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Background: Accurate risk stratification of uveal melanoma (UM) patients is important for determining the interval and frequency of surveillance. Loss of BAP1 expression has been shown to be strongly associated with UM-related death and metastasis., Methods: In this study of 164 enucleated UMs, we assessed the prognostic role of preferentially expressed antigen in melanoma (PRAME) expression and Ki67 proliferation index measured by digital quantitation using QuPath programme in patients with BAP1-positive and BAP1-loss UMs., Results: In univariate analyses with log-rank tests and Kaplan-Meier curves, PRAME further stratified only overall survival (OS) in BAP1-positive and BAP1-loss tumour groups. However, Ki67 further stratified both OS and disease-free survival (DFS) in BAP1-positive and BAP1-loss tumour groups. In multivariate analyses, Ki67 percentage and BAP1 were independent survival predictors for both OS and DFS, whereas PRAME was not a significant covariate. In model comparisons, combined Ki67 and BAP1 performed better than combined PRAME and BAP1 in risk-stratifying patients for both OS and DFS. Ki67 was better than PRAME in risk stratification of BAP1-positive UMs. Low Ki67 index correlated with significantly prolonged DFS in BAP1-loss UMs., Conclusion: A panel of Ki67 and BAP1 could be a helpful risk stratification strategy for UM., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is poor. Lymphocyte activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are the two promising immune checkpoint targets. Therefore, our aim was to explore at how these proteins were expressed in tumour tissue and serum, as well as their prognostic implications in UM., Methods: The expression of LAG3, CTLA-4, CD3, CD4, CD8 and FOXP3 was determined by immunohistochemistry in 54 enucleated UM tissue samples. mRNA expression level of LAG3 and CTLA-4 was determined by quantitative real-time PCR and corroborated by western blotting. Furthermore, soluble form of LAG3, CTLA-4 and CCR8 expression in serum was measured in 40 UM patients using ELISA., Result: The expression of LAG3, CTLA-4, CD3, CD4, CD8 and FOXP3 was observed in 30%, 33%, 41%, 35%, 50% and 39% of the cases, respectively. Loss of nBAP1 expression was significantly correlated with CD8+expression (p=0.012) but not with tumour infiltrating lymphocytes. LAG3 and CTLA-4 mRNA levels were higher in UM compared with normal uveal tissues. Higher LAG3 expression with CD8+expression was associated with lower metastasis-free survival (MFS) (p=0.049), but not with CTLA-4 in UM patients. MFS rate was reduced in patients having lower levels of CCR8 protein (p=0.050) and increased level of LAG3 protein (p=0.001)., Conclusion: Our findings suggest that higher levels of LAG3 in UM with histopathologically high-risk parameters predict high metastatic potential and that it could be used as a targeted immunotherapy alone or in combination with PD-1/PD-L1 blockade agents., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Largest basal diameter (LBD) appears to have independent prognostic value in uveal melanoma (UM)., Methods: All patients undergoing plaque brachytherapy or enucleation for UM involving the choroid and/or ciliary body between 2012 and 2019., Results: A total of 348 patients with a mean age of 60±14 years were included and followed for a mean of 40±26 months (3.3±2.2 years). On multivariate analysis, LBD >12 mm remained a significant independent predictor of metastasis for both class 1 (HR 21.90; 95% CI 2.69 to 178.02; p=0.004) and class 2 (HR 2.45; 95% CI, 1.03 to 5.83; p=0.04) tumours. Four prognostic groups were created: group 1 (class 1, LBD <12 mm), group 2 (class 1, LBD ≥12 mm), group 3 (class 2, LBD <12 mm) and group 4 (class 2, LBD ≥12 mm). Life tables were used to calculate the 3-year and 5-year metastasis-free survival: group 1 (98 and 98%), group 2 (86 and 86%), group 3 (81 and 62%) and group 4 (54 and 47%). Compared with the reference category (group 1), the Cox proportional hazard model demonstrated a significant worsening of survival for each progressive category (group 2 (HR 21.59; p=0.004), group 3 (HR 47.12, p<0.001), and group 4 (HR 114.24; p<0.001)). In our dataset, the four-category Cox model performed poorer compared with the American Joint Committee on Cancer (AJCC) and gene expression profile (AJCC+GEP) in the Akaike's information criteria (AIC) (297 vs 291), fit better with the Bayesian information criteria (BIC) (309 vs 313) and performed similarly with the Harrel's C (0.86 (95% CI 0.80 to 0.91) vs 0.89 (0.84 to 0.94), respectively)., Conclusions: Combination of GEP and LBD allows separation of patients into four easy-to-use prognostic groups and was similar to a model combining AJCC stage with GEP., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To determine whether the GNAQ/11 mutation correlated with the outcome of patients with uveal melanoma (UM) when genetic heterogeneity was considered., Methods: We performed a retrospective study of sixty-seven patients with UM. The heterogeneity of GNAQ/11 was examined by using droplet digital PCR. The correlation between metastasis and heterogeneity of the GNAQ/11 mutation was analysed. Disease free survival curves were constructed using the Kaplan-Meier method, and the Wilcoxon log-rank test was used to compare the curves., Results: The GNAQ/11 mutation ratio was varied between each case. Among these patients, 28.35% of them harboured homogeneous mutation of GNAQ/11, 62.69% present heterogeneous mutation and 8.96% didn't present either GNAQ or GNA11 mutation. The tumour with heterogeneous mutation of GNAQ/11 has a higher metastatic rate than that with homogeneous mutation (13/29 vs 1/18, p=0.027). In Kaplan-Meier analysis, metastasis-free survival was not significantly associated with either homogeneous or heterogeneous mutation of GNAQ/11., Conclusion: The mutation ratio of GNAQ/11 in UM was quite variable. The tumour with heterogeneous mutation of GNAQ/11 is more likely to develop a poor prognosis than that with homogeneous mutation of GNAQ/11., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background/aims: Proton beam radiotherapy and plaque brachytherapy are commonly applied in primary uveal melanoma (UM); however, their effect on chromosome 3 classification of UM by microsatellite analysis (MSA) for prognostication purposes is unknown, where the tumour is sampled post-irradiation. This study examined the prognostic accuracy of genotyping UM biopsied before or after administration of radiotherapy, by MSA., Methods: 407 UM patients treated at the Liverpool Ocular Oncology Centre between January 2011 to December 2017, were genotyped for chromosome 3 by MSA; 172 and 176 primary UM were sampled prior to and post irradiation, respectively., Results: Genotyping by MSA was successful in 396/407 (97%) of UM samples (196 males, 211 females; median age of 61 years (range 12 to 93) at primary treatment). There was no demonstrable association between a failure of MSA to produce a chromosome 3 classification and whether radiation was performed pre-biopsy or post-biopsy with an OR of 0.96 (95% CI 0.30 to 3.00, p=0.94). There was no evidence of association (measured as HRs) between risk of metastatic death and sampling of a primary UM before administration of radiotherapy (HR 1.1 (0.49 to 2.50), p=0.81). Monosomy 3 (HR 12.0 (4.1 to 35.0), p<0.001) was significantly associated with increased risk of metastatic death., Conclusions and Relevance: This study revealed that successful genotyping of UM using MSA is possible, irrespective of irradiation status. Moreover, we found no evidence that biopsy prior to radiotherapy increases metastatic mortality., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Uveal melanoma represents ∼85% of all ocular melanomas and up to 50% of patients develop metastatic disease. Metastases are most frequently localised to the liver and, as few patients are candidates for potentially curative surgery, this is associated with a poor prognosis. There is currently little published evidence for the optimal management and treatment of metastatic uveal melanoma and the lack of effective therapies in this setting has led to the widespread use of systemic treatments for patients with cutaneous melanoma. Uveal and cutaneous melanomas are intrinsically different diseases and so dedicated management strategies and therapies for uveal melanoma are much needed. This review explores the biology of uveal melanoma and how this relates to ongoing trials of targeted therapies in the metastatic disease setting. In addition, we consider the options to optimise patient management and care., Competing Interests: RDC received non-financial support from AstraZeneca, during the conduct of this work; RDC has received grants, personal fees and non-financial support from AstraZeneca, personal fees from Janssen, personal fees from Thompson Reuters, personal fees from Merck, personal fees from Biogen Idec, personal fees from Aura Biosciences, grants from Melanoma Research Foundation, grants from Melanoma Research Alliance, grants from NIH and grants from ASCO, outside the submitted work. PDN reports personal fees from AstraZeneca, outside the submitted work., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background/aims: Iris melanomas (IM) are rare and have a lower mortality than posterior uveal melanomas (UM). Our aims were to determine the prevalence of genetic changes associated with prognosis of posterior UM, in both treated and non-treated IM., Methods: Retrospective database review and molecular analysis of all patients diagnosed with IM at the Liverpool Ocular Oncology Centre (LOOC) between 1993 and 2015. Archival pathology specimens of confirmed IM cases were analysed for chromosomal alterations, using multiplex ligation-dependent probe amplification (MLPA) or microsatellite analysis (MSA) depending on DNA yield, and BRAF mutation status., Results: 5189 patients were diagnosed with intraocular melanoma at LOOC from 1993 to 2015. Of these, 303 (5.8%) patients were diagnosed with IM. Tissue samples were available for 26 IM cases. Twelve of these cases had biopsies taken post-proton beam radiotherapy (PBR). Histological subtyping showed 14 IM being spindle, 2 epithelioid and 10 were of mixed cell type. Twenty of the 26 IM cases (77%) analysed genetically were classified as either disomy 3 (n=16) or monosomy 3 (n=4). Chromosome 6p gain was detected in 4/18 (22%) IM, and polysomy 8q in 6%. BRAF mutations were not detected in any of the four IM cases examined. One patient with IM died from metastatic disease: this tumour was disomy 3 with 6p and 8q gains. All other patients were alive with no evidence of metastases at study closure., Conclusions: Chromosomal aberrations seen in posterior UM can also be demonstrated using MLPA or MSA in both treatment naïve and PBR-treated IM. Most IM display a low-metastatic risk chromosomal profile., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Purpose: To report outcomes and complications of fine-needle aspiration biopsy (FNAB) of uveal melanoma performed for diagnostic and prognostic purposes., Methods: Prospective interventional case series of 150 consecutive patients with a clinical diagnosis of uveal melanoma. The FNAB approach (transcorneal (TCO), transscleral (TSC) and transvitreal (TVT) was primarily determined by the location of the tumour. The FNAB was performed using a 25-gauge needle using a previously published technique. Prognostication was done using fluorescent in situ hybridisation detection of monosomy of chromosome 3., Results: FNAB was obtained via TCO (8), TSC (71) and TVT (64) approach and impression smear in seven cases. Diagnostic yield was 92%. False-negative results were observed in 8%. Diagnostic yield was significantly correlated to biopsy approach (TCO 100%, TSC 96%, TSV 86%; p=0.029) and tumour size (basal diameter >5.0 mm; height >2.5 mm). Persistent haemorrhage (subretinal haemorrhage or vitreous) requiring surgical intervention (1%) and rhegmatogenous retinal detachment (1%) were rare. Endophthalmitis, hypotony, tumour recurrence, episcleral seeding were not observed over the average follow-up of 37 months. Prognostication could be performed in 85% of cases. Overall, only 47% of eligible patients enrolled into the adjuvant therapy trial., Conclusions: FNAB for uveal melanoma with 25-gauge needle is a safe procedure that can yield diagnostic and prognostic information in vast majority of cases (92% and 85%, respectively). Even so, only about half of the eligible cases eventually enrolled into the adjuvant therapy trial. Possibility of negative FNAB yield should be considered when counselling patients with small tumours. Alternative means of diagnostic biopsy and methods of prognostication need to be assessed for small tumours., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Aim: The detection of circulating tumour cells in the bloodstream before and after surgical manipulation, and the qualitative detection of potential shedding of tumour cells during surgical manipulation of patients with uveal melanoma., Methods: 202 patients treated for a newly diagnosed uveal melanoma were included in the study. Blood samples were acquired 24 h before and 30 min after the basic surgical steps. Detection of potential circulating melanoma cells was extrapolated from the presence of tyrosinase and MelanA/Mart1 transcripts by reverse transcription PCR., Results: Based on the measurement of tyrosinase transcripts, as a result of the first and second surgical manipulation there were three and zero transitions from negative to positive respectively, while there were two and one transitions from positive to negative, respectively. According to MelanA/Mart1 transcripts, there were 19 and 5 transitions from negative to positive respectively, and 15 and 2 transitions from positive to negative, respectively. No statistically significant differences were documented, concerning the presence of circulating tumour cells in the blood samples acquired before and after the first surgical manipulation or the second one., Conclusion: The change in the percentage of patients with detected tumour cells in their bloodstream was not statistically significant. The frequent shifts from negative to positive samples as well as from positive to negative samples comparing preoperative to postoperative samples indicates discontinuous shedding or variation due to measurements close to the threshold of detection. As a conclusion, the surgical manipulation does not seem to have a measurable contribution to the spread of melanoma cells in the bloodstream., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background/aim: The study's aim was to compare chromosome 3 aberrations of choroidal melanoma (CM) as determined by multiplex ligation dependent probe amplification (MLPA) or microsatellite analysis (MSA) in intraocular tumour biopsies with those results obtained from subsequent endoresection/enucleation of the same CM., Methods: A retrospective cohort of 28 patients with CM seen between 2007 and 2014 at the Liverpool Ocular Oncology Centre was analysed. Prognostic genetic testing, for chromosome 3 status, was performed on all tumour specimens, either by MLPA or MSA, depending on DNA yield. In nine cases genetic testing was performed on a sample taken after radiotherapy; four of these had genetic information pre- and post-radiotherapy., Results: Fourteen biopsy specimens were analysed by MLPA and 14 by MSA. Twenty-seven endoresection or enucleation specimens were analysed by MLPA, and a single enucleation specimen by MSA. Chromosome 3 data showed prognostic concordance for the patient-matched samples in all 28 cases including 4 cases where samples were taken pre pre- and post radiotherapy. Thirteen cases were classified as monosomy 3 and 12 as disomy 3. Two cases had a loss of chromosome arm 3q in both samples and a single case showed loss of 3p in the biopsy sample with complete monosomy 3 in the subsequent enucleation sample taken 5 months later., Conclusions: Intraocular biopsy of CM yields similar prognostic information to larger surgical specimens. Initial evidence, that genetic testing can be successfully conducted post radiotherapy, is also provided., Trial Registration Number: NITRO trial, ISRCTN35236442., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aims: Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features., Methods: We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes., Results: Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes., Conclusions: Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: To correlate the metabolic activity of primary uveal melanoma on positron emission tomography (PET)/CT scan with known clinical and pathological prognostic factors., Methods: A retrospective cohort analysis of eyes enucleated for uveal melanoma that underwent preoperative imaging with a PET/CT scan was performed. Tumour dimensions were recorded and classified using Collaborative Ocular Melanoma Study (COMS) and American Joint Committee on Cancer (AJCC) Tumour - Nodes - Metastases (TNM) criteria. Metabolic activity was determined by measurement of the maximal standardised uptake value (SUVmax) on PET/CT scans. SUVmax of >2.5 and >4 was also used as cut-off value for metabolic positivity. Chromosome 3 and 8 status was determined using fluorescence in situ hybridisation analysis. Pearson correlation, χ(2) test and non-parametric tests were used. p<0.05 was considered statistically significant., Results: Seventy-six uveal melanomas were imaged preoperatively with a PET/CT scan. Overall 92% of tumours had a SUVmax >2.5 and 67% had a SUVmax >4. Monosomy 3 was found in 35 melanomas, of which 94% had an SUVmax >2.5 and 80% had an SUVmax >4. Only 57% of disomy 3 melanomas had an SUVmax >4. SUVmax was significantly increased in tumours with monosomy 3 (p=0.043) but not in tumours with chromosome 8 gain (p=0.49). SUVmax and increasing tumour size were positively correlated (p<0.05). Using the AJCC criteria, there was a significant difference in SUVmax among prognostic groups (p=0.024). There was no correlation with histopathological cell type (p=0.923)., Conclusions: Metabolic activity of uveal melanoma on PET/CT scan is positively correlated with monosomy 3, increasing tumour size and TNM prognostic groups. No association with chromosome 8 gain or histopathology cell type was noted. SUVmax >4 is a relative but not an absolute indicator of monosomy 3 status., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aim: To determine whether BAP1 gene and protein expression associates with different prognostic parameters in uveal melanoma and whether BAP1 expression correctly identifies patients as being at risk for metastases, following enucleation of the primary tumour., Methods: Thirty cases of uveal melanoma obtained by enucleation between 1999 and 2004 were analysed for a variety of prognostic markers, including histological characteristics, chromosome aberrations obtained by fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) analysis and gene expression profiling. These parameters were compared with BAP1 gene expression and BAP1 immunostaining., Results: The presence of monosomy of chromosome 3 as identified by the different chromosome 3 tests showed significantly increased HRs (FISH on isolated nuclei cut-off 30%: HR 11.6, p=0.002; SNP analysis: HR 20.3, p=0.004) for death due to metastasis. The gene expression profile class 2, based on the 15-gene expression profile, similarly provided a significantly increased HR for a poor outcome (HR 8.5, p=0.005). Lower BAP1 gene expression and negative BAP1 immunostaining (50% of 28 tumours were immunonegative) were both associated with these markers for prognostication: FISH cut-off 30% monosomy 3 (BAP1 gene expression: p=0.037; BAP1 immunostaining: p=0.001), SNP-monosomy 3 (BAP1 gene expression: p=0.008; BAP1 immunostaining: p=0.002) and class 2 profile (BAP1 gene expression: p<0.001; BAP1 immunostaining: p=0.001) and were themselves associated with an increased risk of death due to metastasis (BAP1 gene expression dichotomised: HR 8.7, p=0.006; BAP1 immunostaining: HR 4.0, p=0.010)., Conclusions: Loss of BAP1 expression associated well with all of the methods currently used for prognostication and was itself predictive of death due to metastasis in uveal melanoma after enucleation, thereby emphasising the importance of further research on the role of BAP1 in uveal melanoma., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: This study investigated the capacity of genetic analysis of uveal melanoma samples to identify high-risk patients and discusses its clinical implications., Methods: Patients with posterior uveal melanoma were prospectively enrolled. Tumour samples were derived from enucleated globe, fine-needle aspirates or endoresection. Chromosome 3 and 8 status was determined by array comparative genomic hybridisation (array-CGH). Patients were followed after treatment to detect metastasis., Results: Four groups were classified by array-CGH. Patients were divided into disomy 3 and normal chromosome 8 (D3/8nl), disomy 3 and 8q gain (D3/8g), monosomy 3 and normal chromosome 8 (M3/8nl) and monosomy 3 and 8 or 8q gain (M3/8g). Median follow-up was 28 months (range: 1-147 months). At the end of the study, 128 patients (33.7%) had developed metastasis and 96 patients had died. Univariate Cox proportional hazard analysis showed that factors associated with metastasis included basal tumour diameter p=0.0007, tumour thickness p=0.01, mixed/epithelioid cell type p=0.0009 and genomic data p<0.0001. High-risk profile was more strongly associated with metastasis than the other prognostic factors p<0.001. Multivariate Cox modelling analysis showed that the status of chromosomes 3 and 8 were the only two variables that independently contributed to prognosis: monosomy 3 alone p=0.001 and monosomy 3 and 8q gain p<0.0001., Conclusions: Array-CGH allowed identification of three prognostic groups with low, intermediate and high risk of developing metastasis. Array-CGH is a reliable and inexpensive method for uveal melanoma prognosis. This method is now currently used in France., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Uveal melanoma (UM) is the most common primary cancer of the eye in adults. About half of the patients are at risk of developing metastatic disease resulting in a poor clinical prognosis. Metastatic progression is strongly associated with loss of one chromosome 3 in the tumour (monosomy 3). The tumour suppressor gene BAP1 was found to be recurrently mutated in UM with monosomy 3. Familial UM is rare and amounts to about 0.6-6% of all patients with melanoma. However, BAP1 germline mutations have been identified in rare hereditary tumour syndromes, including cases with UM. One may assume that UM may be part of these hereditary conditions with predisposition to malignant cancers., Methods: The patients underwent complete ophthalmological workup and enucleation due to UM. Microsatellite analysis was performed to determine the chromosome 3 status of the tumours. Sanger sequencing of all coding exons of the BAP1 gene was performed in blood DNA of the patients., Results: Here we report on two family members (mother and son) diagnosed with UM. In both patients, a cosegregating BAP1 germline mutation (c.299 T>C) was found. The mutant BAP1 allele was retained in the tumour of the son showing monosomy 3. The son further developed urothelial carcinoma and liver metastasis, the mother was affected by the UM and cholangiocellular carcinoma., Conclusions: [corrected] We detected a cosegregating BAP1 germline mutation in two family members with UM. This suggests that, consistent with a classic tumour suppressor model, carriers of damaging mutations in BAP1 are predisposed to UM. However, as BAP1 germline mutations have been found to cause other cancer syndromes as well, there must be other factors that decide about the type of tumour emerging from BAP1 inactivation.

Objective: Intraocular melanocytoma is a rare naevus variant that can be located at the optic disc or within the uvea, and belongs to the group of non-epithelial-associated melanocytic lesions. We wanted to gain an understanding of the role of GNAQ, GNA11 and BRAF V600E in the pathogenesis of uveal melanocytoma and in cases of transformation to uveal melanoma and also to perform a differential immunohistochemical study comparing melanocytoma with uveal melanoma., Methods and Results: Two patients were identified with melanocytoma, one of which had transformed to melanoma. In the latter case, the melanocytoma exhibited an immunophenotype that featured nuclear p27 and no HMB45 staining, with very low Cyclin D1 expression compared with the melanoma that featured little nuclear but more cytoplasmic p27 positivity, much higher Cyclin D1 expression and HMB45 positivity. The melanocytomas were negative for CD68 allowing distinction from melanophages. Both melanocytomas and the melanoma harboured mutations in GNAQ, with no mutations of GNA11 or BRAF V600E., Conclusions: GNAQ mutations are present in uveal melanocytomas and in a case of transformation to melanoma, implicating GNAQ-dependent mitogen activation signals, in the pathogenesis of uveal melanocytoma. This assists in explaining why a proportion of uveal melanocytoma can transform to uveal melanoma, known to harbour high-frequency GNAQ mutations at exon 5, codon 209.

Aims: To characterise a histologically unusual paediatric uveal melanoma by gene expression and karyotypic profiling and assess prognosis., Methods: The tumour was studied by histopathology, karyotype analysis, single nucleotide polymorphism and gene expression profile analysis for correlation with clinical outcome., Results: The tumour had predominantly epithelioid histology. Karyotype analysis showed none of the poor prognosis features normally associated with uveal melanoma. single nucleotide polymorphism analysis revealed no imbalance at chromosome 3. Gene expression profiling indicated low risk disease., Conclusions: We report a child remaining relapse-free 6 years after diagnosis of a very rare uveal melanoma, with poor prognosis epithelioid histology, but gene expression profiling that accurately predicted low risk disease.

Aim: To study the GNAQ mutational status in a series of uveal melanomas and evaluate possible associations with mitogen-activated protein kinase (MAPK) pathway protein expression and tumour proliferation markers., Methods: Mutational analysis was performed by PCR/sequencing of exon 5 of the GNAQ gene in a series of 22 uveal melanomas in which total and phosphorylated extracellular signal-regulated kinase (ERK) 1/2 overexpression without coexistent BRAF and NRAS mutations had previously been observed. Expression of the cell cycle markers (Ki-67, cyclin D1 and p27) was evaluated by immunohistochemistry. The association between GNAQ mutational status, ERK1/2, phospho-ERK1/2, Ki-67, cyclin D1 and p27 expression levels and the clinicopathological prognostic parameters of uveal melanomas was also assessed., Results: GNAQ mutations were found in 36% of uveal melanomas. No associations were found between the GNAQ mutational status and prognostic parameters, the expression of ERK1/2, pERK1/2 and cell cycle markers., Conclusion: The results of this study suggest that GNAQ mutated uveal melanomas do not exhibit a higher deregulation of proliferation or higher activation of the MAPK signalling pathway than uveal melanomas without GNAQ overactivation.

Uveal melanomas (UM) are aggressive ocular tumours that spread to the liver. They are characterised by alterations of chromosome 3 and 8, which are highly predictive of a poor prognosis. Unfortunately, being able to identify those patients with aggressive disease has not, as yet, translated into improved survival. Recently, mutations of guanine nucleotide-binding protein G(q) subunit alpha (GNAQ, or G-alpha-q), which effectively turn it into a dominantly acting oncogene, have been identified in approximately half of UM. These mutations are specific to UM and other non-cutaneous melanomas, and are not found in normal tissues, thus making them potential therapeutic targets. Here, the authors review the background to GNAQ in UM and explore what makes it such an interesting target for the future treatment of patients.

Background: Uveal melanomas can develop in the choroid, ciliary body and iris. In choroidal and ciliary body melanomas, specific chromosomal changes correlate with metastatic disease. Iris melanomas have a better prognosis than choroidal melanomas, and it would be interesting to know if they share chromosomal changes. In addition, iris melanomas might harbour UV-induced mutations of tumour suppressor genes, such as PTEN and CDKN2A., Methods: Twenty iris melanomas were analysed for chromosome 1p, 3, 6, 8, 9p and 10q abnormalities using fluorescence in situ hybridisation. These results were correlated to clinical follow-up data using statistical analyses., Results: (Partial) loss of chromosome 3 was observed in nine iris melanomas, and gain of 8q was present in seven tumours. Loss of chromosome 9p was demonstrated in seven tumours, but no deletions of the PTEN region on chromosome 10 were found. Three patients died of metastatic disease, and one patient developed liver metastases, but is still alive. Univariate analysis indicated a lower disease-free survival for patients with diffuse growing melanomas (p=0.01), melanomas that lost a copy of chromosome 3 (p=0.03), or invading the ciliary body (p=0.01). In a multivariate analysis, none of the correlations were significant., Conclusion: Loss of chromosome 3 as well as loss of chromosomal region 9p21 (that entails tumour suppressor gene CDKN2A) plays a role in iris melanoma. A firm correlation with disease-free survival could not be established, possibly due to the small sample size.

Background: Primary melanoma of the iris, for reasons unknown has a lower metastatic rate compared with primary ciliary-body melanoma. Six histology cases of ciliary-body melanoma were identified that had spread onto the iris surface and into the stroma, representing a change in tumour microenvironment from aqueous humour non-exposure (ciliary-body component) to aqueous humour exposure (iris surface component). This provided an ideal paradigm for investigating the effects of different environments on melanoma., Method: Conventional light microscopy was performed on stained paraffin sections of the identified cases, followed by immunohistochemistry to cell cycle proteins p27 and Cyclin D1. Fluorescence in situ hybridisation (FISH) analysis was conducted on the paraffin sections for changes of chromosomes 3 and 8, associated with poor uveal melanoma prognosis., Results: Iris surface melanoma cells were smaller compared with the adjacent deeper iris stromal melanoma cells and with those in the ciliary body. Fewer iris surface melanoma cells expressed Cyclin D1 protein, but more expressed p27 protein, compared with the larger iris stromal melanoma cells (paired Wilcoxon signed ranks test: Cyclin D1 p = 0.028; p27 p = 0.046) and with the ciliary-body melanoma cells (paired Wilcoxon signed ranks test: Cyclin D1 p = 0.028; p27 p = 0.028). With FISH, chromosome 3 and 8 alterations were less common among the iris surface melanoma cells than the deeper iris stromal melanoma cells and the ciliary-body melanoma cells, which were consistently characterised by a relative genetic imbalance for chromosomes 3 and 8., Conclusions: These data suggest that there are tumour-modulatory factors within the anterior chamber environment that probably select populations of ciliary-body melanoma cells, with a less aggressive, better-differentiated status. Furthermore, it may help explain why iris melanomas generally have a less aggressive course than ciliary-body and choroidal melanomas.

Background: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours., Aim: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC., Methods: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks., Results: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed., Conclusions: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

Aim: In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour-related mortality., Methods: The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence-verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques--hierarchical cluster analysis and multidimensional scaling--were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10,000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded., Results: Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real-time quantitative PCR., Conclusions: Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.

Background: Malignant melanoma arising from different body compartments may be associated with differing aetiological factors and clinical behaviour, and may manifest diverse molecular genetic profiles. Although many studies have focused on cutaneous melanoma, little is known of mucosal and other types of melanoma. In particular, malignant melanoma of soft parts is different from other melanomas in many respects, yet manifests a common melanocytic differentiation. Mutation of BRAF is now known to be common in cutaneous melanomas, and raises possible new therapeutic options of anti-RAF treatment for these patients. Few data are available for non-cutaneous melanomas., Aims: To study the incidence of BRAF and NRAS mutations in melanomas arising in diverse internal organs., Methods: Fifty one melanomas from various internal organs were investigated for BRAF and NRAS mutation by direct DNA sequencing., Results: BRAF and NRAS mutations were found in two and five mucosal melanomas arising from the aerodigestive and female genital tracts (n = 36). Their occurrence is mutually exclusive, giving a combined mutation incidence rate of 19.4% in mucosal melanomas. Both BRAF and NRAS mutations were absent in malignant melanoma of soft parts (n = 7). BRAF mutation was also absent in uveal melanoma (n = 6), but was seen in two of five cutaneous melanomas. The incidence of BRAF or combined BRAF/NRAS mutations in all non-cutaneous groups was significantly lower than published rates for cutaneous melanomas., Conclusion: Each melanoma subtype may have a unique oncogenetic pathway of tumour development, and only a small fraction of non-cutaneous melanomas may benefit from anti-RAF treatment.

Background/aim: Interferons (IFN) are currently being used to treat melanoma, including some patients with uveal melanoma. IFN is thought to inhibit tumour growth through downregulation of the c-myc oncogene; the overexpression of which has been shown to be associated with resistance in cell lines. The aim of this study was to investigate the relation between c-myc gene expression and IFN sensitivity in a series of uveal melanomas in a short term chemosensitivity assay., Methods: Tumours from 45 patients with uveal melanoma who had undergone enucleation were studied. The ATP chemosensitivity assay was used to study sensitivity to IFN-alpha-2b in freshly isolated cells from each tumour. Flow cytometry was used to assess c-myc expression in formalin fixed material from the primary specimens., Results: There was a wide range of IFN sensitivity between the specimens whereas c-myc expression was universal and present in 80% of the tumour cells in 80% of the specimens. Higher c-myc expression was associated with IFN-alpha resistance as measured by the maximum percentage of inhibition (p = 0.05) and there was a trend with the IFN sensitivity index (p = 0.07)., Conclusions: These results demonstrate that tumours with high c-myc expression are also associated with IFN resistance. Future research is required to explore the potential of c-myc gene manipulation combined with IFN therapy.

Background/aims: Uveal melanoma is the commonest malignancy of the eye, with a high proportion of patients dying of metastatic disease. Tumours showing a loss of chromosome 3 and gains of chromosome 8 are associated with a worse prognosis. The efficiency of fluorescence in situ hybridisation (FISH) in determining copy numbers of these chromosomes was assessed in individual tumours and related to patient survival., Methods: 33 fresh frozen samples were analysed with centromeric probes for chromosomes 3 and 8. Patient outcomes were divided into two groups: (1) absence of genetic abnormalities (no genetic imbalance) and (2) presence of genetic abnormalities (genetic imbalance). The log rank test was used to compare survival, which was represented by Kaplan-Meier survival curves., Results: Of the 33 tumours analysed, 16 showed evidence of genetic imbalances. Of these 16 tumours, 14 patients had died by the end of the study, with 10 having died of liver metastases. Of the tumours without evidence of genetic imbalances, five patients had died by the end of the study, although none had died as a result of either liver metastases or from the primary uveal melanoma. The difference in survival between the two groups was highly significant (p<0.0001)., Conclusion: The authors have shown that FISH analysis for chromosome 3 and 8 is a reliable and efficient technique in the analysis of fresh frozen tumour specimens and is valuable in the prediction of prognosis in individuals with uveal melanomas.

Background/aims: Accumulating evidence indicates that telomerase activity is repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity has a role in carcinogenesis and immortalisation. To date, telomerase in uveal melanoma and, whether, it may have a role in the development or progression of these tumours has not been described. The expression patterns and the activity of telomerase were investigated in 14 uveal melanoma and these results were correlated with histological and immunohistological features of these tumours., Methods: A modified PCR based telomeric repeat amplification protocol (TRAP) assay was used to demonstrate telomerase activity in 14 uveal melanomas. In addition, in situ hybridisation was used to demonstrate the expression pattern of the telomerase RNA component (hTR) at the single cell level in eight of these globes., Results: The TRAP assay revealed moderate telomerase activity in all uveal melanomas examined. In situ hybridisation visualised a moderate to high upregulation of hTR in the melanoma cells but not in the admixed reactive cells. There was no correlation among tumour location, cell type, or growth fraction and the amount of telomerase activity. In addition, the cells of the germinative zone of the lens demonstrated a strong hTR expression., Conclusion: Telomerase activity is upregulated in uveal melanomas. The expression of hTR was located to the tumour cells and not the reactive tumour infiltrating cells. Strong telomerase expression was also demonstrated in cells of the germinative zone of the lens.

Aims: Overexpression of c-myc protein has independent prognostic significance in a variety of primary and metastatic cutaneous melanomas which suggests a possible role for this gene in melanomagenesis. We have therefore examined the importance of this oncogene in uveal melanoma and studied the coexpression of two other gene products, Bcl-2 and p53, which might contribute to its effect., Methods: The percentage of cells positive for nuclear c-myc expression was estimated by flow cytometric analysis of nuclei extracted from paraffin blocks. The expression of Bcl-2 and p53 protein was assessed by immunohistochemistry. A total of 71 tumours were studied and the results compared with survival with a mean follow up period of 6 years., Results: c-myc was expressed in > 50% of the cells by 70% of the tumours, and was independently associated with improved survival in a Cox multiple regression-model. Although Bcl-2 was expressed by the majority of the cells in 67% tumours, it was without effect on prognosis. None of the cases studied showed convincing positivity for p53. Analysis of coexpression showed that the best survival was seen in c-myc+/Bcl-2+ tumours and the worst in c-myc-/Bcl-2-tumours., Conclusion: The finding of improved rather than reduced survival in c-myc positive tumours is at variance with skin melanoma. There was no evidence to suggest that c-myc was modulated by upregulation of Bcl-2 or p53 inactivation/mutation. Although Bcl-2 is unlikely to have any effect on tumour growth or metastasis, it could contribute to the general lack of susceptibility to apoptosis in these tumours.

Aims/background: The authors have developed transgenic mouse strains at the Arizona Cancer Center using a tyrosinase promoter to target expression of the mutated T24 Ha-ras gene in melanin producing cells. Histopathology and electron microscopy (EM) were performed to characterise the intraocular tumours observed phenotypically., Methods: Transgenic TPras mice (n = 8) and normal, age matched control mice (n = 6) were sacrificed at 3 weeks, 6 weeks, 7 weeks, 4 months, 5 months, 9 months, and 11 months. Six were processed in formalin for light microscopic examination and eight in a glutaraldehyde/formalin solution for electron microscopic examination., Results: Six of the TPras mice were found to have bilateral pigmented melanocytic/RPE proliferations of the uveal tract. The cytological characteristics of the tumours included low nuclear to cytoplasmic ratios (N:C ratios), bland nuclei, and abundant intracytoplasmic melanin. By EM two populations of cells were identified, including spindle-shaped cells with round to oval melanin granules and cuboidal cells with apically located, cigar-shaped, melanin granules, and basement membrane formation. A 3 week and an 11 month old TPras mouse had a higher grade, bilateral, melanocytic proliferation of the uveal tract which, although not metastatic, was morphologically melanoma. Cytological features included increased N:C ratios, nuclear pleomorphism, and prominent nucleoli. The uveal tract was normal in both eyes in all of the control animals., Conclusion: Pigmented intraocular tumours developed in transgenic strains of mice that express a mutated Ha-ras gene in melanin producing cells. The morphology was most consistent with a melanoma in two of the mice and a benign melanocytic/RPE proliferation in the remaining mice.

Aims: To predict 5 year survival in patients with uveal malignant melanomas DNA indices were studied., Methods: Using 45 paraffin embedded uveal malignant melanomas, the DNA index and S phase fraction of each tumour were the predictor variables recorded., Results: Using the Cox proportional hazards model, aneuploid tumours and tumours which had an S phase fraction greater than 4% were significant predictors of early death. In order to demonstrate a biological gradient between a larger DNA index and shorter survival time, linear regression and transformed linear regression models were used. However, no such gradient could be demonstrated., Conclusion: Although this study shows promise for the use of DNA studies in the prognosis of uveal malignant melanoma, the exact role of these techniques remains to be determined.

Background: For uveal melanoma it has been demonstrated that aneuploidy correlates with worse clinical outcome. However, a striking variation in incidence of aneuploidy is reported for uveal melanomas., Methods: Flow cytometry was used to study retrospectively DNA-ploidy of 132 uveal melanomas on paraffin embedded material. Thirty five patients received 2 x 4 Gy doses of irradiation 24 and 48 hours before enucleation. Correlation between DNA-ploidy and histopathological grading, largest tumour diameter, tumour height, tumour location, scleral invasion, and TNM classification was assessed. Survival analysis methods were used to investigate the predictive value of these variables on clinical outcome., Results: Of the tumours 37% were aneuploid and 63% were diploid. Intratumour ploidy heterogeneity was minimal (92% concordance). A strong correlation (p = 0.009) was found between DNA-ploidy and cell type. No correlation was found between DNA-ploidy and other conventional prognostic variables. Irradiated melanomas were significantly more aneuploid than non-irradiated tumours (p < or = 0.01)., Conclusion: In survival analysis DNA-ploidy and the largest tumour diameter were significant in predicting metastatic outcome (p < or = 0.03 and 0.01 respectively); histological cell type and tumour location were of borderline significance.

The cause of uveal melanoma is unknown. In a few cases, however, factors are found in association with the disease which may play some part in the aetiology. One such factor is inheritance. Twelve families have been reported with adequate documentation during the last century in which two or more members have had uveal melanomas. At least some of these may be the result of an inherited disorder. On available data inheritance is most likely autosomal dominant with partial expressivity or incomplete penetrance. This report describes two more families each of which have two members with uveal melanomas.