High- and intermediate-risk susceptibility variants in melanoma families from the Mediterranean area: A multicentre cohort from the MelaNostrum Consortium.

BACKGROUND: Most of large epidemiological studies on melanoma susceptibility have been conducted on fair skinned individuals (US, Australia and Northern Europe), while Southern European populations, characterized by high UV exposure and dark-skinned individuals, are underrepresented. OBJECTIVES: We report a comprehensive pooled analysis of established high- and intermediate-penetrance genetic variants and clinical characteristics of Mediterranean melanoma families from the MelaNostrum Consortium. METHODS: Pooled epidemiological, clinical and genetic (CDKN2A, CDK4, ACD, BAP1, POT1, TERT, and TERF2IP and MC1R genes) retrospective data of melanoma families, collected within the MelaNostrum Consortium in Greece, Italy and Spain, were analysed. Univariate methods and multivariate logistic regression models were used to evaluate the association of variants with characteristics of families and of affected and unaffected family members. Subgroup analysis was performed for each country. RESULTS: We included 839 families (1365 affected members and 2123 unaffected individuals). Pathogenic/likely pathogenic CDKN2A variants were identified in 13.8% of families. The strongest predictors of melanoma were ≥2 multiple primary melanoma cases (OR 8.1; 95% CI 3.3-19.7), >3 affected members (OR 2.6; 95% CI 1.3-5.2) and occurrence of pancreatic cancer (OR 4.8; 95% CI 2.4-9.4) in the family (AUC 0.76, 95% CI 0.71-0.82). We observed low frequency variants in POT1 (3.8%), TERF2IP (2.5%), ACD (0.8%) and BAP1 (0.3%). MC1R common variants (≥2 variants and ≥2 RHC variants) were associated with melanoma risk (OR 1.4; 95% CI 1.0-2.0 and OR 4.3; 95% CI 1.2-14.6, respectively). CONCLUSIONS: Variants in known high-penetrance genes explain nearly 20% of melanoma familial aggregation in Mediterranean areas. CDKN2A melanoma predictors were identified with potential clinical relevance for cancer risk assessment.

Development and validation of a minimally invasive and image-guided tape stripping method to sample atopic skin in children.

BACKGROUND: Molecular skin profiling techniques, typically performed on skin samples taken by punch biopsy, have enhanced the understanding of the pathophysiology of atopic dermatitis (AD), thereby enabling the development of novel targeted therapeutics. However, punch biopsies are not always feasible or desirable, and novel minimally invasive methods such as skin tape stripping have been developed. AIM: To develop, optimize and validate a novel tape stripping method guided by noninvasive in vivo skin imaging to sample atopic skin in children. METHODS: Skin tape stripping-based procedures were compared and optimized using data from 30 healthy controls (HCs: 5 adults, 25 children) and 39 atopic children. Evaluations were guided by high-resolution photography, reflectance confocal microscopy, optical coherence tomography and transepidermal water loss measurements. We assessed and compared adverse events (AEs), the time needed to perform the sampling and the cDNA levels obtained from the tapes. RESULTS: Tape stripping methods based on previously described protocols resulted in erosions in all participants and required a median time of 65 min to perform (range 60-70 min), but provided good cDNA yield. Shorter durations appeared less invasive but provided lower cDNA yield. The final optimized tape stripping protocol, using 11 tapes of 22 mm in diameter, each applied twice for 5 s with 90° rotation, did not produce significant AEs, was completed within a median time of 7 min (range 5-15 min) and provided good cDNA yield both in HCs and atopic children. CONCLUSION: Our minimally invasive method is safe and reliable, and provides reproducible acquisition of cDNA in atopic children. In addition, it enables rapid sample collection, a crucial factor in clinical practice.

Synchronous primary cutaneous melanomas: a descriptive study of their clinical features, histology, genetic background of the patients and clinical outcomes.

BACKGROUND: Around 0.5% of cutaneous melanoma (CM) patients will present with synchronous melanomas when first seen. Moreover, 26-40% of patients with multiple primary melanomas present with synchronous lesions. OBJECTIVES: To assess the prevalence, clinical and histopathological characteristics, germline mutations and outcome in patients with synchronous melanoma. METHODS: Clinical and histopathological data from 4703 melanoma patients were included. Clinical, histological and genetic mutational status information was analysed. Kaplan-Meier curves were used to investigate survival outcomes. RESULTS: A total of 144 patients (3.06%) presented simultaneously with two or more primary melanomas. During follow-up, 25.7% of patients with synchronous melanoma developed a new primary melanoma compared to 8.6% of patients diagnosed with single melanoma (P < 0.001). Germinal CDKN2A mutations were identified in 10.7% of patients with synchronous melanomas and genetic variants in MC1R in 72%. No significant differences in all survival outcomes between patients with synchronous melanomas and single melanomas were found. CONCLUSION: Synchronous melanomas are more frequent than previously reported and are more frequent in older patients compared to single melanomas. Moreover, these patients have a higher risk of developing a new primary melanoma during follow-up and have higher rates of germline susceptibility variants. Nevertheless, these findings were not associated with worse outcomes.

M-CSF as a therapeutic target in BRAFV600E melanoma resistant to BRAF inhibitors.

BACKGROUND: Disseminated BRAFV600E melanoma responds to BRAF inhibitors (BRAFi) but easily develops resistance with poor prognosis. Secretome plays a pivotal role during tumour progression causing profound effects on therapeutic efficacy. Secreted M-CSF is involved in both cytotoxicity suppression and tumour progression in melanoma. We aimed to analyse the M-CSF contribution in resistant metastatic melanoma to BRAF-targeted therapies. METHODS: Conditioned media from melanoma cells were analysed by citoarray. Viability and migration/invasion assays were performed with paired melanoma cells and tumour growth in xenografted SCID mice. We evaluated the impact of M-CSF plasma levels with clinical prognosis from 35 metastatic BRAFV600E-mutant melanoma patients. RESULTS: BRAFi-resistant melanoma cells secretome is rich in pro-tumour cytokines. M-CSF secretion is essential to induce a Vemurafenib-resistant phenotype in melanoma cells. Further, we demonstrated that M-CSF mAb in combination with Vemurafenib and autophagy blockers synergistically induce apoptosis, impair migration and reduce tumour growth in BRAFi-resistant melanoma cells. Interestingly, lower M-CSF plasma levels are associated with better prognosis in metastatic melanoma patients. CONCLUSIONS: Secreted M-CSF induces a BRAFi-resistant phenotype and means worse prognosis in BRAFV600E metastatic melanoma patients. These results identify secreted M-CSF as a promising therapeutic target toward BRAFi-resistant melanomas.

Germline ATM variants predispose to melanoma: a joint analysis across the GenoMEL and MelaNostrum consortia.

PURPOSE: Ataxia-Telangiectasia Mutated (ATM) has been implicated in the risk of several cancers, but establishing a causal relationship is often challenging. Although ATM single-nucleotide polymorphisms have been linked to melanoma, few functional alleles have been identified. Therefore, ATM impact on melanoma predisposition is unclear. METHODS: From 22 American, Australian, and European sites, we collected 2,104 familial, multiple primary (MPM), and sporadic melanoma cases who underwent ATM genotyping via panel, exome, or genome sequencing, and compared the allele frequency (AF) of selected ATM variants classified as loss-of-function (LOF) and variants of uncertain significance (VUS) between this cohort and the gnomAD non-Finnish European (NFE) data set. RESULTS: LOF variants were more represented in our study cohort than in gnomAD NFE, both in all (AF = 0.005 and 0.002, OR = 2.6, 95% CI = 1.56-4.11, p < 0.01), and familial + MPM cases (AF = 0.0054 and 0.002, OR = 2.97, p < 0.01). Similarly, VUS were enriched in all (AF = 0.046 and 0.033, OR = 1.41, 95% CI = 1.6-5.09, p < 0.01) and familial + MPM cases (AF = 0.053 and 0.033, OR = 1.63, p < 0.01). In a case-control comparison of two centers that provided 1,446 controls, LOF and VUS were enriched in familial + MPM cases (p = 0.027, p = 0.018). CONCLUSION: This study, describing the largest multicenter melanoma cohort investigated for ATM germline variants, supports the role of ATM as a melanoma predisposition gene, with LOF variants suggesting a moderate-risk.

Dermoscopy comparative approach for early diagnosis in familial melanoma: influence of MC1R genotype.

BACKGROUND: MC1R polymorphisms interact with CDKN2A mutations modulating melanoma risk and contribute to a less suspicious clinical and dermoscopic appearance of melanomas. Different strategies, including dermoscopic comparative approach and digital monitoring, are used for the melanoma diagnosis in this context. OBJECTIVE: To analyse the diagnostic accuracy of the morphologic approach and comparative approach in dermoscopy, and to detect melanoma in familial melanoma (FamMM) patients according to different genetic backgrounds. METHODS: Two independent readers evaluated 415 lesions belonging to 25 FamMM: 26 melanomas (62% in situ, 36% early invasive) and 389 naevi, blinded for dermoscopic and histopathologic diagnosis, following two different steps. First step-Randomized: all lesions were randomly located in one single folder. Second step-Comparative approach: the lesions were clustered by patient. Sensitivity, specificity and number needed to excise (NNE) for melanoma diagnosis were calculated for both diagnostic strategies. Sensitivity and specificity were also assessed regarding the genetic background. RESULTS: The comparative approach showed lower sensitivity compared to the morphologic approach (69.2 and 73.1 vs. 76.9 both readers) but better specificity (95.9 and 95.1 vs. 84.3 and 90.2, respectively). NNE was better in the comparative approach. The readers had more difficulties diagnosing lesions from CDKN2A mutation carriers with red hair colour (RHC) MC1R variants. CONCLUSION: The comparative approach can be useful in high-risk patients to decrease the NNE. Early melanomas in CDKN2A carriers with RHC polymorphisms are more difficult to diagnose even with the comparative approach and benefit from the detection of changes during digital dermoscopy monitoring for early diagnosis.

Inherited MC1R variants in patients with melanoma are associated with better survival in women.

BACKGROUND: Women have a better melanoma prognosis, and fairer skin/hair colour. The presence of inherited MC1R variants has been associated with a better melanoma prognosis, but its interaction with sex is unknown. OBJECTIVES: To evaluate the relationship between germline MC1R status and survival, and determine any association with sex. METHODS: This was a cohort study including 1341 patients with melanoma from the Melanoma Unit of the Hospital Clinic of Barcelona, between January 1996 and April 2018. We examined known sex-related prognosis factors as they relate to features of melanoma and evaluated the sex-specific role of MC1R in overall and melanoma-specific survival. Hazard ratios (HRs) were calculated using univariate and multivariate Cox logistic regression. RESULTS: Men showed lower overall survival than women (P < 0·001) and the presence of inherited MC1R variants was not associated with better survival in our cohort. However, in women the presence of MC1R variants was associated with better overall survival in the multivariate analysis [HR 0·57, 95% confidence interval (CI) 0·38-0·85; P = 0·006] but not in men [HR 1·26, 95% CI 0·89-1·79; P = 0·185 (P-value for interaction 0·004)]. Analysis performed for melanoma-specific survival showed the same level of significance. CONCLUSIONS: Inherited MC1R variants are associated with improved overall survival in women with melanoma but not in men. Intrinsic sex-dependent features can modify the role of specific genes in melanoma prognosis. We believe that survival studies of patients with melanoma should include analysis by sex and MC1R genotype. What's already known about this topic? Inherited MC1R variants have been associated with a better melanoma prognosis, but their interaction with sex is unknown. What does this study add? MC1R variants are related to better overall survival and melanoma-specific survival in women but not in men. What is the translational message? These differences between the sexes could imply future changes in melanoma follow-up and treatment strategies. This provides a basis for understanding the interaction between sex-related genes and germline variants in cancer.

Detection of cell-free circulating BRAFV 600E by droplet digital polymerase chain reaction in patients with and without melanoma under dermatological surveillance.

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 (cfBRAFV 600E ) in plasma has emerged as a biomarker for monitoring prognosis and treatment response in patients with melanoma. OBJECTIVES: To quantify cfBRAFV 600E 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 cfBRAFV 600E 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. cfBRAFV 600E was detected in 71% of patients with stage IV melanoma and 15% with stage III, and in 1·4% of individuals without melanoma. No cfBRAFV 600E mutation was detected in disease-free patients with melanoma. Individuals without melanoma had lower cfBRAFV 600E levels than patients with melanoma. We established a variant allelic frequency of 0·26% or 5 copies mL-1 of cfBRAFV 600E 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 cfBRAFV 600E in individuals without melanoma, and supports the clinical diagnostic value of plasma cfBRAFV 600E 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.

POT1 germline mutations but not TERT promoter mutations are implicated in melanoma susceptibility in a large cohort of Spanish melanoma families.

BACKGROUND: Germline mutations in telomere-related genes such as POT1 and TERT predispose individuals to familial melanoma. OBJECTIVES: To evaluate the prevalence of germline mutations in POT1 and TERT in a large cohort of Spanish melanoma-prone families (at least two affected first- or second-degree relatives). METHODS: Overall, 228 CDKN2A wild-type melanoma-prone families were included in the study. Screening of POT1 was performed in one affected person from each family and TERT was sequenced in one affected patient from 202 families (26 families were excluded owing to DNA exhaustion/degradation). TERT promoter sequencing was extended to an additional 30 families with CDKN2A mutation and 70 patients with sporadic multiple primary melanoma (MPM) with a family history of other cancers. RESULTS: We identified four families with potentially pathogenic POT1 germline mutations: a missense variant c.233T>C (p.Ile78Thr); a nonsense variant c.1030G>T (p.Glu344*); and two other variants, c.255G>A (r.125_255del) and c.1792G>A (r.1791_1792insAGTA, p.Asp598Serfs*22), which we confirmed disrupted POT1 mRNA splicing. A TERT promoter variant of unknown significance (c.-125C>A) was detected in a patient with MPM, but no germline mutations were detected in TERT promoter in cases of familial melanoma. CONCLUSIONS: Overall, 1·7% of our CDKN2A/CDK4-wild type Spanish melanoma-prone families carry probably damaging mutations in POT1. The frequency of TERT promoter germline mutations in families with melanoma in our population is extremely rare.

Genetic susceptibility to cutaneous melanoma in southern Switzerland: role of CDKN2A, MC1R and MITF.

BACKGROUND: 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 seven patients (four families) with a founder effect, whereas CDKN2A A148T was detected in seven cases (five families) and seven healthy donors (odds ratio 2·76, 95% confidence interval 0·83-9·20). At least one MC1R melanoma-associated polymorphism was detected in 32 patients (78%) and 97 healthy donors (66%), with more than one polymorphism in 12 patients (29%) and 25 healthy donors (17%). The MITF variant p.E318K was identified in four patients from three additional pedigrees (7%) 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.