Identification of clinically-relevant genetic alterations in uveal melanoma using RNA sequencing

IntroductionUveal melanoma is a lethal intraocular tumour, in which the presence of certain genetic alterations correlates with the risk of metastatic dissemination and patient survival. RNA data is typically used to transcriptionally characterise tumours and their micro-environment. In this study, we tested the detectability of all key genetic alterations in uveal melanoma from RNA sequencing data. MethodsCohort-wide gene expression profiling was used to classify tumours at the transcriptional level. In individual samples, copy number alterations affecting chromosomes 3 and 8q were analysed by measuring expressed allelic imbalances of heterozygous common single nucleotide polymorphisms. Mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX were identified by screening of hotspot regions and by evaluating their transcriptional effects. All findings were cross-validated with DNA-derived data in a training cohort of 80 primary uveal melanomas studied by The Cancer Genome Atlas (TCGA) initiative, and in five prospectively analysed cases from our institution. ResultsUnsupervised gene expression profiling strongly correlated to the presence of chromosome 3 alterations, but was not reliable in identifying other (clinically-)relevant genetic alterations. However, the presence of both chromosome 3 and 8q copy number alterations could be successfully inferred from expressed allelic imbalances in most tumours. The majority of mutations were adequately recognised at the RNA level by their nucleotide changes (all genes), alternative splicing around the mutant position (BAP1) and transcriptome-wide aberrant splice junction usage (SF3B1). Notably, in the TCGA cohort we detected previously unreported mutations in BAP1 (n=3) and EIF1AX (n=5), that were missed by the original DNA sequencing. In our prospective cohort, all mutations and copy number alterations were successfully identified at the RNA level by combining the described approaches. ConclusionIn addition to providing gene expression levels and profiles, RNA from uveal melanomas presents insights into the expressed tumour genotype and its phenotypic consequences. Such complete analysis of transcriptional data may augment or even substitute current DNA-based approaches, and has potential applicability in both research and clinical practice.