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

Keywords: chromosome; clinical samples; copy number; mutation; next-generation sequencing; prognostication; uveal melanoma.

Figure 1

Flowchart of 117 UM specimens examined in the present study: n = 76 frozen-resection (2 post-irradiation); n = 27 Biopsy (24 post-irradiation); n = 14 FFPE. Four patients were lost to follow-up and excluded from survival analysis. n = 55 were D3, and n = 59 were M3 or ID3. Proportion of cases with the genetic alteration listed are highlighted by the coloured boxes. Each box represents 5% of UM patients examined.

Figure 2

Kaplan–Meier survival curves estimate survival in UM patients stratified by: (A) SF3B1 wild-type/mutation status in D3-UM n = 51 (Log Rank, p = 0.027); (B) BAP1 wild-type/mutation status n = 113 (Log Rank, p  <  0.001); (C) extra copies of chr 8q in M3/ID3-UM n = 59 (Log Rank, p = 0.014) and (D) SF3B1 wild-type/mutation status in D3-UM n = 51 and extra copies of chr 8q in M3/ID3-UM n = 59 (Log Rank, p  <  0.001). Number of events indicates the number of deaths due to metastatic melanoma. Log Rank tests were used to compare survival across groups.