Methylated CfDNA may distinguish between high- and intermediate-risk uveal melanoma: a pilot study

Uveal melanoma (UM) is a highly aggressive malignancy with a metastatic risk that depends on the molecular subclass. This subclass can be determined through molecular characterization of tumor-derived tissue. With eye-sparing treatments, tumor tissue is rarely available for molecular testing. We hypothesized that minimal invasive biomarkers such as methylated cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) can be used for prognosis and monitoring of patients.

Plasma cfDNA was isolated from healthy blood donors (HBDs, N = 19) and UM patients (N = 22). Plasma was collected at baseline (localized disease, N = 13) and during follow-up (metastatic disease, N = 9) from independent patients with high metastatic risk (HR, N = 11) (monosomy 3 and/or BAP1-mutated tumor) or intermediate metastatic risk (IR, N = 11) (disomy 3 and/or SF3B1-mutated tumor). Methylation signatures were determined using genome-wide LpnPI-based methylated DNA sequencing (MeD-seq). Samples with a CpG/reads ratio < 20% (N = 3) were excluded. IchorCNA was used to estimate the tumor fraction. cfDNA samples with detectable tumor fraction (N = 2) were analyzed separately from the other cfDNA samples without detectable tumor fraction (N = 18) to reduce noise in downstream analyses. Differentially methylated regions (DMRs) were identified between the following predefined subgroups: UM (N = 11) vs. HBDs (N = 19), and HR (N = 10) vs. IR (N = 7). To visualize clustering, principal component analysis (PCA) and hierarchical clustering was performed on the DMRs with fold change > 2.0. Gene set enrichment analysis (GSEA, Z-score > 2.0 and p < 0.05) was performed to evaluate biological relevance.

Distinct clustering was observed between UM and HBDs samples, and between HR and IR samples, although outliers were present in the latter comparison. GSEA implicated eight canonical pathways including the S100 Family Signaling Pathway and RAF/MAP kinase cascade, which are linked to tumorigenesis and immune processes.

This pilot study reports on cfDNA methylation signatures that differentiates UM patients from HBDs, and may distinguish between intermediate and high risk UM subgroups, supporting its prognostic potential. However, its role in monitoring disease progression requires further validation. Independent replication studies are warranted to confirm our findings and evaluate the clinical applicability in UM.