Jolanda Vaarwater

Patient survival in uveal melanoma is not affected by oncogenic mutations in GNAQ and GNA11.

Background:Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas.Methods:Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas. The mutation status was correlated with disease-free survival (DFS) and other parameters.Results:None of the tumours harboured a GNAQ exon 4 mutation. A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours. Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209. Six tumours did not show any mutations in exons 4 and 5 of these genes. Univariate analyses showed no correlation between DFS and the mutation status.Conclusion:GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.

Chemokine receptor CCR7 expression predicts poor outcome in uveal melanoma and relates to liver metastasis whereas expression of CXCR4 is not of clinical relevance.

PURPOSEnTo 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.nnnMETHODSnPrimary 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.nnnRESULTSnStrong 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.nnnCONCLUSIONSnOur 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.

The Prognostic Value of Extraocular Extension in Relation to Monosomy 3 and Gain of Chromosome 8q in Uveal Melanoma

PURPOSEnTo 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.nnnMETHODSnRetrospective 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.nnnRESULTSnIn 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.nnnCONCLUSIONSnLarger 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.

Risk factors associated with secondary enucleation after fractionated stereotactic radiotherapy in uveal melanoma

To evaluate risk factors for secondary enucleation after fractionated stereotactic radiotherapy (fSRT) in uveal melanoma.

Metastatic disease in polyploid uveal melanoma patients is associated with BAP1 mutations

PURPOSEnMost 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.nnnMETHODSnIn 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.nnnRESULTSnTwenty-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).nnnCONCLUSIONSnThe 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.

Correlation of Gene Mutation Status with Copy Number Profile in Uveal Melanoma

Copy number variations (CNV), gene expression profiling, and the recurrent gene mutations in BAP1, SF3B1, or EIF1AX, can be used to stratify uveal melanoma patients. Recently, Decatur et al described an association between specific gene mutations and gene expression profiles. In this report, we describe the relationship between mutational status of BAP1, SF3B1, and EIF1AX and CNV patterns by using conventional karyotyping data to explore the nature of these CNVs. In total, 277 patients from the Rotterdam Ocular Melanoma Study group (ROMS) were included in this study. Details of the patient population and methods are described in the Supplementary Methods (available at www.aaojournals.org). For 207 samples, single nucleotide polymorphism array data and mutational status were available. These patients were divided into 4 subgroups: patients with (1) immunohistochemically BAP1-negative tumors (BAP1), (2) SF3B1-mutated tumors (SF3B1), and (3) EIF1AX-mutated tumors (EIF1AX). Patients in which the tumors were immunohistochemically BAP1 positive and contained no SF3B1 or EIF1AX mutations were classified as (4) no recurrent mutations (NRM) tumors. This group was further split into subgroups with and without the loss of heterozygosity (LOH) of the BAP1 locus (NRMLOHþ and NRM , respectively). We visualized sample distribution of the single nucleotide polymorphism array data across the predefined groups to obtain chromosomal patterns (Fig 1A) and found that BAP1 tumors (n 1⁄4 84) were characterized by loss of chromosome 3 (95% of cases) and gain of the entire long (q) arm of chromosome 8 (80%). Gain of chromosome 8q was often accompanied by loss of chromosome 8p (26%). Loss of chromosomes 1p, 6q, and 16q was also observed frequently (25%, 19%, and 20%, respectively). SF3B1 tumors (n 1⁄4 42) were characterized by gain of chromosomes 6p (85%) and 8q (73%), and loss of chromosomes 6q (52%) and 11q (45%). In addition, loss of chromosome 1p (36%) and gain of chromosome 9q (24%) were present in SF3B1 tumors. BAP1 uveal melanomas (UMs) showed gains or losses of entire chromosomes or chromosome arms, whereas SF3B1 UMs were characterized by structural variants of the distal ends of the chromosomes (e.g., gain of chromosome 8q23qter, 8q13qter, 6p12.3pter or loss of 6q16.1qter and 11q22qter). EIF1AX tumors (n 1⁄4 26) showed gain of chromosome 6p (often 6p21.33pter) in 65% of the cases. No other recurrent CNVs were observed in EIF1AX UMs. NRM tumors (n 1⁄4 28) were characterized by gains of the distal ends of chromosome 6p (53%) and 8q (28%). NRMLOHþ UMs (n 1⁄4 19) were characterized by monosomy 3, because we stratified for LOH of BAP1. These samples showed a very similar CNV profile as the BAP1 UMs because these tumors also contained the typical gain of the entire arm of chromosome 8q, often accompanied by loss of chromosome 8p, and gain of the entire arm of chromosome 6p with loss of chromosome 6q. To substantiate the finding that BAP1 UMs contain mainly entire chromosome or chromosome arm CNVs and SF3B1 UMs smaller, partial chromosome CNVs, we calculated the percentages of aneuploidy and the number of copynumber (CN) events for all groups. These results (Fig 1B) showed significantly greater percentages of aneuploidy in BAP1 tumors compared with SF3B1 tumors (11.8% and 9.3%, respectively; P 1⁄4 0.024). EIF1AX tumors and NRM UMs harbored the least percentage of aneuploidy (1.7% and 4.3%, respectively), which were significantly lower than in BAP1 and SF3B1 tumors (P < 0.001 for all). As for CN events, SF3B1 UMs harbored the most CN events, although this was not significantly higher than BAP1 UMs (P 1⁄4 0.074) in the single nucleotide polymorphism array data when corrected for multiple testing. However, both groups harbored more CN events than EIF1AX and NRM UMs (P 0.01 for all). Furthermore, cytogenetic data and mutational status of 70 patients were used for analysis of the different types of chromosomal anomalies. Translocations or partial chromosome arm CNVs were categorized as chromosomal structural variants (CSVs) and were numerated manually. Although cases were limited, SF3B1 UMs strongly associated with >3 CSVs per tumor compared with BAP1 (P 1⁄4 0.002; Fig 1D). Moreover, 70% (7 of 10) of the UMs with >3 CSVs harbored an SF3B1 mutation, showing that tumors with multiple CSVs are most likely to be SF3B1 mutated. Another type of chromosomal anomaly found in UMs were isochromosomes. These were observed in 74% (25/34) of all BAP1 tumors (Table S1, available at www.aaojournals.org), whereas both SF3B1 and EIF1AX UMs harbored no isochromosomes. Remarkably, beside isochromosomes 8q, isochromosome 6p was recurrent in BAP1 UMs. Other chromosomal changes in BAP UMs were almost exclusively translocations with breakpoints in the centromeric regions (and thus entire chromosome arms), whereas SF3B1 tumors contained more structural aberrations with recurrent distal breakpoints on chromosomes 6 and 8, suggesting that different mechanisms play a role in causing these anomalies. These novel insights in the chromosomal patterns and different types of chromosomal anomalies show that UMs with different mutated genes represent distinct molecular classes. We and others have shown that stratification based on BAP1, SF3B1, and EIF1AX mutations reveal that patients with these mutated genes have a high, intermediate, or low risk of developing metastases, respectively. A limitation in this study was the use of BAP1 immunohistochemistry to replacedin partdBAP1 sequencing. For example, we expect BAP1 mutations in the NRMLOHþ group, because these tumors revealed similar CNVs as the BAP1 UMs. We are currently investigating this using deep sequencing and Western blotting. Furthermore, in this study all except 2 tumors were obtained from enucleated eyes; therefore, the tumor population is skewed toward larger tumors. These tumors tend to have a worse prognosis than eyes that undergo eye-conserving therapy. Nevertheless, this limitation does not affect the clinical significance of our observations that UMs have different molecular classes. In conclusion, in this report we show that patients with UMs harbor mutation-specific chromosomal patterns in the tumor. These

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.

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

PURPOSEnUveal 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.nnnDESIGNnMulticenter, retrospective case series.nnnPARTICIPANTSnPatients diagnosed with iris melanoma or iris nevi who underwent surgical intervention as primary or secondary treatment.nnnMETHODSnNext-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.nnnMAIN OUTCOME MEASURESnMutation and copy number status were analyzed. Results of BAP1 immunohistochemistry were used for survival analysis.nnnRESULTSnIn 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.nnnCONCLUSIONSnMutations 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 nevixa0can harbor BAP1 mutations and may be designated iris melanocytic tumors of uncertain malignant potential.