Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas

Abstract

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.

Keywords: DNA methylation; PRAME; chromosomal instability; preferentially expressed antigen in melanoma; uveal melanoma.

Figure 1. Defining the threshold for PRAME+ expression status

(A) PRAME mRNA expression plotted from lowest to highest expression for 678 uveal melanoma samples measured by qPCR with a LOESS model (second degree, family = ”Gaussian”, spanning 0.4, fitting by least-squares). (B) Predicted PRAME mRNA expression for an additional “hypothetical” 678 samples based on the LOESS model. (C) Predicted slope change between each of these predicted points. (D) The same process depicted in panels A–C was repeated separately for the RNA-Seq data from 80 TCGA uveal melanoma samples in order to generate a predicted slope change plot. For both datasets, the threshold for PRAME+ (red) was defined as the point where the slope sustainably rose above baseline (blue).

Figure 2. Summary of PRAME expression status measured by qPCR

(A) PRAME expression status with respect to gene expression profile classification in 678 uveal melanomas. (B) PRAME expression status in 454 Class 1 uveal melanomas with respect to 1A/1B sub-classification.

Figure 3. Prognostic significance of PRAME expression status in uveal melanoma

(A) Kaplan-Meier survival plot showing metastasis-free survival for Class 1 and Class 2 tumors combined, with respect to PRAME expression status. (B) Kaplan-Meier survival plot showing metastasis-free survival for Class 2 tumors only, with respect to PRAME expression status. (C) Kaplan-Meier plot showing melanoma-specific mortality for Class 1 and Class 2 tumors combined, with respect to PRAME expression status. (D) Kaplan-Meier survival plot showing melanoma-specific mortality for Class 2 tumors only, with respect to PRAME expression status.

Figure 4. Association of PRAME expression with chromosomal gains and losses

The bar graphs depict chromosomal gains and losses that were significantly associated with PRAME+ tumors when Class 1 and Class 2 tumors were analyzed together, and when each class was analyzed separately. PRAME+ (red), PRAME− (blue).

Figure 5. Transcriptional activation of PRAME is associated with hypomethylation of the PRAME promoter in uveal melanoma

(A) The only normal adult human tissue that expresses high levels of PRAME mRNA is testis. Data were obtained through the GTEx Portal [41]. (B) Locations of 12 CpG sites (blue bars) within or near the PRAME promoter that exhibited significantly decreased methylation in PRAME+ uveal melanomas (n = 41) compared to PRAME- samples (n = 39) at a significance level of FDR < 0.05. (C) Scatter plots showing the relationship between PRAME mRNA expression levels (obtained from TCGA RNA-Seq data) and PRAME promoter methylation (obtained from TCGA Infinium HumanMethylation450 BeadChip data) using two representative methylation probes (cg17648213 and cg27303185). Spearman's rank correlation coefficient was used to determine P-values. Graphs depicting the other 10 differentially methylated probes are in Supplementary Figure S1. (D) Methylation data for the cg27303185 methylation probe was plotted for normal tissues obtained from Marmal-aid [40]. A separate panel (right) depicts PRAME+ and PRAME- uveal melanomas samples for comparison. RPKM, reads per kilobase of transcript per million mapped reads; CPM, counts per million.