Christina L. Decatur

Uveal melanoma (UM) is characterized by a small number of recurrent genetic alterations that determine metastatic propensity. BAP1 loss and PRAME expression define the dominant prognostic axes in UM, yet how they promote malignant progression remains unclear. We profiled 190,535 cells from normal uvea, uveal nevus, primary and metastatic UM using single-cell transcriptomics, T cell receptor sequencing, spatial transcriptomics and isogenic perturbation models. Normal melanocytes, nevus cells and UM cells formed a transcriptional continuum marked by loss of differentiation and emergence of neural crest-like, stress-responsive, hypoxic-glycolytic and immune-interacting states. BAP1 loss and PRAME expression imposed distinct but convergent immunoregulatory programs, inducing interferon and TNF–NF-κB signaling and MHC-I expression, with HLA-E showing the strongest response. These alterations were accompanied by macrophage and CD8 ⁺ T cell remodeling. PRAME-enriched tumor regions formed spatially organized niches enriched for macrophages and plasma cells. These findings define BAP1 loss and PRAME expression as distinct but convergent axes of tumor–immune coevolution and nominate HLA-E as a candidate mediator of immune resistance.

Uveal melanoma (UM) is an aggressive eye cancer that frequently results in metastatic death despite successful primary tumor treatment. Subclinical micrometastasis is thought to occur early, when tumors are small and difficult to distinguish from benign nevi. However, the early genetic evolution of UM is poorly understood, and biomarkers for malignant transformation are lacking. Here, we perform integrated genetic profiling of 1140 primary UMs, including 131 small tumors. A clinically available 15-gene expression profile (15-GEP) prospectively validated by our group is more accurate than driver mutations for predicting patient survival. Small tumors are significantly more likely to be in earlier stages of genetic evolution than larger tumors. Further, the 15-GEP support vector machine discriminant score predicts small tumors undergoing transformation from low-risk Class 1 to high-risk Class 2 profile. These results shed light on the early genetic evolution of UM and move us closer to a molecular definition of malignant transformation in this cancer type.

Uveal melanoma (UM) is an aggressive cancer of the eye that frequently results in metastatic death. UMs are most likely to metastasize when they are small, at a time when they are difficult to distinguish from benign nevi and often observed without treatment. Unfortunately, little is known about the early genetic evolution of UM or potential biomarkers to indicate small tumors undergoing malignant transformation. Here, we performed targeted next generation sequencing for the 7 canonical UM driver mutations in 1140 primary UMs, including 131 small early-stage tumors. We found that the evolutionary burst of genetic aberrations that determines the archetypal UM subtypes and metastatic propensity has already occurred by the time most small tumors are biopsied, although a significantly larger proportion of small tumors are still evolving compared to larger tumors. We found that the 15-gene expression profile (15-GEP) support vector machine discriminant score was the best indicator of tumors in transition from low-risk Class 1 to high-risk Class 2 signature. While BAP1 , SF3B1 and EIF1AX mutations were associated with poor, intermediate and good prognosis, respectively, mutation analysis was inferior to the prospectively validated 15-GEP + PRAME expression classifier for predicting metastasis-free and overall survival. These results provide a more complete picture of genetic evolution in UM, and they move us closer to a molecular definition of malignant transformation in this cancer type.

PURPOSE: Validated 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 METHODS: This 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). RESULTS: 15-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. CONCLUSION: In 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.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.

BAP1KDvsWT_HyperMethDecGE: All significantly hypermethylated probes from hypermethylated/downregulated genes (p < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KDvsWT_HypoMethIncGE: All significantly hypomethylated probes from hypomethylated/upregulated genes (p < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMethDecGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypermethylated/downregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMethIncGE_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for hypomethylated/upregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMethDecGE_KEGG: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypermethylated/downregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMethIncGE_KEGG: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for hypomethylated/upregulated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_TCGA_HyperMethDecGE_Gene: All significantly hypermethylated probes (FDR < 0.05) from hypermethylated/downregulated genes shared in common between the shBAP1KD (p < 0.05) and TCGA (FDR < 0.05) datasets. BAP1KD_TCGA_HypoMethIncGE_Gene: All significantly hypomethylated probes (FDR < 0.05) from hypomethylated/upregulated genes shared in common between the shBAP1KD (p < 0.05) and TCGA (FDR < 0.05) datasets.

BAP1KDvsWT_HyperMethyl: All significantly hypermethylated probes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KDvsWT_HypoMethyl: All significantly hypomethylated probes (FDR < 0.05) with an absolute value (log2(RNA fold change)) > 1 and absolute value (methylation Delta Beta value) > 0.01 in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMeth_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for top 1000 hypermethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMeth_ChrLoc: Chromosomal loci enriched (FDR < 0.05) for top 1000 hypomethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HyperMeth_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for top 1000 hypermethylated genes in BAP1KDvsWT 92.1 and Mel202 cells. BAP1KD_HypoMeth_KEGG_REACTOME: Functional (KEGG and REACTOME) pathways enriched (FDR < 0.05) for for top 1000 hypomethylated genes in BAP1KDvsWT 92.1 and Mel202 cells.

TCGA_Methyl_DMP: All significantly methylated probes (FDR < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from the 80 TCGA cases. JWH_Methyl_DMP: All significantly methylated probes (p < 0.05) in Class 2 uveal melanomas compared to Class 1 uveal melanomas from 12 cases of the senior author (JWH). TCGA_D12_PC1_PC2_Methyl: The 67,261 probes shared between the top 20% most variable methylated probes from the TCGA and JWH datasets used in the principal component analyses. The percentage contribution to PC1 and PC2 are displayed for all probes. TCGA_D12_Probes_Top5000PC1: The 204 probes shared between the TCGA and JWH datasets out of the Top 5000 shared most variable methylated probes contributing to PC1 in the principal component analyses.

BAP1 knockdown alters the epigenomic profile in uveal melanoma (UM) cells. (A) Western blot of tetracycline-inducible 92.1 and Mel202 shBAP1 UM cells without (-) and with (+) induction by doxycycline. (B) Multidimensional scaling (MDS) plot of the 1000 most variably methylated probes in the BAP1KD cell experiment, comparing the tetracycline-induced BAP1KD cells (BAP1KD, red) to the un-induced BAP1 control cells (BAP1WT, green). (C) Hierarchical clustering dendrogram, using all methylation probes, plotting the induced (BAP1KD) and uninduced (BAP1WT) 92.1 and Mel202 biological replicates.