Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma

Abstract

Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with 'high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.

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

“…EIF1AX mutations were detected in the present study in 19% of UM, which is consistent with that reported by other groups [11,14,18,25]. Interestingly, two UM demonstrated mutations in both EIF1AX and SF3B1 despite previous reports describing that these occur in a mutually exclusive manner [11,25].…”

“…The frequency of BAP1 mutations in the present study was 43% in total, occurring in 82% of M3-UM; these data are consistent with the findings of others [11,14,19,25]. The presence of a BAP1 mutation in UM was associated with a worse survival.…”

“…The frequency of SF3B1 mutations in UM ranges in the literature from 11-34% [14,25], and in this study SF3B1 mutations occurred in 21% of cases. SF3B1 mutations are reported to occur mainly in D3-UM associated with late onset metastasis and decreased survival (22).…”

Intraocular biopsy of uveal melanoma Risk assessment and identification of genetic prognostic markers

“…New targeted essays are developed so that next‐generation sequencing can allow for a simultaneous assessment of selected gene mutations and chromosomal copy number (Smit et al. ).…”

Chromosomal rearrangements in uveal melanoma: Chromothripsis

“…Mutation analysis of GNAQ, GNA11, EIF1AX, SF3B1, and BAP1 was performed with Sanger sequencing and Ion Torrent next-generation sequencing (NGS; Thermo Fisher Scientific, Waltham, MA) as described before. 32 UM without a GNAQ or GNA11 mutation were sequenced for PLCB4 and CYSLTR2. If the tumor did not harbor a mutation in EIF1AX, SF3B1 or BAP1, mutation analysis for SRSF2 was performed.…”