Identification of Early-Onset Metastasis in SF3B1 Mutated Uveal Melanoma

Abstract

Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor SF3B1, and whilst patients with such SF3B1 mutations generally have an intermediate risk on developing metastatic disease, a third of these patients develop early metastasis within 5 years after diagnosis. We therefore investigated whether clinical and/or genetic variables could be indicative of short progression-free survival (PFS < 60 months) or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1^mut) UM patients. We collected 146 SF3B1^mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1^mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1^mut group vs. the long PFS group (mean: 14.7 (±3.7 SD, p = 0.001). Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1^mut-specific canonical transcripts (e.g., a low expression of ABHD6 indicative for early-onset metastatic disease) or distinct expression of SF3B1^mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1^mut UM.

Keywords: RNA-seq; SF3B1 mutation; aberrant splicing; early metastasis; uveal melanoma.

Figure 1

Flowchart visualizing study design and aims. (A) Data from SF3B1^mut UM from 11 cohorts were collected and described with stratification criteria. (B) Differential gene expression analysis (DGE) was performed and the ROMS, and TCGA-UVM data were intersected to investigate SF3B1^mut specific transcripts. (C) DGE was performed on SF3B1^mut-only samples using the PFS stratification criteria to investigate transcript expression characteristic for early onset (PFS < 60) and late onset (PFS ≥ 60 months). Finally, in silico results were validated in vitro.

Figure 2

Kaplan–Meier survival curve of all SF3B1^mut UM. Grey area indicates PFS < 60 months. Dashed gray line indicates median progression free survival percentage with corresponding time from either diagnosis or treatment, dependent on the description in the original papers. Blue line shows progression-free survival with a confidence interval of 95% indicated by the blue area and censored data indicated by vertical bar.

Figure 3

Differential gene expression in SF3B1^mut UM. (A) Volcano plot of the differential expression analysis between SF3B1^mut (n = 12) and SF3B1^wt (n = 14) UM within the ROMS cohort. Genes significantly down-regulated (blue) and up-regulated (red) in SF3B1^mut UM are shown. Gene names for the top 50 genes (based on descending -log₁₀ q-value) and top 20 (based on |log₂ fold change|) for both directions are shown. Genes that were found to be differentially expressed in both cohorts (a and b; n = 617) are highlighted by a dark green outer circle. The x-axis displays the log₂ fold-change and y-axis displays the adjusted p-value (q) on a -log₁₀ scale. The total amount of tested genes is shown on top. (B) Same as a), except for the differential expression analysis between SF3B1^mut (n = 15) and SF3B1^wt (n = 61) UM within the TCGA-UVM cohort. (C) Venn diagram displays differential gene expression results for ROMS, ROMS and TCGA, and TCGA cohorts.

Figure 4

Differential gene expression between SF3B1^mut with short PFS < 60 (Early-onset) vs. SF3B1^mut with a long PFS ≥ 60 months (Late-onset). Overview of the most differentially expressed genes between SF3B1^mut with an PFS < 60 from the combined (ROMS and TCGA-UVM) cohort. ROMS samples are depicted with closed circles, and TCGA-UVM are depicted with open circles. The boxplots show the variance stabilizing transformation (VST-transformed and batch corrected) expression per PFS category and metastatic status for (A) ABHD6, for (B) CSRNP1, for (C) BT2G, and for (D) TAGLN. All results show p-adjusted value < 0.05.

Figure 5

Increased levels of ABHD6 can be associated with SF3B1^mut UM with late-onset metastatic disease (PFS ≥ 60 months). (A) RT-qPCR performed in triplicates using CHMP2A expression as a normalizer of 10 primary UM (see Supplementary Figure S5). (B) ABHD6 IHC on all SF3B1^mut UM samples that were available and stratified for early- and late-onset metastatic disease. Red error bars represent standard deviation and red dot represents mean. Wilcoxon rank sum test was used to evaluate statistical difference of delta Ct values and IHC scores shown in scatterplots in panel A and B. p-value < 0.05 was considered statistically significant. (1*–7 and S17) ABHD6 IHC staining of eight primary UM samples, which are a selection of samples in panel A and B. The corresponding delta Ct values and IHC values with regard to histology are represented in Supplementary Figure S5. (40× magnification). (*) indicates the control sample with a PFS < 60 months.

Figure 6

SF3B1^mut UM is characterized by aberrant splicing events. Unsupervised clustering (Euclidean distances and Ward.D2 method) on the differential exon usages between SF3B1^mut samples in the ROMS cohort with PFS < 60 months and PFS ≥ 60 months. Values are mean-centered read counts represented with a z score of differentially expressed splicing events in genes (blue, low expression; red, high expression). Top bars represent PFS-status (yellow; PFS < 60 months and purple; PFS ≥ 60 months). Asterisk (*) indicates novel splicing aberrations that are either donors or acceptors.