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Purpose\ud Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach.\ud \ud Methods\ud Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays.\ud \ud Results\ud We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70.\ud \ud Conclusion\ud This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

BACKGROUND The p.V600E mutation in the BRAF protein is the most frequent mutation in cutaneous melanoma and is a recurrent alteration found in common benign naevi. Analysis of the cell-free BRAF c.1799T>A, p.V600E mutation (cfBRAFV600E ) in plasma has emerged as a biomarker for monitoring prognosis and treatment response in patients with melanoma. OBJECTIVES To quantify cfBRAFV600E levels in plasma from patients with melanoma and from patients without melanoma undergoing regular follow-up of their melanocytic lesions, in order to assess the clinical significance of the test. METHODS We quantified cfBRAFV600E by droplet digital polymerase chain reaction in plasma from 146 patients without melanoma undergoing continuous dermatological screening, from 26 stage III and seven stage IV patients with BRAF-mutant melanoma, and from 32 patients with melanoma who were free of disease for 3 or more years. RESULTS Among disease-free patients and individuals without melanoma, 52% presented a high naevus count (> 50) and 49% had clinically atypical naevi. cfBRAFV600E was detected in 71% of patients with stage IV melanoma and 15% with stage III, and in 1·4% of individuals without melanoma. No cfBRAFV600E mutation was detected in disease-free patients with melanoma. Individuals without melanoma had lower cfBRAFV600E levels than patients with melanoma. We established a variant allelic frequency of 0·26% or 5 copies mL-1 of cfBRAFV600E as the optimal cutoff value for identifying patients with melanoma with > 99% specificity. CONCLUSIONS This study suggests that naevus-related factors do not influence the detection of cfBRAFV600E in individuals without melanoma, and supports the clinical diagnostic value of plasma cfBRAFV600E quantification in patients with melanoma. What's already known about this topic? The analysis of the BRAF c.1799T>A (p.V600E) mutation in cell-free (cf)DNA has emerged as a potential biomarker for monitoring prognosis and treatment response in patients with metastatic BRAFV600E melanoma. The BRAFV600E alteration is a common genetic alteration found in benign proliferations such as melanocytic naevi. No information exists about the impact of the number of common acquired naevi or the presence of clinically atypical naevi in cfBRAFV600E detection in an individual. What does this study add? The cfBRAFV600E mutation is detected in plasma from a reduced number of individuals without melanoma undergoing continuous dermatological follow-up. A high number of naevi or the presence of clinically atypical naevi are factors that do not influence cfBRAFV600E detection in an individual. Both total cfBRAF concentration and cfBRAFV600E frequency are effective biomarkers in patients with advanced melanoma but not in patients at early stages or with micrometastases. What is the translational message? Detection of cfBRAFV600E in an individual is not influenced by naevus-related factors. cfBRAFV600E is a robust and reliable biomarker that can be used in dermatological surveillance programmes.

Summary Background Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer‐prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. Objectives To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. Methods We conducted RNA‐Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. Results Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA‐Seq data. In addition, enzyme‐linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. Conclusions Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury‐sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development.Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix‐synthetic capacity.This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor‐β signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention.It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB., Linked Comment: https://doi.org/10.1111/bjd.18104. https://doi.org/10.1111/bjd.18271 available online

Background Melanomas harboring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy (RCM). BRAF mutational status is crucial for the management of metastatic melanoma. Objective To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumors has been also analyzed for the presence of possible confocal features that might be linked with BRAF status. Methods Retrospectively acquired dermoscopic and confocal images of melanoma patients in tertiary referral academic centers: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskall-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. Results Dermoscopically the presence of irregular peripheral streaks and ulceration were positive predictor of BRAF mutated melanomas with a statistically significance value while dotted vessels were more represented in wild type melanomas. None of the evaluated RCM features were correlated with genetic profiling. Conclusions Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF mutated melanoma while dotted vessels are suggestive for wild type melanoma. This article is protected by copyright. All rights reserved.

SummaryBackground Nearly 10% of all cases of cutaneous melanoma (CM) occur in patients with a personal or family history of the disease. Objectives To obtain information about genetic predisposition to CM in Ticino, the southern region of Switzerland, a zone with moderate-to-high CM incidence. Methods We identified germline mutations in highly CM-associated genes (CDKN2A and CDK4) and low/medium-penetrance variants (MC1R and MITF) in patients with multiple primary CMs or individuals with one or more CM and a positive family history for CM or pancreatic cancer among first- or second-degree relatives. Healthy blood donors (n = 146) were included as a control group. Results From July 2010 to July 2012, 57 patients (41 pedigrees) were included. Twenty-six were melanoma-prone families (with at least two cases) and 15 had multiple CMs. Pancreatic cancer was found in six families. The CDKN2A mutation p.V126D was identified in five patients (four families) with a founder effect, whereas CDKN2A A148T was detected in seven cases and seven healthy donors (odds ratio 3.39, 95% confidence interval 1.12–10.23; P = 0.031). At least one MC1R melanoma-associated polymorphism was detected in 39 patients (81%) and 97 healthy donors (66%), with more than one polymorphism in 16 patients (33%) and 25 healthy donors (17%). The MITF variant p.E318K was identified in four patients from three additional pedigrees (8%) and one healthy control (0.7%). Conclusions Inclusion criteria for the Ticino population for genetic assessment should follow the rule of two (two affected individuals in a family or a patient with multiple CMs), as we detected a CDKN2A mutation in almost 10% of our pedigrees (four of 41), MITF p.E318K in 7% (three of 41) and a higher number of MC1R variants than in the control population. This article is protected by copyright. All rights reserved.

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma.

Summary Background The identification of BRAF mutations in melanoma led to the development and implementation of new and effective therapies. Few clinical and histological features have been associated with this mutational status. Objectives The main objective of this study was to investigate clinical, histopathological and dermoscopic characteristics of primary melanomas according to BRAF or NRAS mutational status. Methods An observational retrospective study including melanoma dermoscopy images assessed for somatic mutations in BRAF and NRAS. Results Seventy-two patients were included, 30 women (42%) and 42 men (58%), mean age was 59 ± 15·51 years. BRAF-mutated melanomas were more frequently located on the trunk (n = 18, 64% for BRAF-mutated vs. n = 11, 29% for wild-type melanomas, P = 0·013). Histological ulceration was associated with the presence of BRAF mutations [odds ratio (OR) 3·141; 95% confidence interval (CI) 1·289–7·655; P = 0·002]. The Breslow index tended to be thicker in BRAF-mutated compared with wild-type (P = 0·086). BRAF mutations were present in 28 (39%) patients and only four cases were positive for NRAS mutations (6%), BRAF and NRAS mutations being mutually exclusive. The presence of dermoscopic peppering was associated with MAPK mutations (BRAF and NRAS) (OR 1·68; 95% CI 1·089–2·581; P = 0·015). Dermoscopic ulceration was also associated with BRAF mutations excluding acral and facial melanomas (OR 2·64; 95% CI 1·032–6·754). Conclusions This study showed a correlation between BRAF and NRAS status and dermoscopic findings of ‘peppering’ as an expression of regression and melanophages in the dermis, suggesting a morphological consequence of immune behaviour in BRAF-mutated melanomas.

Background Patients with familial melanoma or multiple primary melanoma represent a high-risk population to hereditary melanoma. Mutations in susceptibility genes, such as CDKN2A, CDK4 and MC1R, have been associated with the development of melanoma. Objectives The purpose of this study was to determine the genotypic background of patients with familial and/or multiple melanoma in southern Brazil. Methods This study analysed 33 cases (5 patients with multiple primary melanoma and 28 patients from families with at least two well documented cases) and 29 controls. Genomic analysis of CDKN2A and CDK4 genes by PCR-SSCP analysis and sequencing and direct sequencing of MC1R were performed in all individuals. Results No functional mutations in CDKN2A or CDK4 were detected in the 62 individuals. Infrequent variants in polymorphic loci of CDKN2A gene were identified in 15 participants (24.2%) and 24/33 (72.8%) cases and 19/27 (70.4%) controls reported at least one infrequent variant in MC1R (P = 0.372). Furthermore, a non-significant tendency towards an association between melanoma risk and MC1R variants G274A and C451T and a non-significant linear tendency to the number of infrequent high-risk variants in MC1R were observed. Conclusions These results suggest that in southern Brazilian population, CDKN2A or CDK4 germinal alterations may have a weaker influence than previously thought and environmental risk factors may play a central role in melanoma susceptibility. However, considering the tendency observed for gene MC1R, low-penetrance genes may be a relevant aetiological factor in southern Brazil with fair skin population and high sunlight exposure.

Reflectance confocal microscopy (RCM) is a noninvasive imaging technique that has been shown to enhance secondary evaluation of melanocytic lesions by allowing for in vivo visualization of microscopic structures and cellular details of the epidermis and superficial dermis at almost histologic resolution. RCM has been used to image various skin conditions, including benign and malignant pigmented lesions, amelanotic melanoma, BCC, and actinic keratosis.

Background: Actinic keratoses (AKs) are considered to be a ‘field of cancerization’ consisting of a histologically abnormal epithelium adjacent to tumour tissue. Treatment of the ‘field of canceri-zation’ is important for the prevention of neoplasm progression. UV radiation, especially UVB, produce genotoxic photoproducts such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photo-products (6-4PPs) in DNA, being major players in skin cancerization. The potential use of DNA photolyases in skin cancer prevention is increasingly being demonstrated. Topical application of a liposome formulation containing CPD photolyase onto human skin provides protection against UV-B-induced damages. Objectives: To assess the effects of topical application of a medical device (Eryf-AK) containing a DNA-repair enzyme, photolyase, encapsulated in liposomes and UV filters, on cancerization field in actinic keratosis (AK). Methods: 13 AK patients were included. Clinical, dermoscopic, and reflectance confocal microscopy (RCM) assessments, as well as skin biopsies, before and after a 4-week treatment were performed. Patients used Eryf-AK twice daily or only a sunscreen (3:1) with a similar sun protection factor (SPF) for one month. Results: Erythema and scaling improved with Eryf-AK. RCM showed a reduction in scaling, detached corneocytes and polygonal nucleated cells in the stratum corneum (p=0.004, p=0.018, and p=0.021), an improvement of the atypical honeycomb pattern, and a decreased number of round nucleated cells at the spinous granulous layer (p

Importance The improved knowledge of clinical, morphologic, and epidemiologic heterogeneity of melanoma in the context of multiple primary and familial melanomas may improve prevention, diagnosis, and prognosis of melanoma. Objective To characterize reflectance confocal microscopy (RCM) morphologic patterns of melanomas in multiple primary and familial melanomas. Design, Setting, and Participants In this cross-sectional, retrospective study, patients in a hospital-based referral center were recruited from March 1, 2010, through August 31, 2013; data analysis was conducted from September 1, 2013, through May 31, 2014. Consecutive primary melanomas, documented by dermoscopic and confocal examination, from multiple primary and familial melanomas with known CDKN2A mutational status were studied. Main Outcomes and Measures Epidemiologic, genetic, dermoscopic, and histologic data were evaluated according to an RCM morphologic classification: dendritic cell, round cell, dermal nest, combined, and nonclassifiable types. Results Fifty-seven melanomas from 50 patients (28 women [56%] and 49 white patients [98%]) were included: 23 dendritic cell (40%), 21 round cell (37%), 2 dermal nests (4%), 2 combined (4%), and 9 nonclassifiable (16%). The median (SD) age of the participants was 53.0 (16.9) years (interquartile range, 41.8-71.2 years), and the median (SD) age at the first melanoma was 46.0 (17.1) years (interquartile range, 35.8-61.5 years). Dendritic cell melanoma was characterized by older age at diagnosis, phototypes 2 and 3, more intense solar exposure, and moderate to severe solar lentigines; it was the most prevalent confocal type in facial lesions and was associated with the lentigo maligna histologic subtype. Round cell melanomas were identified more often in the familial context and in individuals with phototype 1 skin types; RCM features, such as junctional thickening, dense dermal nests, and nucleated cells within papillary dermis, were more frequently found in this subtype. Dermal nest and combined melanoma were associated with the absence of pigmented network on dermoscopy and thicker tumors on histologic analysis. Nonclassifiable type was associated, by RCM, with the absence of pagetoid cells on confocal examination and lower frequency of marked atypia on melanocytes in the basal cell layer; it presented with lower ABCD Total Dermoscopy Scores and RCM scores compared with the other types. CDKN2A mutation carriers may develop any RCM type of melanoma. Conclusions and Relevance Different routes to develop melanoma can be identified according to RCM morphologic classification, with dendritic cell melanomas being associated with chronic sun damage and round cell melanoma with early age at onset and phototype 1 in the context of multiple primary and familial melanomas. The morphologic expression of melanomas via dermoscopy and confocal examination varies according to differences in tumor stage and biological behavior.

Summary Objective. To determine the prognostic value of detecting tyrosinase transcripts in melanoma sentinel lymph nodes (SLNs). Methods. Reverse transcription (RT) PCR for tyrosinase mRNA was performed on negative SLNs of 76 patients with melanoma. Results. Tyrosinase mRNA was found in 39 patients (51.3%). After a median follow-up period of 51 months, significant differences were found in overall survival (OS) but not in disease-free survival (DFS). The 5-year OS and DFS rates were 97.2% and 80%, respectively, for RT-PCR tyrosinase-negative (TN) patients vs. 78.67% and 66.24% for RT-PCR tyrosinase-positive (TP) patients (P = 0.019 and P = 0.38, respectively). Of four progressing patients in the TN group, three relapsed with subcutaneous, soft-tissue or lymph-node metastases, while seven out of nine progressing patients in the TP group relapsed at visceral sites. Conclusions. No significant differences in DFS were found by RT-PCR tyrosinase expression analysis at melanoma SLNs. Significant differences in OS could be related to a different pattern of relapse and must be confirmed after a longer follow-up time.