Aberrant DNA methylation of uveal melanoma drivers as an excellent prognostic tool

Abstract Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between -0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, DNA methylation values of nine genes, HTR2B , AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2 , and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUC ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining A HNAK2, CALHM2, and IL12RB and Signature 2 A HNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (HR = 4.38, 95% CI 1.30-16.41, HR = 5.59, 95% CI 1.30-16.41; HR = 3.43, 95% CI 1.30-16.41, HR = 4.61, 95% CI 1.30-16.41 and HR = 4.95, 95% CI 1.39-17.58, respectively). Conclusions: Our results demonstrate a significant role of DNA methylation aberrancy in UM progression. The advantages of DNA as a biological material and excellent prediction accuracies of methylation markers open the perspective for their more extensive clinical use.