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Integrative Copy Number Analysis of Uveal Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3052 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Integrative Copy Number Analysis of Uveal Melanoma Reveals Novel Candidate Genes Involved in Tumorigenesis Including a Tumor Suppressor Role for PHF10/BAF45a Hima Anbunathan1, Ruth Verstraten1,2, Arun D. Singh3, J. William Harbour4, and Anne M. Bowcock1,2,5 Abstract Purpose: Uveal melanoma is a primary malignancy of the clusters of genes in focal copy number regions whose expres- eye with oncogenic mutations in GNAQ, GNA11, or CYSLTR2, sion was associated with metastasis and worse overall sur- and additional mutations in BAP1 (usually associated with vival. This included genes from Chr 1p36, 3p21, and 8q24.3. LOH of Chr 3), SF3B1,orEIF1AX. There are other character- At Chr 6q27, we identified two tumors with homozygous istic chromosomal alterations, but their significance is not deletion of PHF10/BAF45a and one with a frameshift muta- clear. tion with concomitant loss of the wild-type allele. Down- Experimental Design: To investigate genes driving chro- regulation of PHF10 in uveal melanoma cell lines and tumors mosomal alterations, we integrated copy number, transcrip- altered a number of biological pathways including develop- tome, and mutation data from three cohorts and followed up ment and adhesion. These findings provide support for a role key findings. for PHF10 as a novel tumor suppressor at Chr 6q27. Results: We observed significant enrichment of transcripts Conclusions: Integration of copy number, transcriptome, on chromosomes 1p, 3, 6, 8, and 16q and identified seven and mutation data revealed novel candidate genes playing a shared focal copy number alterations (FCNAs) on Chr 1p36, role in uveal melanoma pathogenesis and a potential tumor 2q37, 3, 6q25, 6q27, and 8q24. Integrated analyses revealed suppressor role for PHF10. Introduction profiles (GEPs), some of which are predictive of metastatic risk (3, 4). Mutations in GNAQ, GNA11, CYSLTR2, and PLCB4 Uveal melanoma is the most common primary intraocular that constitutively activate Gaq signaling are seen in almost all malignant tumor diagnosed in approximately six cases per mil- tumors in a mutually exclusive manner (5–8). Inactivating muta- lion per year. Approximately 40% of patients develop metastatic tions in BAP1 at chromosome 3p21 (9) are found in tumors with melanoma to the liver within 10 years (1). Advances made in the loss of heterozygosity for Chr 3 (LOH3) and a GEP predictive of local methods of treatment of primary uveal melanoma have not metastatic risk (class 2 tumors; ref. 10), and mutations in SF3B1 or led to an improvement in survival and after metastasis there is a EIF1AX are found in tumors with disomy 3 and a GEP associated median survival time of less than 6 months (2). Uveal melanomas with an intermediate and low likelihood of metastasis, respec- display characteristic genomic signatures including recurrent tively (class 1 tumors; refs. 4, 11, 12). Whole-genome sequencing chromosomal aberrations, gene mutations, and gene expression has also revealed potential mutations in other genes (13). In addition to LOH3, uveal melanomas are characterized by chromosomal alterations that can include Chr 1p loss, 6p gain, 6q 1National Heart and Lung Institute, Imperial College, London, United Kingdom. loss, 8p loss, 8q gain, and 16q loss (14–20). These DNA copy 2Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, number aberrations (CNAs) are an important category of genetic New York, New York. 3Department of Ophthalmic Oncology, Cole Eye Institute, alterations that can lead to development and progression of 4 Cleveland Clinic, Cleveland, Ohio. Bascom Palmer Eye Institute, Sylvester cancers by affecting gene dosage, sometimes amplifying genes Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, Univer- conferring a proliferative or metastatic advantage or leading to sity of Miami Miller School of Medicine, Miami, Florida. 5Departments of Der- matology and Genetics & Genome Sciences, Icahn School of Medicine at Mount loss of function of tumor suppressor genes. However, the signif- Sinai, New York, New York. icance of CNAs in most cancers, including in uveal melanoma is poorly understood. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). A global characterization of copy number, transcriptome, mutation, and methylation status in uveal melanoma has been Corresponding Author: Anne M. Bowcock, Icahn School of Medicine at Mount described (20). This confirmed much previous work and showed Sinai, One Gustave L. Levy Place, Box 1130, New York, NY 10029. Phone: 212-659- 8256; Fax: 212-987-2240; E-mail: [email protected] four distinct molecular subtypes of CNA, each associated with a varying degree of metastatic risk. Here, we describe copy number Clin Cancer Res 2019;25:5156–66 analysis of 182 tumor samples from three different cohorts doi: 10.1158/1078-0432.CCR-18-3052 followed by integration of transcriptomic, methylation, and Ó2019 American Association for Cancer Research. mutation data to look for genes driving copy number loss or 5156 Clin Cancer Res; 25(16) August 15, 2019 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3052 Copy Number Analysis Reveals Novel UM Candidate Genes to an assay for chemotaxis as described elsewhere (27). Collagen Translational Relevance invasion (ECM552 Millipore) and adhesion assays (ECM545 Here we describe integration of copy number, transcrip- Millipore) were performed according to the manufacturer's tomic, epigenomic, and mutational data in uveal melanoma instructions. across three independent datasets. Although loss of one copy of chromosome 3 is usually accompanied by loss-of-function Western blot analysis mutations in BAP1, the significance of other chromosomal This was performed with standard approaches by querying a set changes such as loss of Chr 1p, 6q, and 8p and gain of Chr 6p of proteins identified following PHF10 knockdown in cell lines. and 8q are not clear. We describe candidate genes on altered The following antibodies were used: PHF10 (ab154637, Abcam), chromosomes affecting uveal melanoma pathogenesis and JAZF1 (ab80329, Abcam), SMAD2 (ab40855, Abcam), and patient survival. We also provide evidence for a tumor sup- PCGF3/5 (ab201510, Abcam). pressor role for PHF10 on Chr 6q27. Its loss affects early pathways in cell development such as transcriptional regula- Statistical analysis tion as well as adhesion and migration. This knowledge will be Three replicates per cell line were used for PHF10 knock- important as we progress toward a more comprehensive downs. Intergroup differences were assessed with a one-way molecular diagnosis of uveal melanoma and determination ANOVA with Bonferroni post hoc test. Results were expressed as of prognosis. Besides providing important insights in the mean and SD or in boxplots. The Kaplan–Meier method and development of uveal melanoma, these studies provide novel log-rank test were used to compare the survival plots and a therapeutic targets for this cancer. univariate Cox proportion hazard model was used to compare the effects of high and low expression levels of select genes on overall survival. Time was computed for days. Survival was defined as the elapsed interval until date of last follow-up or gain, extending earlier studies investigating the relationship with date of death. An effect was considered significant at an FDR chromosomal alterations and gene expression in uveal melanoma q-value less than 0.05. with microarrays (21). We describe candidate genes on chromo- somes 1p, 8q, and 6q and provide evidence for a tumor suppressor role for PHF10 mapping to Chr 6q27. Functional studies provided Results insights into the consequences of loss of PHF10. Broad copy number alterations in uveal melanoma Genomic DNA samples from 182 primary enucleated uveal melanomas (87 with matched normal DNA) from three different Materials and Methods cohorts (Supplementary Table S2A) were profiled on three dif- Data source and analysis ferent high-resolution SNP platforms encompassing >700 K Data from 182 primary uveal melanoma tumor samples were probes with a median interprobe spacing of 24 to 18 Kb. The obtained from three different cohorts: Washington University analysis pipeline is described in Supplementary Fig. S1 and (WU), Cleveland Clinic (CC), and TCGA. All of the samples were revealed a high degree of nonrandom CNAs across samples from enucleated specimens obtained from adult patients after they had all cohorts. Twelve broad events affected >10% of all samples. This provided written informed consent. Studies were conducted in included Chr 1q gain, 6p gain, 8p gain, 8q gain, 21q gain and Chr accordance with recognized ethical guidelines (e.g., Declaration 1p loss, 3p loss, 3q loss, 6q loss, 8p loss, 9p loss, and 16q loss of Helsinki, CIOMS, Belmont Report, U.S. Common Rule) and (Supplementary Table S2B; Supplementary Fig. S2A). Chr 8q gain were approved by an institutional review board. The data analysis and LOH3 were detected in more than 50% of all samples, workflow and methods of analysis of SNP arrays (copy number), consistent with previous studies (15, 28). Ploidy estimates RNA sequencing (RNA-seq), and exome sequencing are described revealed an average of 2.4n in 103 tumors from the WU and CC in Supplementary Fig. S1. cohorts (Supplementary Fig. S2C) due to a minority of tumors being tetraploid that was more common in class 2 versus class 1 PHF10 knockdown and RNA sequencing tumors consistent with BAP1 deficiency being the prognostic Established cell lines derived from primary uveal melanomas predictor for patients with polyploid tumors (29). [Mel202 (22), 92-1 (23) and Mel290 (24) (described in Supple- Unsupervised hierarchical clustering of all uveal melanoma mentary Table S1)] were subjected to PHF10 knockdown with samples (N ¼ 182) revealed four distinct clusters similar to those siRNA (Santa Cruz Biotechnology, sc-95343) or a siRNA control described elsewhere (ref.
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