PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma

Abstract

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.

Figure 1

Gene expression profiling of Class 1 primary uveal melanomas based on metastatic status. (A) Heatmap showing the 50 most highly up-regulated and down-regulated genes in 5 Class 1 primary uveal melanomas that metastasized (Class1^met+ tumors, indicated by diamonds) versus 8 that did not (Class1^met− tumors). Gene expression profiles were obtained using Illumina Human HT12v4 BeadChip arrays and analyzed with Significance Analysis of Microarrays (SAM). Blue = decreased gene expression, red = increased gene expression in Class1^met+ versus Class1^met− tumors. (B) Unsupervised principal component analysis of the same dataset, showing differential clustering of Class1^met+ (red) and Class1^met− (blue) tumors. Red and blue ellipsoids represent 95% confidence intervals for each respective group. (C) Circos plot of the same dataset demonstrating genes that are significantly up-regulated (red) or down-regulated (blue) in Class1^met+ compared to Class1^met− tumors with respect to chromosomal location. (D) Box-and-whisker plots of PRAME mRNA expression in Class1^met+ versus Class1^met− tumors ≥1 year of follow-up using the Illumina Human HT12v4 BeadChip array dataset. (E) Box-and-whisker plots of PRAME mRNA expression in Class1^met+ versus Class1^met− tumors with ≥3 years of follow-up using qPCR. (F) Representative examples of immunohistochemical staining for PRAME in Class1^PRAME+ and Class1^PRAME− tumors, 100x magnification. Blue, PRAME nuclear staining (arrow); brown, intrinsic melanin pigment; red, nuclear counterstain. (G) Analysis of PRAME mRNA expression with respect to Class 1A versus Class 1B designation. In box-and-whiskers plots, the central box represents the 25^th to 75^th percentiles, the middle line represents the median, the horizontal bars represent the minimum and maximum values, except for “far out” values, indicated by red boxes and defined as values larger than the upper 75% plus 3 times the interquartile range.

Figure 2

Survival analysis in patients with Class 1 primary uveal melanoma. (A) Kaplan-Meier plot showing metastasis-free survival in 39 patients with Class1^PRAME− (blue) and 25 patients with Class1^PRAME+ (red) uveal melanomas. (B) Kaplan-Meier plot showing metastasis-free survival in 16 patients with Disomy3^PRAME− (blue) and 9 patients with Disomy3^PRAME+ (red) uveal melanomas.

Figure 3

Functional and chromosome copy number analyses in Class 1 primary uveal melanoma. (A) Gene Set Enrichment Analysis (GSEA) of annotated gene sets exhibiting significant enrichment for genes that were up-regulated in Class1^PRAME+ tumors relative to Class1^PRAME− tumors. (B) High density Affymetrix Genome-Wide Human SNP 6.0 Array analysis of Class1^PRAME+ and Class1^PRAME− tumors with respect to metastatic status. Blue indicates copy number loss, and red, copy number gain.

Figure 4

Molecular hypothesis of tumor progression in uveal melanoma, highlighting the role of PRAME reported herein. Increased PRAME mRNA expression in Class 1 uveal melanomas is associated with transcriptional up-regulation of key genes involved in chromosome maintenance and stability, many of which are located on chromosome 1q and 6p and contain regulatory elements for transcription factors that interact with PRAME, including the retinoic acid receptor and NFY complexes. These observations suggest a possible feed-forward mechanism in which progressively increasing PRAME expression and specific chromosomal gains mutually reinforce one another to promote Class 1 tumor progression. There was a significant association of this pathway with SF3B1 mutations, which were mutually exclusive with EIF1AX mutations, suggesting a bifurcated pathway. This Class 1 metastatic pathway was distinct from the more common pathway leading to metastasis through the bi-allelic loss of BAP1 and acquisition of the Class 2 gene expression profile.