread first to regional lymph nodes, UM metastasizes almost exclusively via the hematogenic route to the liver. We investigated whether these different metastatic patterns might be attributable to differential hem- and lymphangiogenic characteristics of CM and UM cells., Methods: Human CM (CM2005.1, CRMM1, CRMM2) and UM (Mel270, Mel290, OM431) cell lines were analyzed for VEGF-A, -C, and -D expression by RT-PCR and ELISA. The influence of CM- or UM-conditioned medium on blood (BEC) and lymphatic (LEC) endothelial cell proliferation and migration was measured using 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) and scratch assays, respectively., Results: Vascular endothelial growth factor-A, -C and -D mRNA, and VEGF-A and -D protein were expressed by all CM and UM cell lines, while VEGF-C protein was only expressed by UM cell lines. The CM- and UM-conditioned medium did neither differentially affect BEC (P = 0.86) and LEC (P = 0.90) proliferation, nor BEC (P = 0.56) and LEC (P = 0.90) migration., Conclusions: Conjunctival melanoma cell lines did not show a higher prolymphangiogenic potential, and UM cell lines did not show a higher prohemangiogenic potential. Accordingly, other mechanisms within the tumor microenvironment might account for the diverging metastatic patterns of conjunctival versus uveal melanomas.

Purpose: Monosomy 3 (M3) and abnormalities of chromosome 8 associate with poor prognosis in uveal melanomas (UM). Although M3 has been the subject of more in-depth studies, none have intensively focused on chromosome 8. To elucidate the potential role of chromosome 8 abnormalities, array comparative genomic hybridization (aCGH) was performed on primary UM., Methods: A specifically-designed custom high-resolution array was developed focusing on changes most implicated in UM. Probes for chromosome 8 had a mean spacing of 2.3 kb while chromosomes infrequently affected had a mean spacing of 36.6 kb. A series of 75 UM, including one formalin-fixed paraffin sample were analyzed, and where possible control DNA extracted from the patient's own peripheral blood was used., Results: The most common copy number abnormalities were chromosome 8 (75%) and M3 (51%), with M3 and gain of the long arm of chromosome 8 (8q+) associated in 41% of cases. Also identified were partial deletions of chromosome 3 (3%) and regional 8q+ (23%), and the intensive coverage of chromosome 8 revealed small focal deletions and amplifications affecting both arms. The most significant predictor of prognosis was M3/8q+ having a hazard ratio of 10.1 (P < 0.0001)., Conclusions: Neither 8p deletion nor focal changes affecting chromosome 8 were linked to outcome. The most significant indicator was M3/8q, and multiple 8q+ associated with shorter survival. Studying UM with this technology provides a powerful robust tool for predicting prognosis while considering other genetic changes, allowing the future incorporation of such data as it becomes clinically significant.

Purpose: Monosomy 3 (M3) or the presence of a specific RNA expression profile, known as class 2, is strongly associated with death from uveal melanoma (UM). Given the important role of epigenetic processes in cancer development and progression, we compared the transcriptional profiles of a selection of epigenetic regulators between primary UM with a good and a bad prognosis., Methods: Transcriptional levels of 59 epigenetic regulator genes were measured by quantitative PCR (qPCR) in 20 UM, 12 with monosomy of chromosome 3 (M3) and 8 with disomy of chromosome 3 (D3). Validation was performed in an independent cohort. Expression levels were compared to clinicopathological characteristics, including class type. Bisulfite sequencing was used to evaluate the role of DNA methylation in gene silencing., Results: In the first set of tumors, general downregulation of transcription of the genes encoding epigenetic regulatory enzymes was seen in association with M3. The 10 genes with the highest differential expression between M3 and D3 were selected and were analyzed in a second set of tumors. In the validation set, significantly lower levels of KAT2B (P = 0.008), HDAC11 (P = 0.009), KMT1C (P = 0.05), KDM4B (P = 0.003), KDM6B (P = 0.04), and BMI-1 (P = 0.001) transcripts were found in tumors with M3/class 2. Methylation of C-phosphate-G (CpG) residues was not observed on the putative regulatory regions of KAT2B, KDM4B, or KDM6B., Conclusions: Expression levels of a number of histone-modifying genes and polycomb family members are significantly lower in uveal melanoma with monosomy 3/class 2, supporting a general dysregulation of epigenetic modifiers in UM with a bad prognosis., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression., Methods: TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi., Results: Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%)., Conclusions: We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Somatic mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1 have been identified in uveal melanoma (UM). The aim of this study was to determine whether mutations in these genes in primary tumors were associated with metastases in individuals diagnosed with UM., Methods: A total of 63 UM cases who developed a metastasis within 48 months of primary treatment and 53 UM controls who were metastasis-free over a similar time period were selected for the study. Primary UM cases were screened for mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1. The association of these mutations with tumor characteristics, chromosome 3 copy number, and metastatic status was analyzed by logistic regression to estimate the odds of developing metastasis within 48 months., Results: As expected, tumor diameter, thickness, cilio-choroidal location, and chromosome 3 monosomy were all significantly (P < 0.02) associated with the presence of metastasis. In univariate analysis, GNA11 (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.1-5.5) and BAP1 (OR 6.3, 95% CI 2.7-14.4) mutations were positively associated and EIF1AX mutation (OR 0.13, 95% CI 0.034-0.47) was inversely associated with metastatic status at 48 months after UM treatment. After adjustment for covariates, a chromosome 3 monosomy/BAP1-mutation/EIF1AX-wild-type (WT) mutation profile was strongly associated (OR 37.5, 95% CI 4.3-414) with the presence of metastasis compared with a chromosome 3 disomy/BAP1-WT/EIF1AX mutation profile., Conclusions: The results suggest that knowledge of mutations in BAP1 and EIF1AX can enhance prognostication of UM beyond that determined by chromosome 3 and tumor characteristics. Tumors with chromosome 3 disomy/BAP1-WT/EIF1AX-WT have a 10-fold increased risk of metastasis at 48 months compared with disomy-3/BAP1-WT/EIF1AX mutant tumors., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines., Methods: Four different cell lines were treated with AICAR (1-4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot., Results: Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5'-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5'-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation., Conclusions: Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Tumor-derived VEGF-A, apart from expediting sufficient vascularization, subsequent tumor growth, and metastatic spread, can act on malignant cells themselves provided that VEGF receptors 1 or 2 (VEGF-R1, -R2) are co-expressed. The study goal was to investigate whether such autocrine VEGF-A signaling exists in uveal melanoma (UM)., Methods: Primary (MEL-270, OM-431) and metastatic (OMM-2.3, OMM-2.5) UM cell lines were analyzed for VEGF-A, VEGF-R1, and VEGF-R2 expression by RT-PCR, ELISA (VEGF-A protein), and immunocytochemistry (VEGF receptors). Proliferation of UM cells incubated with neutralizing anti-VEGF-A antibody bevacizumab (≤ 2.5 mg/mL), or VEGF-A (≤ 100 ng/mL) was assessed by bromodeoxyuridine (BrdU) ELISA. It was measured by real-time PCR, whether VEGF-A (100 ng/mL) modulated the expression ratio of VEGF-A itself and its antiangiogenic antagonist pigment epithelium-derived factor (PEDF)., Results: All UM cells expressed VEGF-A, VEGF-R1, VEGF-R2 mRNA, and protein. In each cell line, the proliferation was stimulated by VEGF-A or inhibited by blocking VEGF-A, or both: bevacizumab significantly decreased the proliferation in MEL-270 (P = 0.005), OMM-2.3 (P = 0.001), and OMM-2.5 (P = 0.011). Increased VEGF-A signaling significantly raised the proliferation in MEL-270, OM-431 (P < 0.001, respectively), and OMM-2.3 (P = 0.043) in a dose-dependent manner but did not significantly change the VEGF-A/PEDF mRNA expression ratio., Conclusions: Autocrine VEGF-A signaling seems to be present in UM, sustaining the proliferation of both primary and metastatic UM cells. Apparently, VEGF-A signaling in UM cells neither acts retroactively on VEGF-A expression, in the sense of a feedback loop, nor contributes to a pro-angiogenic shift of the VEGF-A/PEDF ratio.

Purpose: Uveal melanoma (UM) tumors require large doses of radiation therapy (RT) to achieve tumor ablation, which frequently results in damage to adjacent normal tissues, leading to vision-threatening complications. Approximately 50% of UM patients present with activating somatic mutations in the gene encoding for G protein αq-subunit (GNAQ), which lead to constitutive activation of downstream pathways, including protein kinase C (PKC). In this study, we investigated the impact of small-molecule PKC inhibitors bisindolylmaleimide I (BIM) and sotrastaurin (AEB071), combined with ionizing radiation (IR), on survival in melanoma cell lines., Methods: Cellular radiosensitivity was determined by using a combination of proliferation, viability, and clonogenic assays. Cell-cycle effects were measured by flow cytometry. Transcriptomic and proteomic profiling were performed by quantitative real-time PCR, reverse-phase protein array analysis, and immunofluorescence., Results: We found that the PKC inhibitors combined with IR significantly decreased the viability, proliferation, and clonogenic potential of GNAQ(mt), but not GNAQ(wt)/BRAF(mt) cells, compared with IR alone. Combined treatment increased the antiproliferative and proapoptotic effects of IR in GNAQ(mt) cells through delayed DNA-damage resolution and enhanced induction of proteins involved in cell-cycle arrest, cell-growth arrest, and apoptosis., Conclusions: Our preclinical results suggest that combined modality treatment may allow for reductions in the total RT dose and/or fraction size, which may lead to better functional organ preservation in the treatment of primary GNAQ(mt) UM. These findings suggest future clinical trials combining PKC inhibitors with RT in GNAQ(mt) UM warrant consideration.

Purpose: To identify the prognostic value of extraocular extension in enucleated uveal melanoma (UM) patients and to correlate extraocular extension to chromosomal aberrations, metastasis-free survival (MFS), and clinico-histopathological risk factors., Methods: Retrospective study of patients with UM treated with enucleation between 1987 and 2011. Melanoma-related metastasis and death were recorded. Statistical analysis (log-rank test or Cox regression analysis) was performed to correlate MFS with tumor characteristics, extraocular extension, episcleral diameter of the extraocular extension, cell type, extracellular matrix patterns, inflammation, loss of chromosome 3, and gain of chromosome 8q., Results: In 43 (12%) of 357 patients, extraocular extension was observed. In this subset of patients, we noted a reduced survival of 70 months (105.5 months, P = 0.010) compared with patients without extraocular extension (175.8 months). Patients with gain of chromosomal region 8q in UM with extraocular extension had an increased risk of metastatic disease (P < 0.001). In multivariate Cox proportional hazard analysis, largest basal tumor diameter (P = 0.001), extracellular matrix patterns (P = 0.009), episcleral diameter of the extraocular extension (P = 0.016), loss of chromosome 3 (P < 0.001), and gain of 8q (P < 0.001) were independent predictors for MFS., Conclusions: Larger episcleral diameter of the extraocular extension and additional gain of chromosome 8q in extraocular extension UM correlates to a worse prognosis. MFS is significantly reduced in UM with a large basal tumor diameter, extracellular matrix patterns, loss of chromosome 3, and gain of chromosome 8q.

Purpose: To evaluate the relationship between rate of flattening of posterior uveal melanomas (PUMs) over the first 6 months following I-125 plaque radiotherapy and gene expression profile (GEP) class of tumor cells obtained by fine needle aspiration biopsy (FNAB) prior to treatment., Methods: Retrospective analysis of relationship between GEP of PUM cells obtained by FNAB at or prior to treatment and rate of tumor flattening following I-125 plaque radiotherapy. Impact of initial tumor thickness was minimized by pairing cases so baseline tumor thickness in subgroups was matched to within ± 0.5 mm. Paired t-testing compared mean tumor thickness in GEP subgroups at 3- and 6-months post treatment assessments., Results: Our initial group consisted of 269 patients. Seventy-seven tumors (28.6%) were GEP class 2. Twenty-seven of these were treated by I-125 plaque radiotherapy post-FNAB and returned for post treatment evaluations at 3 and 6 months. A matched GEP class 1 tumor was identified for 25 class 2 cases. Matched tumor pairs ranged in thickness from 2.5 to 11.5 mm at baseline. Mean tumor thickness at baseline in the GEP 1 subgroup was 5.8 and 5.9 mm in the GEP 2 subgroup. Three-months post plaque, mean tumor thickness was 4.5 mm in class 1 cases and 4.6 mm in class 2 cases (paired t = 0.31, P = 0.76). The 6-month post-plaque, mean tumor thickness was 4.0 mm in each subgroup (paired t = 0.25, P = 0.81)., Conclusions: Our study showed a lack of association between the GEP class and the rate of flattening of posterior uveal melanomas following I-125 plaque radiotherapy of PUMs.

Purpose: To examine the prognostic relevance of expression of the chemokine receptors CCR7 and CXCR4 and its ligand CXCL12 in uveal melanoma in nonmetastatic and metastatic patients with correlation to liver metastasis and overall survival., Methods: Primary uveal melanoma specimens from 19 patients with correlating liver metastasis specimens and 30 primary uveal melanoma specimens of patients without metastasis were collected between the years 1988 and 2008. Expression of CCR7, CXCR4, and CXCL12 were studied using immunohistochemistry. Single nucleotide polymorphism (SNP) arrays were used to examine gains or losses of chromosomes 1, 3, 6, and 8 and the regions of CCR7 (17q12-q21.2), CXCR4 (2q21), and CXCL12 (10q11.1) genes., Results: Strong cytoplasmic staining for CCR7 correlated with the presence of epithelioid cells (P = 0.037), tumor thickness (P = 0.011), lymphocytic infiltration (P = 0.041), and necrosis (P = 0.045). Nuclear staining for CXCR4 correlated with lymphocytic infiltration (P = 0.017). CXCL12 showed no correlation to histologic parameters. Single nucleotide polymorphism analyses showed no copy number variations in the regions of CCR7, CXCR4, or CXCL12. Strong expression of CCR7 was observed in 76% of the metastatic patients and 0% of nonmetastasis patients. In multivariate analysis, CCR7 staining was inversely correlated to overall survival and disease-free survival, whereas CXCR4 nuclear staining was not., Conclusions: Our data suggest that CCR7 plays a role in uveal melanoma metastasis and is associated with poor survival. CCR7 and its involved related pathways are of prognostic value in uveal melanoma and may prove to be a target for therapeutic intervention.

Purpose: Uveal melanoma (UM) was a fatal malignancy in 40% to 50% of cases. The aim of this study is to evaluate the independent contributions of chromosome 1, 3, 6, and 8 abnormalities for prognostication of metastasis, and to define multichromosome copy number aberration (CNA) signatures that can be used to evaluate risk., Methods: A series of 320 UM were analyzed for chromosome 1, 3, 6, and 8 abnormalities using whole genome single-nucleotide polymorphism arrays. Results for changes in six chromosomal regions were analyzed using univariate and multivariate Cox proportional hazard modeling to identify significant predictors of metastasis and CNA signatures., Results: Univariate Cox analysis indicated that losses of chromosome 3, 1p, 6q, and 8p and gain of 8q, as well as sex, source of tumor tissue (fine-needle aspiration biopsy [FNAB] compared with tumor from an enucleated eye), tumor basal diameter and height, and ciliary body involvement were all significant predictors of poor metastatic outcome. In the multivariate analysis, loss of chromosome 3 and 8p remained significant after adjusting for the effects of all other variables, as did sex, tissue source, and basal diameter. Multivariate analysis of the joint effects of changes in the six chromosomal regions showed that six signatures, including chromosome 3-loss, 1p-loss, 8p-loss, and/or 8q-gain had hazard ratios (HR) ranging from 7.90 to 37.25., Conclusions: In UM, tumor size and location, tissue source, and sex were all significantly associated with increased metastasis. In addition, chromosome 3-loss and 8p-loss were found to be independent predictors of poor metastatic outcome and CNA signatures were identified that can add a specific HR value for classification of risk categories.

Purpose: MicroRNA-124a (miR-124a), an abundant microRNA in the central neuron system, has been linked to tumor progression. Here, we investigated the role of miR-124a in uveal melanoma development., Methods: Expression of miR-124a in uveal melanoma cells was examined using real time RT-PCR. The effect of miR-124a on cell proliferation, migration, and invasion was analyzed using MTS assay, flow cytometry, and transwell experiments. The ability of miR-124a to repress tumor growth was tested in vivo. Target genes of miR-124a were first predicted by bioinformatics, confirmed using a luciferase assay, and their expression determined by Western blotting. DNA methylation and histone modification of miR-124a was analyzed by methylation-specific PCR and ChIP assay. Finally, epigenetic drugs were used to alter the expression of miR-124a., Results: miR-124a expression was downregulated in both uveal melanoma cells and clinical specimens. Transient transfection of miR-124a into uveal melanoma cells inhibited cell growth, migration, and invasion. Moreover, introduction of miR-124a suppressed in vivo growth of tumor. Potential targets of miR-124a were found to include CDK4, CDK6, cyclin D2, and EZH2. Knockdown of EZH2 by siRNA resulted in inhibition of uveal melanoma cell migration and invasion. In addition, miR-124a expression was found to be regulated via epigenetic mechanisms, with its expression restored when cells were treated with a DNA hypomethylating agent, 5-aza-2'-deoxycytidine, and a histone deacetylase inhibitor, trichostatin A., Conclusions: Our results demonstrated that miR-124a could function as a potent tumor suppressor by regulation of multiple targets, and was epigenetically silenced in the development of uveal melanoma.

Purpose: We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system., Methods: DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology., Results: Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11., Conclusions: The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.

Purpose: RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. We investigated the effect of RASSF1A re-expression on the UM phenotype in vivo and in vitro., Methods: The phenotypic effect of methylation-induced inactivation of RASSF1A in UM was explored using a stable RASSF1A-expressing UM-15 clone. RASSF1A expression was assessed using QRT-PCR. Proliferation was evaluated in vitro using MTT assays. Additionally, athymic NOD/SCID mice were injected subcutaneously or intraocularly with RASSF1A-expressing and -non-expressing UM-15 clones, and euthanized when tumors reached a volume of 1500 mm(3), or at 56 or 46 days, respectively. Tumor tissues, eyes, and livers were analyzed histologically., Results: In vitro analysis confirmed the lack of RASSF1A expression and full methylation of the RASSF1A promoter region in the UM-15 cell line, which was reversible following treatment with 5-Aza-2-deoxycytidine. Cells expressing exogenous RASSF1A showed slower proliferation than controls and regained sensitivity to cisplatin. Compared to mice injected with control cells, mice treated with UM-15 cells expressing exogenous RASSF1A did not acquire intraocular tumors, and their subcutaneous tumors were relatively delayed and small. Neither group had liver metastases., Conclusions: UM cells reduced tumorigenicity in the presence of activated RASSF1A. RASSF1A apparently has an important role in the development of UM, and its reactivation might be applied in the development of new treatments.

Purpose: Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties., Methods: Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice., Results: Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation., Conclusions: Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo.

Purpose: In contrast to many other malignancies, in uveal melanoma (UM) the presence of an immune infiltrate is associated with a bad prognosis. An analysis of the different functional phenotypes of tumor-infiltrating leukocytes (TIL) and a comparison with the genetic background of the tumors may help to explain this apparent anomaly., Methods: We performed a comprehensive immunohistochemical study by evaluating the density of CD8(+) and CD4(+) T lymphocytes, forkhead box p3 (Foxp3(+)) regulatory T cells (Tregs), and CD68(+) and CD68(+)CD163(+) macrophages in 43 cases of UM in relation to tumor characteristics. Expression of the chemokines CCL2, CCL17, and CCL22 in cultured human UM cells and peripheral blood monocytes was analyzed by quantitative PCR (qPCR)., Results: The presence of TILs was highly variable between tumors and was dominated by CD8(+) T cells with fewer CD4(+) T cells and Tregs. When tumors were infiltrated by immune cells, the infiltrate generally comprised all different subsets of lymphocytes (P < 0.001) and M2 macrophages (P < 0.001). Different T-cell ratios did not influence clinical outcome. In addition, the presence of TIL correlated with the loss of one chromosome 3 (P < 0.04). UM cells express CCL2 and CCL22, two chemokines known to mediate trafficking of immune cells to the tumor., Conclusions: All studied subtypes of tumor-infiltrating immune cells were collectively increased and showed an association with monosomy of chromosome 3 suggesting that tumor intrinsic factors control the leukocyte influx, possibly through local chemokine secretion.

Purpose: Almost all uveal melanomas showing chromosome 3 loss (i.e., monosomy 3) are fatal. Randomized clinical trials are therefore needed to evaluate various systemic adjuvant therapies. Conventional trial designs require large numbers of patients, which are difficult to achieve in a rare disease. The aim of this study was to use existing data to estimate how sample size and study duration could be reduced by selecting high-risk patients and adopting multistage trial designs., Methods: We identified 217 patients with a monosomy 3 melanoma exceeding 15 mm in basal diameter; these patients had a median survival of 3.27 years. Several trial designs comparing overall survival were explored for such a population. A power of 0.90 to detect a hazard ratio of 0.737 was set, and recruitment of 16 patients per month was assumed., Results: A suitable single-stage study would require 960 patients and a duration of 76 months. A two-stage design with an interim analysis based on 852 patients after 53.3 months would have a 50% probability of stopping because no statistically significant treatment effect is seen. Encouraging but inconclusive results would require a further 108 patients and prolongation of the study to 77.2 months. A multistage design would have a 43% probability of stopping before 47 months having recruited 759 patients., Conclusions: Prospects for clinical studies of systemic adjuvant therapy for uveal melanoma are enhanced by multistage trial designs enrolling only high-risk patients.