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PURPOSEValidated and accurate prognostic testing is critical for precision medicine in uveal melanoma (UM). Our aims were to (1) prospectively validate an integrated prognostic classifier combining a 15-gene expression profile (15-GEP) and PRAME RNA expression and (2) identify clinical variables that enhance the prognostic accuracy of the 15-GEP/ PRAME classifier.MATERIALS AND METHODSThis study included 1,577 patients with UM of the choroid and/or ciliary body who were enrolled in the Collaborative Ocular Oncology Group Study Number 2 (COOG2) and prospectively monitored across 26 North American centers. Test results for 15-GEP (class 1 or class 2) and PRAME expression status (negative or positive) were available for all patients. The primary end point was metastasis-free survival (MFS).RESULTS15-GEP was class 1 in 1,082 (68.6%) and class 2 in 495 (31.4%) patients. PRAME status was negative in 1,106 (70.1%) and positive in 471 (29.9%) patients. Five-year MFS was 95.6% (95% CI, 93.9 to 97.4) for class 1/ PRAME (-), 80.6% (95% CI, 73.9 to 87.9) for class 1/ PRAME (+), 58.3% (95% CI, 51.1 to 66.4) for class 2/ PRAME (-), and 44.8% (95% CI, 37.9 to 52.8) for class 2/ PRAME (+). By multivariable Cox proportional hazards analysis, 15-GEP was the most important independent predictor of MFS (hazard ratio [HR], 5.95 [95% CI, 4.43 to 7.99]; P < .001), followed by PRAME status (HR, 1.82 [95% CI, 1.42 to 2.33]; P < .001). The only clinical variable demonstrating additional prognostic value was tumor diameter.CONCLUSIONIn the largest prospective multicenter prognostic biomarker study performed to date in UM to our knowledge, the COOG2 study validated the superior prognostic accuracy of the integrated 15-GEP/ PRAME classifier over 15-GEP alone and clinical prognostic variables. Tumor diameter was found to be the only clinical variable to provide additional prognostic information. This prognostic classifier provides an advanced resource for risk-adjusted metastatic surveillance and adjuvant trial stratification in patients with UM.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8 + T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.

Importance: There has been speculation on the pathogenesis of unilateral multifocal uveal melanoma, but there remains no convincing explanation. Genetic analysis suggests that unilateral multifocal uveal melanoma may represent intraocular metastasis with increased risk of systemic metastasis., Objective: To evaluate the pathogenesis of unilateral multifocal uveal melanoma., Design, Setting, and Participants: This clinical case series was conducted in tertiary academic ocular oncology referral centers and included patients with unilateral multifocal uveal melanoma., Main Outcomes and Measures: Gene expression and mutation profiling of tumor samples., Results: Four patients (all male; age range, 54-77 years) who were diagnosed with uveal melanoma were treated with plaque brachytherapy, and subsequently developed a second discrete uveal melanoma in the same eye were included. None demonstrated ocular or oculodermal melanocytosis. All 8 tumors available for analysis exhibited class 2 gene expression profiles. In all 4 cases, the initial and subsequent tumors were available for targeted DNA sequencing and identical driver mutations were present in both tumors. Data were collected from September 2015 to August 2018., Conclusions and Relevance: Unilateral multifocal uveal melanoma in the absence of ocular melanocytosis appears to occur preferentially in tumors with the class 2 gene expression profile and a BRCA1-associated protein 1 gene (BAP1) mutation. The presence of identical BAP1 mutations in multiple tumors in the same eye in the absence of a germline BAP1 mutation suggests intraocular metastasis rather than independent primary tumors. These findings indicate that the first site of metastasis can be within the eye itself and suggest that patients with unilateral multifocal uveal melanoma may be at increased risk of systemic metastasis.

Purpose: The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM. Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells., Results: Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development., Conclusions: This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs., (©2019 American Association for Cancer Research.)

Ocular melanocytosis is the most important predisposing condition for the eye cancer uveal melanoma (UM). Here, we present a patient who developed UM arising within ocular melanocytosis who was treated with enucleation (eye removal), which provided an invaluable opportunity to interrogate both the UM and adjacent uveal tissue containing the melanocytosis using whole-exome and deep-targeted sequencing. This analysis revealed a clonal PLCB4 mutation in the melanocytosis, confirming that this is indeed a neoplastic condition and explaining why it predisposes to UM. This mutation was present in 100% of analyzed UM cells, indicating that a PLCB4 -mutant cell gave rise to the UM. The earliest aberrations specific to the tumor were loss of Chromosomes 1p, 3, and 9p, which were present in virtually all tumor cells. A mutation in BAP1 arose later on the other copy of Chromosome 3 in a tumor subclone, followed by a gain of Chromosome 8q. These findings provide a mechanistic explanation for the well-known clinical association between ocular melanocytosis and UM by showing that this predisposing condition introduces the first "hit" and thereby increases the stochastic likelihood of acquiring further aberrations leading to UM., (© 2019 Durante et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: To compare the prognostic accuracy of gene expression profiling (GEP) combined with PRAME status vs the clinical Tumor-Node-Metastasis (TNM) staging in patients with uveal melanoma (UM)., Design: Retrospective cohort study., Methods: The study included 240 consecutive patients with UM. Tumors were assessed for GEP status (Class 1 or Class 2) using a validated 15-gene assay and PRAME expression status using quantitative polymerase chain reaction. TNM staging was according to the American Joint Committee on Cancer 8th edition. Statistical analysis included univariate and multivariate Cox proportional hazard models. Metastasis was the primary endpoint., Results: GEP was Class 1 in 128 (53.3%) cases and Class 2 in 112 (46.7%) cases. PRAME status was negative in 157 (65.4%) cases and positive in 83 (34.6%) cases. TNM was stage I in 26 (10.8%) cases, IIA in 67 (27.9%) cases, IIB in 50 (20.8%) cases, IIIA in 59 (24.6%) cases, and IIIB in 38 (15.8%) cases. Metastatic disease was detected in 59 (24.6%) cases after median follow-up of 29 months (mean 42 months; range 1-195 months). Variables associated with metastasis included (in order of decreasing significance): GEP class (P = 1.5 × 10 -8 ), largest basal tumor diameter (P = 2.5 × 10 -6 ), PRAME status (P = 2.6 × 10 -6 ), and TNM stage (P = 3.7 × 10 -6 ). The prognostic accuracy of an optimized 3-category GEP/PRAME model (P = 8.6 × 10 -14 ) was superior to an optimized TNM model (P = 1.3 × 10 -5 )., Conclusions: In UM, molecular prognostic testing using GEP and PRAME provides prognostic accuracy that is superior to TNM staging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.

Background: We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter., Results: Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter., Materials and Methods: Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA., Conclusions: PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Importance: Frequent mutations have been described in the following 5 genes in uveal melanoma (UM): BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. Understanding the prognostic significance of these mutations could facilitate their use in precision medicine., Objective: To determine the associations between driver mutations, gene expression profile (GEP) classification, clinicopathologic features, and patient outcomes in UM., Design, Setting, and Participants: Retrospective study of patients with UM treated by enucleation by a single ocular oncologist between November 1, 1998, and July 31, 2014., Main Outcomes and Measures: Clinicopathologic features, patient outcomes, GEP classification (class 1 or class 2), and mutation status were recorded., Results: The study cohort comprised 81 participants. Their mean age was 61.5 years, and 37% (30 of 81) were female. The GEP classification was class 1 in 35 of 81 (43%), class 2 in 42 of 81 (52%), and unknown in 4 of 81 (5%). BAP1 mutations were identified in 29 of 64 (45%), GNAQ mutations in 36 of 81 (44%), GNA11 mutations in 36 of 81 (44%), SF3B1 mutations in 19 of 81 (24%), and EIF1AX mutations in 14 of 81 (17%). Sixteen of the mutations in BAP1 and 6 of the mutations in EIF1AX were previously unreported in UM. GNAQ and GNA11 mutations were mutually exclusive. BAP1, SF3B1, and EIF1AX mutations were almost mutually exclusive with each other. Using multiple regression analysis, BAP1 mutations were associated with class 2 GEP and older patient. EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement. SF3B1 mutations were associated with younger patient age. GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter. GNA11 mutations were not associated with any of the analyzed features. Using Cox proportional hazards modeling, class 2 GEP was the prognostic factor most strongly associated with metastasis (relative risk, 9.4; 95% CI, 3.1-28.5) and melanoma-specific mortality (relative risk, 15.7; 95% CI, 3.6-69.1) (P < .001 for both). After excluding GEP class, the presence of BAP1 mutations was the factor most strongly associated with metastasis (relative risk, 10.6; 95% CI, 3.4-33.5) and melanoma-specific mortality (relative risk, 9.0; 95% CI, 2.8-29.2) (P < .001 for both)., Conclusions and Relevance: BAP1, SF3B1, and EIF1AX mutations occur during UM tumor progression in an almost mutually exclusive manner and are associated with different levels of metastatic risk. These mutations may have value as prognostic markers in UM.

Purpose: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors., Experimental Design: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR., Results: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1(PRAME-), 38% for Class1(PRAME+), and 71% for Class 2 tumors. Median metastasis-free survival for Class1(PRAME+) patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1(PRAME+) tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003)., Conclusions: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM., (©2016 American Association for Cancer Research.)