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

Abstract

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in <i>GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1</i> and <i>EIF1AX</i>, 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 <i>BAP1</i> but harbored <i>SF3B1</i> mutations, novel frameshift deletions in <i>SF3B1</i> and <i>EIF1AX</i>, as well as a <i>PLCB4</i> mutation outside of the hotspot on exon 20 coinciding with a <i>GNAQ</i> mutation in some UM. We observed samples that harboured mutations in both <i>BAP1</i> and <i>SF3B1</i>, and <i>SF3B1</i> and <i>EIF1AX</i>, respectively. Novel mutations were also identified in <i>TTC28, KTN1, CSMD1</i> and <i>TP53BP1</i>. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. <i>BAP1</i> and <i>SF3B1</i> mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.