Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in and , most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for but harbored mutations, novel frameshift deletions in and , as well as a mutation outside of the hotspot on exon 20 coinciding with a mutation in some UM. We observed samples that harboured mutations in both and , and and , respectively. Novel mutations were also identified in and . NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. and mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.