Defining the Molecular Genetics of Dermoscopic Naevus Patterns.

Melanocytic naevi are common melanocytic proliferations that may simulate the appearance of cutaneous melanoma. Naevi commonly harbour somatic mutations implicated in melanomagenesis but in most cases lack the necessary genomic alterations required for melanoma development. While the mitogen-activated protein kinase pathway and ultraviolet radiation strongly contribute to naevogenesis, the somatic mutational landscape of dermoscopic naevus subsets distinguishes some of the molecular hallmarks of naevi in relation to melanoma. We herein discuss the classification of naevi and theories of naevogenesis and review the current literature on the somatic alterations in naevi and melanoma. This review focusses on the clinical-dermoscopic-pathological and genomic correlation of naevi that shapes the current understanding of naevi.

MicroRNA expression profiling of cutaneous squamous cell carcinomas and precursor lesions.

Background: Actinic keratoses (AK) are pre-malignant skin lesions caused by chronic sun exposure. Progression from an AK to intraepidermal carcinoma (IEC) and a cutaneous squamous cell carcinoma (SCC) is well known but the rate of transformation to an invasive SCC is highly variable. Since no definitive biomarkers are available, treatment decisions are made ad hoc. Objectives: To fully characterise our AK to SCC progression series, we performed microRNA (miRNA) microarray expression profiling of normal and photodamaged skin, as well as AKs, IEC, and invasive SCCs. Methods: The study recruited 27 patients who donated fresh biopsies of normal skin, photodamaged skin, AK, IEC, and SCC (n = 67 specimens). All miRbase (v.21) miRNAs were profiled to identify miRNAs related to SCC progression. miRNAs were validated using qRT-PCR and in vitro phenotypic assays. Results: There were 234 robustly expressed miRNAs across the tissue collection, which resulted in 20 miRNA that were differentially expressed ((cor)p ≤ 0.05 and ≥ 10 fold) between normal skin and SCC. Hierarchical clustering all samples illustrated that AKs, IEC, and SCCs were largely indistinguishable, which confirms the premalignant status of an AK. A panel of miRNAs showed significant dysregulation between normal and photodamaged skin and AK. Importantly, we found miR-34a-5p and miR-31-5p had significant differential expression between AKs and IEC and IEC and SCC respectively. Phenotypic assays determined that the miR-31 duplex had opposing effects on SCC cell lines which suggests that dysregulation of this duplex may be related to the dynamic control of progression of transformed keratinocytes. Conclusions: This study confirmed the continuum of AK with IEC and SCC highlighting that miRNA expression plays a role in keratinocyte transformation. Development of our putative miRNA biomarker candidates is warranted to aid in clinical management of patients experiencing high AK load to determine the most appropriate treatment.

Genome-Scale DNA Methylation Analysis Identifies Repeat Element Alterations that Modulate the Genomic Stability of Melanocytic Nevi.

Acquired melanocytic nevi grow and persist in a stable form into adulthood. Using genome-wide methylation profiling, we evaluated 32 histopathologically and dermoscopically characterized nevi to identify the key epigenetic regulatory mechanisms involved in nevogenesis. Benign (69% globular and 31% nonspecific dermoscopic pattern) and dysplastic (95% reticular/nonspecific dermoscopic pattern) nevi were dissimilar, with only two shared differentially methylated loci. Benign nevi showed an increase in both genome-scale methylation and methylation of Alu/LINE-1 retrotransposable elements, a marker of genomic stability, as well as global methylation. In contrast, dysplastic nevi showed evidence for genomic instability through the hypomethylation of Alu/LINE-1 (Alu: P = 0.00019; LINE-1: P = 0.000035). Using dermoscopic classifications, reticular/nonspecific patterned nevi had 59,572 5'-C-phosphate-G-3' differentially methylated loci (Q < 0.05), whereas globular nevi had no significant differentially methylated loci. In reticular/nonspecific patterned nevi, the tumor suppressor PTEN had the greatest proportion of hypermethylated 5'-C-phosphate-G-3' loci in its promoter region than all other assayed gene promoters. The relative activity of reticular/nonspecific nevi was evidenced by 50,720 hypomethylated loci being enriched for accessible chromatin and 8,852 hypermethylated loci strongly enriched, for example, marks of active gene promoters, which suggests that gain of DNA methylation observed in these nevus types plays a role in gene regulation.

RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers.

Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

The "melanoma-enriched" microRNA miR-4731-5p acts as a tumour suppressor.

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the 'cell cycle' pathway and the 'melanosome'. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and the melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.