Genomic and transcriptomic landscape of conjunctival melanoma

Abstract

Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye. Similar to cutaneous melanoma (CM), it originates from melanocytes. Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view. To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues. Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions. This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis. In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type. In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions. Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene. Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM. Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM.

Fig 1. Mutational spectrum of CJM.

(A) The proportions of different SNV changes show that C>T transitions dominate the mutational spectrum. Grey bars on the top indicate the number of non-neutral mutations in coding regions (see Methods). (B) Two main mutational signatures, detected in our dataset and presenting the highest correlation with COSMIC signatures 7 and 30 (v2 March 2015), are shown.

Fig 2. Principal-component analysis (PCA) of the mutational spectrum data.

PCA is showing the similarity between CJM and CM, as well as a possible separation of tarsal vs. bulbar CJM tumors.

Fig 3. Landscape of single nucleotide and copy number variations in key driver genes in CJM, according to the 4-group genetic classification of CM.

Each column represents alterations in 17 known cancer driver genes in one tumor sample; the number of non-neutral coding somatic mutations for each patient is indicated by the grey bars on the top of each column and the frequency of mutation for each gene is shown on the right side of the figure.

Fig 4. The p.P532S mutation in ACSS3 stimulates cell proliferation.

CRMM-1 cells were left untreated or transfected with a plasmid containing a wild-type ACSS3 cDNA (pCMV-ACSS3-wt) or a cDNA containing the mutation p.P532S (pCMV-ACSS3-mut). Proliferation was assessed after 24 hours, by means of the MTT colorimetric test. Data are indicated in arbitrary units, corresponding to background-corrected absorbance values at 570 nm. Asterisks indicate significant p-values (6.91x10^-7 between untreated and pCMV-ACSS3-mut and 3.39x10^-6 between pCMV-ACSS3-wt and pCMV-ACSS3-mut, by t-test).

Fig 5. Landscape of CNVs in CJM.

Sub-chromosomal level CNVs are shown, in relationship to specific characteristics of each tumor.

Fig 6. Transcriptomic classification of CJM.

(A) Heatmap depicting pairwise similarity (Pearson’s correlation coefficients) between the Self Organizing Maps (SOM) portraits of single patients, highlighting the presence of three distinct clusters of samples, based on gene expression similarity (CM06-10-07, CM11-01-09, and CM03-05-04-02-08). CM gene sets mapped to the data allow for the classification of CJM into an “cell cycle high subtype”, a “keratin high subtype”, and a “immune high subtype”. Grey bars on the top of each column indicate the number of non-neutral coding somatic variants in each patient. (B) Overexpression profiles in selected hallmarks of cancer gene sets, scaled in units of the gene set Z-score in the three clusters.