RNA-sequencing of 27 patient-derived tissue samples of primary and metastatic melanomas
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ABSTRACT: Analysis of the gene expression profile characterizing primary and metastatic melanomas. These data were integrated with ChIP-seq H3K27ac profiles obtained on the same patients' cohort (E-MTAB-12244).
Project description:Genetic heterogeneity can provide tumors with opportunities for therapy evasion, however the degree of genetic heterogeneity within metastatic melanomas has not been thoroughly investigated. We therefore isolated DNA from different regions of formalin fixed paraffin embedded metastatic melanoma tissue samples and subjected them to amplicon sequencing-based profiling of mutations in a panel of well known cancer genes using the Ion Ampliseq Cancer Panel.
Project description:DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. Here, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients and compared the methylation status with melanoma tissues from the same individuals. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports and indicated that NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease. Our results highlight the importance of using matched fresh melanoma and melanocyte samples in epigenetic studies. Illumina Infinium 450k Human Methylation Beadchip
Project description:DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. Here, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients and compared the methylation status with melanoma tissues from the same individuals. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports and indicated that NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease. Our results highlight the importance of using matched fresh melanoma and melanocyte samples in epigenetic studies. illumina HumanHT-12_v4 BeadChip
Project description:The dominant genetic signature found in melanoma is C to T mutations due to UV radiation. Nucleotide excision repair (NER) recognises and repairs UV-induced DNA damage. We aimed to determine the effect of high UV exposure on the melanoma transcriptome and key NER transcripts in melanoma tumours in association with clinical and genetic features of the disease. 196 primary or metastatic melanomas were utilised for this study. Solar elastosis and transcriptome data was collected. mRNA transcript levels of NER components XPC, DDB1 and DDB2 were quantified and compared to clinical parameters. Solar elastosis negatively correlated with Breslow thickness (-0.251, p=0.017) and was significantly lower in BRAFV600E melanomas. Lower XPC expression was associated with BRAFV600E and NRASQ61R mutations, earlier age of diagnosis and poorer survival. Transcriptome profiling identified an over-representation of DNA repair processes in high vs low solar elastosis. Further analysis revealed DNA repair and kinase activity related transcripts in the low XPC melanomas. XPC deficiency is associated with the presence of kinase mutations and an aggressive disease phenotype, both of which result from the hypermutability of melanoma.
Project description:We report the distribution of human SATB2 in a primary zebrafish melanoma that overexpresses human SATB2, and H3K27Ac histone marks in zebrafish melanomas overexpressing either EGFP (control) or human SATB2.
Project description:A 122-epigenetic gene signature distinguished low- versus high-risk early stage melanomas. We sought to validate the signature, examined clinical correlates in a cohort of primary melanomas of the skin, and studied the underlying transcriptomic aberrations and changes in chromatin in cell lines representing these two groups. H3K27Ac ChipSeq profiles of primary human melanoma cell lines (n=4) as well as corresponding input samples were generated by deep sequencing using Illumina NextSeq500. Purpose: The goals of this study are to identify H3K27Ac differential peak expression between two groups of melanoma cell lines (Epgn1 and Epgn3) and relate this data to RNASeq. Methods: H3K27Ac ChipSeq was performed on 4 melanoma cell lines by deep sequencing 75bp single end reads using Illumina NextSeq500. Reads were aligned and counts called using____. The sequence reads that passed quality filters were analyzed. Results: Using an optimized data analysis workflow, we mapped about 40 million reads per Chip sample and 60 million reads per input sample to the human genome (build _____). Conclusions: Our study allowed us to identify differentially expressed peaks between two classes of melanoma cell lines and relate this information to differential gene expression. Further in vitro and in vivo assays using some of the candidate genes identified will allow us to more fully understand primary melanoma progression.
Project description:Primary uveal melanomas show multiple genetic alterations. To determine mutational status of six human primary uveal melanomas, we performed whole exome sequencing (WES) and called Single Nucleotide Polimorphism (SNPs) to identify somatic mutations in these human primary uveal melanomas.