Uveal melanoma (UM) is a rare aggressive intraocular tumour with a propensity for liver metastases, occurring in ∼50% of patients. The tumour suppressor BAP1 is considered to be key in UM progression. Herein, we present the largest study to date investigating cellular expression patterns of BAP1 protein in 165 UMs, correlating these patterns to prognosis. Full clinical, histological, genetic, and follow‐up data were available for all patients. BAP1 gene sequencing was performed on a subset of 26 cases. An independent cohort of 14 UMs was examined for comparison. Loss of nuclear BAP1 (nBAP1) protein expression was observed in 54% (88/165) UMs. nBAP1 expression proved to be a significant independent prognostic parameter: it identified two subgroups within monosomy 3 (M3) UM, which are known to have a high risk of metastasis. Strikingly, nBAP1‐positiveM3 UMs were associated with prolonged survival compared to nBAP1‐negative M3 UMs (Log rank, p = 0.014). nBAP1 protein loss did not correlate with a BAP1 mutation in 23% (6/26) of the UMs analysed. Cytoplasmic BAP1 protein (cBAP1) expression was also observed in UM: although appearing ‘predominantly diffuse’ in most nBAP1‐negative UM, a distinct ‘focal perinuclear’ expression pattern – localized immediately adjacent to the cis Golgi – was seen in 31% (18/59). These tumours tended to carry loss‐of‐function BAP1 mutations. Our study demonstrates loss of nBAP1 expression to be the strongest prognostic marker in UM, confirming its importance in UM progression. Our data suggest that non‐genetic mechanisms account for nBAP1 loss in a small number of UMs. In addition, we describe a subset of nBAP1‐negative UM, in which BAP1 is sequestered in perinuclear bodies, most likely within Golgi, warranting further mechanistic investigation.

Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with ‘high-risk’ tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients’ metastatic risk and potentially assess eligibility for new therapies.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

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.

Purpose of the Study: To describe the clinicopathological features, mutational and chromosomal copy number analysis, and 8-year follow-up of a case of bilateral diffuse uveal melanocytic proliferation (BDUMP) associated with clear-cell carcinoma of the endometrium. Methods: Histological evaluation, multiplex ligation-dependent probe amplification (MLPA) analysis and GNAQ/11 mutational analysis were performed in a 67-year-old female patient with the diagnosis of BDUMP. Results: Histological evaluation revealed proliferation of bland spindle cells, diffusely replacing the uveal tract, which showed a proliferation index of less than 1%. There was absence of mutations involving the codon 209 and 183 of GNAQ, and of GNA11. MLPA analysis showed disomy 3 with polysomy 8q for both eyes. The patient died 8 years later of an unrelated condition. Conclusions: Although BDUMP is considered to be a benign proliferative disease, copy number alterations of unknown significance may occur in these lesions.