A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance.

Melanoma heterogeneity is a hurdle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcomes, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker for the real-time tracing of disease evolution. Thus, we aimed to correlate liquid biopsy dynamics with treatment response and progression by devising a multiplatform approach applied to longitudinal melanoma patient monitoring. We conceived an approach that exploits Next Generation Sequencing (NGS) and droplet digital PCR, as well as the FDA-cleared platform CellSearch, to analyze circulating tumor DNA (ctDNA) trend and circulating melanoma cell (CMC) count, together with their customized genetic and copy number variation analysis. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, followed for up to 28 months. BRAF mutations were detected in the plasma of 82% of patients. Single nucleotide variants known or suspected to confer resistance were identified in 70% of patients. Moreover, the amount of ctDNA, both at baseline and during response, correlated with the type and duration of the response itself, and the CMC count was confirmed to be a prognostic biomarker. This work provides proof of principle of the power of this approach and paves the way for a validation study aimed at evaluating early ctDNA-guided treatment decisions in stage IV melanoma. The NGS-based molecular profile complemented the analysis of ctDNA trend and, together with CMC analysis, revealed to be useful in capturing tumor evolution.

Comprehensive genomic profiling on metastatic Melanoma: results from a network screening from 7 Italian Cancer Centres.

BACKGROUND: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma. METHODS: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting. RESULTS: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints. CONCLUSIONS: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research.

Molecular Profile of Subungual Melanoma: A MelaNostrum Consortium Study of 68 Cases Reporting BRAF, NRAS, KIT, and TERT Promoter Status.

BACKGROUND: Subungual melanoma (SM) is an unusual type of melanocytic tumor affecting the nail apparatus. The mutational prevalence of the most prominently mutated genes in melanoma has been reported in small cohorts of SM, with unclear conclusions on whether SM is different from the rest of melanomas arising in acral locations or not. Hence, the molecular profile of a large series of SM is yet to be described. OBJECTIVES: The aim of this study was to describe the molecular characteristics of a large series of SM and their association with demographic and histopathological features. METHODS: Patients diagnosed with SM between 2001 and 2021 were identified from six Spanish and Italian healthcare centers. The mutational status for BRAF, NRAS, KIT, and the promoter region of TERT (TERTp) were determined either by Sanger sequencing or next-generation sequencing. Clinical data were retrieved from the hospital databases to elucidate potential associations. RESULTS: A total of 68 SM cases were included. Mutations were most common in BRAF (10.3%) and KIT (10%), followed by NRAS (7.6%), and TERTp (3.8%). Their prevalence was similar to that of non-subungual acral melanoma but higher in SM located on the hand than on the foot. CONCLUSIONS: To date, this study represents the largest cohort of SM patients with data on the known driver gene mutations. The low mutation rate supports a different etiopathogenic mechanism for SM in comparison of non-acral cutaneous melanoma, particularly for SM of the foot.

Molecular Modeling Unveils the Effective Interaction of B-RAF Inhibitors with Rare B-RAF Insertion Variants.

The Food and Drug Administration (FDA) has approved MAPK inhibitors as a treatment for melanoma patients carrying a mutation in codon V600 of the BRAF gene exclusively. However, BRAF mutations outside the V600 codon may occur in a small percentage of melanomas. Although these rare variants may cause B-RAF activation, their predictive response to B-RAF inhibitor treatments is still poorly understood. We exploited an integrated approach for mutation detection, tumor evolution tracking, and assessment of response to treatment in a metastatic melanoma patient carrying the rare p.T599dup B-RAF mutation. He was addressed to Dabrafenib/Trametinib targeted therapy, showing an initial dramatic response. In parallel, in-silico ligand-based homology modeling was set up and performed on this and an additional B-RAF rare variant (p.A598_T599insV) to unveil and justify the success of the B-RAF inhibitory activity of Dabrafenib, showing that it could adeptly bind both these variants in a similar manner to how it binds and inhibits the V600E mutant. These findings open up the possibility of broadening the spectrum of BRAF inhibitor-sensitive mutations beyond mutations at codon V600, suggesting that B-RAF V600 WT melanomas should undergo more specific investigations before ruling out the possibility of targeted therapy.

Ataxia-Telangiectasia Mutated Loss of Heterozygosity in Melanoma.

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

Per- and polyfluoroalkyl substances (PFAS) exposure in melanoma patients: a retrospective study on prognosis and histological features.

Per- and polyfluoroalkyl substances (PFAS) are endocrine disrupting chemicals which could be associated with cancer development, such as kidney and testicular cancers, pancreatic and hepatocellular carcinoma and thyroid tumor. Available scientific literature offers no information on the role of PFAS in melanoma development/progression. Since 1965, a massive environmental contamination by PFAS has occurred in northeastern Italy. This study compared histopathology and prognosis between melanoma patients exposed (n = 194) and unexposed (n = 488) to PFAS. All patients were diagnosed and/or treated for melanoma at the Veneto Oncological Institute and the University Hospital of Padua (Italy) in 1998-2014. Patients were categorized in exposed or unexposed groups according to their home address and the geographical classification of municipalities affected by PFAS contamination as provided by Veneto Government in 2018. Presence of mitoses was found in 70.5% of exposed patients and 58.7% of unexposed patients (p = 0.005). Median follow-up was 90 months (IQR 59-136). 5-year overall survival was 83.7% in exposed patients and 88.0% in unexposed patients (p = 0.20); 5-year disease-specific survival was 88.0% in exposed patients and 90.9% in unexposed patients (p = 0.50); 5-year disease-free survival was 83.8% in exposed patients and 87.3% in unexposed patients (p = 0.20). Adjusting for imbalanced characteristics at baseline (presence of mitoses), survival was not statistically different between exposed and unexposed patients (overall survival: HR 1.10, 95% CI 0.77 to 1.58, p = 0.57; disease-specific survival: HR 0.99, 95% CI 0.62 to 1.59, p = 0.99; disease-free survival: HR 1.10, 95% CI 0.74 to 1.64, p = 0.62). Although the magnitude of PFAS exposure was not quantifiable, our findings suggested that exposure to PFAS was associated with higher level of mitosis in melanoma patients, but this did not translate into a survival difference. Further studies are required to investigate this relationship and all effects of PFAS on prognosis.

Diagnostics of BAP1-Tumor Predisposition Syndrome by a Multitesting Approach: A Ten-Year-Long Experience.

Germline mutations in the tumor suppressor gene BRCA1-associated protein-1 (BAP1) lead to BAP1 tumor predisposition syndrome (BAP1-TPDS), characterized by high susceptibility to several tumor types, chiefly melanoma, mesothelioma, renal cell carcinoma, and basal cell carcinoma. Here, we present the results of our ten-year experience in the molecular diagnosis of BAP1-TPDS, along with a clinical update and cascade genetic testing of previously reported BAP1-TPDS patients and their relatives. Specifically, we sequenced germline DNA samples from 101 individuals with suspected BAP1-TPDS and validated pathogenic variants (PVs) by assessing BAP1 somatic loss in matching tumor specimens. Overall, we identified seven patients (7/101, 6.9%) carrying six different germline BAP1 PVs, including one novel variant. Consistently, cascade testing revealed a total of seven BAP1 PV carriers. In addition, we explored the mutational burden of BAP1-TPDS tumors by targeted next-generation sequencing. Lastly, we found that certain tumors present in PV carriers retain a wild-type BAP1 allele, suggesting a sporadic origin of these tumors or a functional role of heterozygous BAP1 in neoplastic development. Altogether, our findings have important clinical implications for therapeutic response of BAP1-TPDS patients.

Altitude Effect on Cutaneous Melanoma Epidemiology in the Veneto Region (Northern Italy): A Pilot Study.

The incidence of cutaneous melanoma has been increasing in the last decades among the fair-skinned population. Despite its complex and multifactorial etiology, the exposure to ultraviolet radiation (UVR) is the most consistent modifiable risk factor for melanoma. Several factors influence the amount of UVR reaching the Earth's surface. Our study aimed to explore the relationship between melanoma and altitude in an area with mixed geographic morphology, such as the Veneto region (Italy). We included 2752 melanoma patients who were referred to our centers between 1998 and 2014. Demographics, histological and clinical data, and survival information were extracted from a prospectively maintained local database. Head/neck and acral melanoma were more common in patients from the hills and the mountains, while limb and trunk melanoma were more common in patients living in plain and coastal areas. Breslow thickness, ulceration and mitotic rate impaired with increased altitude. However, the geographical area of origin was not associated with overall or disease-free survival. The geographical area of origin of melanoma patients and the "coast-plain-hill gradient" could help to estimate the influence of different sun exposure and to explain the importance of vitamin D levels in skin-cancer control.

Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family.

While several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations we identified a novel NRAS variant (c.170A > C, p.D57A) in an Italian melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11,273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2 + binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A > C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.

Corrigendum: Melanoma in Adolescents and Young Adults (AYA): Evaluation of the Characteristics, Treatment Strategies, and Prognostic Factors in a Monocentric Retrospective Study.

[This corrects the article DOI: 10.3389/fonc.2021.725523.].

Melanoma in Adolescents and Young Adults: Evaluation of the Characteristics, Treatment Strategies, and Prognostic Factors in a Monocentric Retrospective Study.

The "Veneto Cancer Registry" records melanoma as the most common cancer diagnosed in males and the third common cancer in females under 50 years of age in the Veneto Region (Italy). While melanoma is rare in children, it has greater incidence in adolescents and young adults (AYA), but literature offers only few studies specifically focused on AYA melanoma. The aim of this study was to describe the characteristics, surgical treatment, and prognosis of a cohort of AYA melanoma in order to contribute to the investigation of this malignancy and provide better patient care. This retrospective cohort study included 2,752 Caucasian patients (702 AYA and 2,050 non-AYA patients) from the Veneto Region who were over 15 years of age at diagnosis, and who received diagnosis and/or treatment from our institutions between 1998 and 2014. Patients were divided in adolescents and youth (15-25 years), young adults (26-39 years) and adults (more than 39 years) for the analysis. We found statistically significant differences in gender, primary site, Breslow thickness, ulceration, pathologic TNM classification (pTNM) stage and tumor subtype among the age groups. Disease-specific survival and disease-free survival were also different among the age groups. Our findings suggest that the biological behavior of melanoma in young people is different to that in adults, but not such as to represent a distinct pathological entity. Additional and larger prospective studies should be performed to better evaluate potential biological and cancer-specific differences between AYAs and the adult melanoma population.

The Interplay between Nevi and Melanoma Predisposition Unravels Nevi-Related and Nevi-Resistant Familial Melanoma.

Genetic susceptibility to nevi may affect the risk of developing melanoma, since common and atypical nevi are the main host risk factors implicated in the development of cutaneous melanoma. Recent genome-wide studies defined a melanoma polygenic risk score based on variants in genes involved in different pathways, including nevogenesis. Moreover, a predisposition to nevi is a hereditary trait that may account for melanoma clustering in some families characterized by cases with a high nevi density. On the other hand, familial melanoma aggregation may be due to a Mendelian inheritance of high/moderate-penetrance pathogenic variants affecting melanoma risk, regardless of the nevus count. Based on current knowledge, this review analyzes the complex interplay between nevi and melanoma predisposition in a familial context. We review familial melanoma, starting from Whiteman's divergent pathway model to overall melanoma development, distinguishing between nevi-related (cases with a high nevus count and a high polygenic risk score) and nevi-resistant (high/moderate-penetrance variant-carrier cases) familial melanoma. This distinction could better direct future research on genetic factors useful to identify high-risk subjects.

The Molecular Landscape of Primary Acral Melanoma: A Multicenter Study of the Italian Melanoma Intergroup (IMI).

Acral melanoma (AM) is a rare and aggressive subtype of melanoma affecting the palms, soles, and nail apparatus with similar incidence among different ethnicities. AM is unrelated to ultraviolet radiation and has a low mutation burden but frequent chromosomal rearrangements and gene amplifications. Next generation sequencing of 33 genes and somatic copy number variation (CNV) analysis with genome-wide single nucleotide polymorphism arrays were performed in order to molecularly characterize 48 primary AMs of Italian patients in association with clinicopathological and prognostic features. BRAF was the most commonly mutated gene, followed by NRAS and TP53, whereas TERT promoter, KIT, and ARID1A were less frequently mutated. Gains and losses were recurrently found in the 1q, 6p, 7, 8q, 20 and 22 chromosomes involving PREX2, RAC1, KMT2C, BRAF, CCND1, TERT, and AKT3 genes, and in the 6q, 9, 10, 11q and 16q chromosomes including CDKN2A, PTEN, and ADAMTS18 genes, respectively. This study confirmed the variety of gene mutations and the high load of CNV in primary AM. Some genomic alterations were associated with histologic prognostic features. BRAF mutations, found with a higher rate than previously reported, correlated with a low Breslow thickness, low mitotic count, low CNV of the AMs, and with early-stage of disease.

Gender-specific associations between polymorphisms of the circadian gene RORA and cutaneous melanoma susceptibility.

BACKGROUND: Melanoma is the deadliest of skin cancers and has an increasing annual incidence worldwide. It is a multi-factorial disease most likely arising from both genetic predisposition and environmental exposure to ultraviolet light. Genetic variability of the components of the biological circadian clock is recognized to be a risk factor for different type of cancers. Moreover, two variants of a clock gene, RORA, have been associated with melanoma patient's prognosis. Our aim is to test the hypothesis that specific single nucleotide polymorphisms (SNPs) of the circadian clock genes may significantly influence the predisposition to develop cutaneous melanoma or the outcome of melanoma patients. METHODS: We genotyped 1239 subjects, 629 cases of melanoma and 610 healthy controls in 14 known SNPs of seven selected clock genes: AANAT, CLOCK, NPAS2, PER1, PER2, RORA, and TIMELESS. Genotyping was conducted by q-PCR. Multivariate logistic regression was employed for susceptibility of melanoma assessment, modeled additively. Subgroup analysis was performed by gender. For the female subgroup, a further discrimination was performed by age. For prognosis of melanoma assessment, multivariate Cox proportional hazard regression was employed. The Benjamini-Hochberg method was utilized as adjustment for multiple comparisons. RESULTS: We identified two RORA SNPs statistically significant with respect to the association with melanoma susceptibility. Considering the putative role of RORA as a nuclear steroid hormone receptor, we conducted a subgroup analysis by gender. Interestingly, the RORA rs339972 C allele was associated with a decreased predisposition to develop melanoma only in the female subgroup (OR 0.67; 95% CI 0.51-0.88; P = 0.003) while RORA rs10519097 T allele was associated with a decreased predisposition to develop melanoma only in the male subgroup (OR 0.62; 95% CI 0.44-0.87; P = 0.005). Moreover, the RORA rs339972 C allele had a decreased susceptibility to develop melanoma only in females aged over 50 years old (OR 0.67; 95% CI 0.54-0.83; P = 0.0002). None of the studied SNPs were significantly associated with the prognosis. CONCLUSIONS: Overall, we cannot ascertain that circadian pathway genetic variation is involved in melanoma susceptibility or prognosis. Nevertheless, we identified an interesting relationship between melanoma susceptibility and RORA polymorphisms acting in sex-specific manner and which is worth further future investigation.

Genetic Variants of the TERT Gene, Telomere Length, and Circulating TERT as Prognostic Markers in Rectal Cancer Patients.

Single-nucleotide polymorphisms (SNPs) in the TERT gene can affect telomere length and TERT expression and have been associated with risk and/or outcome for several tumors, but very few data are available about their impact on rectal cancer. Eight SNPs (rs2736108, rs2735940, rs2736098, rs2736100, rs35241335, rs11742908, rs2736122 and rs2853690), mapping in regulatory and coding regions of the TERT gene, were studied in 194 rectal cancer patients to evaluate their association with constitutive telomere length, circulating TERT mRNA levels, response to neoadjuvant chemoradiotherapy (CRT) and disease outcome. At diagnosis, the rs2736100CC genotype was associated with longer telomeres measured pre-CRT, while the rs2736100CC, rs2736108TT and rs2735940AA were associated with greater telomere erosion evaluated post-CRT. The rs2736108CC and rs2853690AA/GG genotypes, respectively associated with lower telomere erosion and lower levels of circulating TERT post-CRT, were also independently associated with a better response to therapy [OR 4.6(1.1-19.1) and 3.0(1.3-6.9)]. Overall, post-CRT, low levels (≤ median value) of circulating TERT and its stable/decreasing levels compared to those pre-CRT, were independently associated with a better response to therapy [OR 5.8(1.9-17.8) and 5.3(1.4-19.4), respectively]. Furthermore, post-CRT, patients with long telomeres (>median value) and low levels of circulating TERT had a significantly lower risk of disease progression [HR 0.4(0.1-0.9) and 0.3(0.1-0.8), respectively]. These findings suggest that TERT SNPs could be a useful tool for improving the selection of patients who could benefit from CRT and support the role of telomere length and circulating TERT mRNA levels as useful markers for monitoring the response to therapy and disease outcome in rectal cancer patients.

MC1R variants and cutaneous melanoma risk according to histological type, body site, and Breslow thickness: a pooled analysis from the M-SKIP project.

Little is known on whether melanocortin 1 receptor (MC1R) associated cutaneous melanoma (CM) risk varies depending on histological subtype and body site, and whether tumour thickness at diagnosis (the most important prognostic factor for CM patients) differs between MC1R variant carriers and wild-type individuals. We studied the association between MC1R variants and CM risk by histological subtype, body site, and Breslow thickness, using the database of the M-SKIP project. We pooled individual data from 15 case-control studies conducted during 2005-2015 in Europe and the USA. Study-specific, multi-adjusted odds ratios were pooled into summary odds ratios (SOR) and 95% confidence intervals (CI) using random-effects models. Six thousand eight hundred ninety-one CM cases and 5555 controls were included. CM risk was increased among MC1R variant carriers vs. wild-type individuals. The increase in risk was comparable across histological subtypes (SOR for any variant vs. wild-type ranged between 1.57 and 1.70, always statistical significant) except acral lentiginous melanoma (ALM), for which no association emerged; and slightly greater on chronically (1.74, 95% CI 1.47-2.07) than intermittently (1.55, 95% CI 1.34-1.78) sun-exposed skin. CM risk was greater for those carrying 'R' vs. 'r' variants; correlated with the number of variants; and was more evident among individuals not showing the red hair colour phenotype. Breslow thickness was not associated with MC1R status. MC1R variants were associated with an increased risk of CM of any histological subtype (except ALM) and occurring on both chronically and intermittently sun-exposed skin.

TERT Promoter Mutations Differently Correlate with the Clinical Outcome of MAPK Inhibitor-Treated Melanoma Patients.

Resistance is a major challenge in the management of mitogen-activated protein kinase inhibitor (MAPKi)-treated metastatic melanoma. Tumor genetic alterations can cause MAPK pathway reactivation, leading to lack of response and poor outcome. Characterization of the mutational profile in patients with melanoma might be crucial for patient-tailored treatment choices. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTprom) lead to increased TERT expression and telomerase activity and are frequent in BRAFV600 mutant melanoma. Reportedly, TERTprom, and BRAFV600 mutations cooperate in driving cancer progression and aggressiveness. We evaluated the effect of the TERTprom status on the clinical outcome in 97 MAPKi-treated melanoma patients. We observed that patients with the c.-146C > T mutation showed a significantly worse progression-free survival (PFS) compared to those carrying the c.-124C > T mutation and a two-fold increased risk of progression (median 5.4 vs. 9.5 months; hazard ratio (HR) 1.9; 95% confidence interval (CI) 1.2-3.2; p = 0.013). This trend was also observed for the overall survival (OS); melanoma patients with the c.-146C > T mutation showed a poorer prognosis compared to those with the c.-124C > T mutation (median 13.3 vs. 25.5 months; HR 1.9, 95% CI 1.1-3.3, p = 0.023). Our results disclose a different correlation of the two TERTprom mutations with MAPKi-treated melanoma patient outcome, highlighting a different impact of the pathway blockade.

Genome-wide association meta-analyses combining multiple risk phenotypes provide insights into the genetic architecture of cutaneous melanoma susceptibility.

Most genetic susceptibility to cutaneous melanoma remains to be discovered. Meta-analysis genome-wide association study (GWAS) of 36,760 cases of melanoma (67% newly genotyped) and 375,188 controls identified 54 significant (P < 5 × 10-8) loci with 68 independent single nucleotide polymorphisms. Analysis of risk estimates across geographical regions and host factors suggests the acral melanoma subtype is uniquely unrelated to pigmentation. Combining this meta-analysis with GWAS of nevus count and hair color, and transcriptome association approaches, uncovered 31 potential secondary loci for a total of 85 cutaneous melanoma susceptibility loci. These findings provide insights into cutaneous melanoma genetic architecture, reinforcing the importance of nevogenesis, pigmentation and telomere maintenance, together with identifying potential new pathways for cutaneous melanoma pathogenesis.