Uveal Melanoma Patients Have a Distinct Metabolic Phenotype in Peripheral Blood.

Uveal melanomas (UM) are detected earlier. Consequently, tumors are smaller, allowing for novel eye-preserving treatments. This reduces tumor tissue available for genomic profiling. Additionally, these small tumors can be hard to differentiate from nevi, creating the need for minimally invasive detection and prognostication. Metabolites show promise as minimally invasive detection by resembling the biological phenotype. In this pilot study, we determined metabolite patterns in the peripheral blood of UM patients (n = 113) and controls (n = 46) using untargeted metabolomics. Using a random forest classifier (RFC) and leave-one-out cross-validation, we confirmed discriminatory metabolite patterns in UM patients compared to controls with an area under the curve of the receiver operating characteristic of 0.99 in both positive and negative ion modes. The RFC and leave-one-out cross-validation did not reveal discriminatory metabolite patterns in high-risk versus low-risk of metastasizing in UM patients. Ten-time repeated analyses of the RFC and LOOCV using 50% randomly distributed samples showed similar results for UM patients versus controls and prognostic groups. Pathway analysis using annotated metabolites indicated dysregulation of several processes associated with malignancies. Consequently, minimally invasive metabolomics could potentially allow for screening as it distinguishes metabolite patterns that are putatively associated with oncogenic processes in the peripheral blood plasma of UM patients from controls at the time of diagnosis.

Molecular Genetics of Conjunctival Melanoma and Prognostic Value of TERT Promoter Mutation Analysis.

The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.

Chromosomal rearrangements in uveal melanoma: Chromothripsis.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the Western world. Recurrent mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, EIF1AX, and SF3B1 are described as well as non-random chromosomal aberrations. Chromothripsis is a rare event in which chromosomes are shattered and rearranged and has been reported in a variety of cancers including UM. SNP arrays of 249 UM from patients who underwent enucleation, biopsy or endoresection were reviewed for the presence of chromothripsis. Chromothripsis was defined as ten or more breakpoints per chromosome involved. Genetic analysis of GNAQ, GNA11, BAP1, SF3B1, and EIF1AX was conducted using Sanger and next-generation sequencing. In addition, immunohistochemistry for BAP1 was performed. Chromothripsis was detected in 7 out of 249 tumors and the affected chromosomes were chromosomes 3, 5, 6, 8, 12, and 13. The mean total of fragments per chromosome was 39.8 (range 12-116). In 1 UM, chromothripsis was present in 2 different chromosomes. GNAQ, GNA11 or CYSLTR2 mutations were present in 6 of these tumors and 5 tumors harbored a BAP1 mutation and/or lacked BAP1 protein expression by immunohistochemistry. Four of these tumors metastasized and for the fifth only short follow-up data are available. One of these metastatic tumors harbored an SF3B1 mutation. No EIF1AX mutations were detected in any of the tumors. To conclude, chromothripsis is a rare event in UM, occurring in 2.8% of samples and without significant association with mutations in any of the common UM driver genes.

Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma.

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.

Genetic Background of Iris Melanomas and Iris Melanocytic Tumors of Uncertain Malignant Potential.

PURPOSE: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Iris melanoma comprises 4% to 10% of all UMs and has a lower mortality rate. The genetic changes in iris melanoma are not as well characterized as ciliary body or choroidal melanoma. The aim of this study was to gain more insight into the genetic background of iris melanoma and iris nevi. DESIGN: Multicenter, retrospective case series. PARTICIPANTS: Patients diagnosed with iris melanoma or iris nevi who underwent surgical intervention as primary or secondary treatment. METHODS: Next-generation sequencing of GNAQ, GNA11, EIF1AX, SF3B1, BAP1, NRAS, BRAF, PTEN, c-Kit, TP53, and TERT was performed on 30 iris melanomas and 7 iris nevi. Copy number status was detected using single nucleotide polymorphisms (SNPs) included in the next-generation sequencing (NGS) panel, SNP array, or fluorescent in situ hybridization. BAP1 immunohistochemistry was performed on all samples. MAIN OUTCOME MEASURES: Mutation and copy number status were analyzed. Results of BAP1 immunohistochemistry were used for survival analysis. RESULTS: In 26 of the 30 iris melanoma and all iris nevi, at least 1 mutation was identified. Multiple mutations were detected in 23 iris melanoma and 5 nevi, as well as mutations in GNAQ and GNA11. Furthermore, 13 of 30 BAP1, 5 of 30 EIF1AX, and 2 of 30 SF3B1 mutations were identified in iris melanoma. No correlation between BAP1 status and disease-free survival was found. The iris nevi showed 1 EIF1AX and 3 BAP1 mutations. Two of the nevi, with a BAP1 mutation, were histologically borderline malignant. Mutations in NRAS, BRAF, PTEN, c-KIT, and TP53 were detected in 6 iris melanomas and 4 iris nevi. CONCLUSIONS: Mutations that are often found in uveal and cutaneous melanoma were identified in this cohort of iris melanomas and iris nevi. Therefore, iris melanomas harbor a molecular profile comparable to both choroidal melanoma and cutaneous melanoma. These findings may offer adjuvant targeted therapies for iris melanoma. There was no prognostic significance of BAP1 expression as seen in choroidal melanoma. Consequently, iris melanoma is a distinct molecular subgroup of UM. Histologic borderline malignant iris nevi can harbor BAP1 mutations and may be designated iris melanocytic tumors of uncertain malignant potential.

Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases.

PURPOSE: To investigate the prevalence and prognostic value of SF3B1 and EIF1AX mutations in uveal melanoma (UM) patients. DESIGN: Case series. PARTICIPANTS: Cohort of 151 patients diagnosed with and treated for UM. METHODS: SF3B1 and EIF1AX mutations in primary tumors were investigated using whole-exome sequencing (n = 25) and Sanger sequencing (n = 151). For the detection of BAP1 mutations, a previously reported cohort of 90 patients was extended using BAP1 sequencing or immunohistochemistry. MAIN OUTCOME MEASURES: The status of SF3B1, EIF1AX, and BAP1 in tumors of patients were correlated to clinical, histopathologic, and genetic parameters. Survival analyses were performed for patients whose tumors had SF3B1, EIF1AX, and BAP1 mutations. RESULTS: Patients with tumors harboring EIF1AX mutations rarely demonstrated metastases (2 of 28 patients) and overall had a longer disease-free survival (DFS; 190.1 vs. 100.2 months; P < 0.001). Within the patient group with disomy 3, UM patients with an SF3B1 mutation had an increased metastatic risk compared with those without an SF3B1 mutation (DFS, 132.8 vs. 174.4 months; P = 0.008). Patients with such a mutation were more prone to demonstrate late metastases (median, 8.2 years; range, 23-145 months). Patients with UM and loss of BAP1 expression had a significantly decreased survival (DFS, 69.0 vs. 147.9 months; P < 0.001). CONCLUSIONS: According to our data, patients with UM can be classified into 3 groups, of which EIF1AX-mutated tumors and tumors without BAP1, SF3B1, or EIF1AX mutations are associated with prolonged survival and low metastatic risk, SF3B1-mutated tumors are associated with late metastasis, and tumors with an aberrant BAP1 are associated with an early metastatic risk and rapid decline in patient DFS.

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.

8q Gain Has No Additional Predictive Value in SF3B1MUT Uveal Melanoma but Is Predictive for a Worse Prognosis in Patients with BAP1MUT Uveal Melanoma.

Purpose: Gain of chromosome 8q has been associated with poor prognosis in uveal melanoma (UM), and an increase in the absolute number of 8q-copies correlated with an even shorter survival. Splicing factor 3b subunit 1 (SF3B1)-mutated (SF3B1MUT) tumors display structural chromosomal anomalies and frequently show a partial gain of chromosome 8qter. A recent subset of SF3B1MUT UM with early-onset metastases has been identified, prompting the investigation of the relationship between survival, 8q gain, and SF3B1MUT UM. Design: Retrospective cohort study. Subjects: Patients diagnosed with UM who underwent enucleation or received a biopsy at the Erasmus MC Cancer Institute or the Rotterdam Eye Hospital, The Netherlands were included. Methods: Fifty-nine patients with SF3B1MUT tumors and 211 patients with BRCA1 associated protein 1 (BAP1)-mutated (BAP1MUT) tumors were included in this study. Copy number status and gene expression were assessed using either a single nucleotide polymorphism array, fluorescence in situ hybridization, and karyotyping, or a combination of these techniques. Disease-free survival was determined and a cut-off of 60 months was used to define early-onset metastatic disease. Main Outcome Measures: Disease-free survival. Results: Forty-eight patients with SF3B1MUT UM (81%) had chromosome 8q gain (3 copies, 78%; 4 copies, 22%). Kaplan-Meier analysis of SF3B1MUT UM did not indicate a difference in survival in patients with or without gain of 8q (P = 0.99). Furthermore, the number of 8q copies was not associated with survival when comparing early (P = 0.97) versus late (P = 0.23) metastases group. In contrast, the presence of 8q gain (86%) was correlated with a decreased survival in BAP1MUT UM (P = 0.013). Conclusions: We did not find a correlation between 8q gain and early-onset metastasis in SF3B1MUT tumors. Gain of 8q has no additional predictive value in SF3B1MUT tumors. In contrast, 8q gain is predictive of a worse prognosis in patients with BAP1MUT tumors. Thus, gain of chromosome 8q has additional predictive value for BAP1MUT tumors, but not for SF3B1MUT tumors. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Protein and mRNA Expression in Uveal Melanoma Cell Lines Are Related to GNA and BAP1 Mutation Status.

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.

The Pediatric and Young Adult Choroidal and Ciliary Body Melanoma Genetic Study, A Survey by the European Ophthalmic Oncology Group.

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%).

Genome-wide aberrant methylation in primary metastatic UM and their matched metastases.

Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.

Identification of Early-Onset Metastasis in SF3B1 Mutated Uveal Melanoma.

Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.

Radiological Patterns of Uveal Melanoma Liver Metastases in Correlation to Genetic Status.

This study reports the role played by the mutation status of Uveal Melanoma (UM) in relation to hepatic metastatic patterns as seen on imaging modalities. Radiological images were obtained from 123 patients treated at the Erasmus Medical Center Rotterdam or the Rotterdam Eye Hospital. Radiological images were derived from either computed tomography or magnetic resonance imaging. Hepatic metastatic patterns were classified by counting the number of metastases found in the liver. Miliary metastatic pattern (innumerable small metastases in the entire liver) was analyzed separately. Mutation status was determined in 85 patients. Median disease-free survival (DFS) and survival with metastases differed significantly between each of the metastatic patterns (respectively, p = 0.009, p < 0.001), both in favor of patients with less hepatic metastases. The mutation status of the primary tumor was not correlated with any hepatic tumor profiles (p = 0.296). Of the patients who had a solitary metastasis (n = 18), 11 originated from a primary BAP1-mutated tumors and one from a primary SF3B1-mutated tumor. Of the patients who had a miliary metastasis pattern (n = 24), 17 had a primary BAP1-mutated tumor and two had a primary SF3B1-mutated tumor. Chromosome 8p loss was significantly more in patients with more metastases (p = 0.045). Moreover, the primary UMs of patients with miliary metastases harbored more chromosome 8p and 1p loss, compared to patients with single solitary metastasis (p = 0.035 and p = 0.026, respectively). In conclusion, our study shows that there is an inverse correlation of the number of metastasis with the DFS and metastasized survival, indicating separate growth patterns. We also revealed that the number and type of metastases is irrelevant to the prognostic mutation status of the tumor, showing that both BAP1- and SF3B1-mutated UM can result in solitary and miliary metastases, indicating that other processes lay ground to the different metastatic patterns.

SRSF2 Mutations in Uveal Melanoma: A Preference for In-Frame Deletions?

Background: Uveal melanoma (UM) is the most common primary ocular malignancy in adults in the Western world. UM with a mutation in SF3B1, a spliceosome gene, is characterized by three or more structural changes of chromosome 1, 6, 8, 9, or 11. Also UM without a mutation in SF3B1 harbors similar chromosomal aberrations. Since, in addition to SF3B1, mutations in U2AF1 and SRSF2 have also been observed in hematological malignancies, UM without a SF3B1 mutation-but with the characteristic chromosomal pattern-might harbor mutations in one of these genes. Methods: 42 UMs were selected based on their chromosomal profile and wildtype SF3B1 status. Sanger sequencing covering the U2AF1 (exon 2 and 7) hotspots and SRSF2 (exon 1 and 2) was performed on DNA extracted from tumor tissue. Data of three UM with an SRSF2 mutation was extracted from the The Cancer Genome Atlas (TCGA). Results: Heterozygous in-frame SRSF2 deletions affecting amino acids 92-100 were detected in two UMs (5%) of 42 selected tumors and in three TGCA UM specimens. Both the UM with an SRSF2 mutation from our cohort and the UM samples from the TCGA showed more than four structural chromosomal aberrations including (partial) gain of chromosome 6 and 8, although in two TCGA UMs monosomy 3 was observed. Conclusions: Whereas in myelodysplastic syndrome predominantly missense SRSF2 mutations are described, the observed SRSF2 mutations in UM are all in-frame deletions of 8-9 amino acids. This suggests that the R625 missense SF3B1 mutations and SRSF2 mutations in UM are different compared to the spliceosome gene mutations in hematological cancers, and probably target a different, as yet unknown, set of genes involved in uveal melanoma etiology.

Aberrant MicroRNA Expression and Its Implications for Uveal Melanoma Metastasis.

Uveal melanoma (UM) is the most frequently found primary intra-ocular tumor in adults. It is a highly aggressive cancer that causes metastasis-related mortality in up to half of the patients. Many independent studies have reported somatic genetic changes associated with high metastatic risk, such as monosomy of chromosome 3 and mutations in BAP1. Still, the mechanisms that drive metastatic spread are largely unknown. This study aimed to elucidate the potential role of microRNAs in the metastasis of UM. Using a next-generation sequencing approach in 26 UM samples we identified thirteen differentially expressed microRNAs between high-risk UM and low/intermediate-risk UM, including the known oncomirs microRNA-17-5p, microRNA-21-5p, and miR-151a-3p. Integration of the differentially expressed microRNAs with expression data of predicted target genes revealed 106 genes likely to be affected by aberrant microRNA expression. These genes were involved in pathways such as cell cycle regulation, EGF signaling and EIF2 signaling. Our findings demonstrate that aberrant microRNA expression in UM may affect the expression of genes in a variety of cancer-related pathways. This implies that some microRNAs can be responsible for UM metastasis and are promising potential targets for future treatment.

Multi-Modality Analysis Improves Survival Prediction in Enucleated Uveal Melanoma Patients.

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.

Molecular cytogenetic analysis of archival uveal melanoma with known clinical outcome.

Uveal melanoma (UM) is the most common primary intraocular tumor in the Western world. Cytogenetically, this tumor is characterized by typical chromosomal aberrations such as loss of 1p, 3, and 6q, and gain of 6p and 8q. Routinely, karyotyping and fluorescent in situ hybridization (FISH) on fresh tumor-biopsies are used to identify chromosomal changes. In addition, archival UM samples can be examined using comparative genomic hybridization (CGH). In the presented study, we used CGH on a series of 46 archival uveal melanomas to identify chromosomal changes. In 44 tumors aberrations were present and classic prognostic markers as loss of 1p (12 tumors, 26.1%), monosomy 3 (26 tumors, 56.5%), loss of 6q (10 tumors, 21.7%), and gain of chromosome arm 8q (27 tumors, 58.7%) were observed. Gain of chromosome arms 18q or 21q was found in three UMs. Multiplex ligation-dependent probe amplification (MLPA), a novel technique in UM, was performed to verify this low number of chromosome 18 and 21 abnormalities, but we could not confirm the previously reported gain of 18q11.2 and 21q11.2 as poor prognostic factors in UM.

Regional deletion and amplification on chromosome 6 in a uveal melanoma case without abnormalities on chromosomes 1p, 3 and 8.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Loss of the long arm and gain of the short arm of chromosome 6 are frequently observed chromosomal aberrations in UM, together with loss of chromosome 1p36, loss of chromosome 3 and gain of chromosome 8. This suggests the presence of one or more oncogenes on 6p and tumor suppressor genes at 6q that are involved in UM development. Both regions, however, have not been well defined yet. Furthermore in other neoplasms gain of 6p and loss of 6q are frequently occurring events. In this case report, we describe the delineation of a partial gain on chromosome 6p and a partial deletion on 6q in a UM with the objective to pinpoint smaller candidate regions on chromosome 6 involved in UM development. Conventional cytogenetics, comparative genomic hybridization (CGH) and fluorescence in-situ hybridization (FISH) were used to delineate regions of loss and gain on chromosome 6 in this UM patient. With conventional cytogenetics a deleted region was found on chromosome 6q that was further delineated to a region ranging from 6q16.1 to 6q22 using CGH and FISH. A region of gain from 6pter to 6p21.2 was also demarcated with CGH and FISH. No other deletions or amplifications on recurrently involved chromosomes were found in this patient. This study indicates the presence of one or more tumor suppressor genes on chromosomal region 6q16.1-6q22 and the presence of one or more oncogenes on chromosomal region 6pter-6p21.2, which are likely to be important in UM and other tumors.