Characterization of genetic factors associated with melanoma susceptibility and prognosis

[eng] Melanoma is the malignant tumor arising from melanocytes and is the most aggressive of the common skin cancers. Melanoma is the tumor with the highest heritability (58%) and around 10% of cases occur in a familial context. Genetic susceptibility can be explained due to the inheritance of low, medium or high-risk variants, or a combination of them. To date, germline mutations in high-risk genes have been detected in 20-30% of melanoma-prone families. Interestingly, there is an important relationship between genetic factors associated with the risk to develop melanoma and genetic factors modulating melanoma outcome. The thesis hypotheses were: a) The proper characterization of known melanoma risk genes in a specific population context will facilitate genetic counseling in melanoma b) The use of genome-wide linkage can allow the identification of new melanoma susceptibility loci in our population. c) Melanoma susceptibility and nevus-related genes can play a role in melanoma prognosis. d) Variants in immune checkpoints genes can play a role in melanoma prognosis. Based on those hypotheses, the aims of this thesis were: 1) To characterize known risk genes in patients at high-risk to develop melanoma in Spain to refine genetic counseling. 2) To identify new familial melanoma loci using genome-wide linkage analysis. 3) To study the role of candidate genes in melanoma prognosis. To answer those aims we designed six studies. In the first study, we evaluated the prevalence of CDKN2A germline mutations in patients at high risk to develop melanoma in our population and characterize families with mutation. We identified a higher prevalence of lung, breast and pancreatic cancer cases in families with mutation. Based on the study results, CDKN2A mutation carriers, besides sun protection advice and dermatologic surveillance, should receive recommendations on avoiding smoking and can be included in early detection programs for pancreatic, lung and breast cancers. In the second study, we evaluated the prevalence of MITF p.Glu318Lys carriers and assessed their characteristics detecting fast-growing melanoma among carriers. Thus, MITF p.Glu318Lys should be given fast-track visits to dermatology as they may be at risk to develop fast-growing melanomas and can be included in early detection programs for renal cancer. In the third study we identified that POT1 is mutated in a subset of melanoma families in Spain, thus genetic testing in melanoma should include the analysis of this gene. In the fourth study we identified a new melanoma locus at 11q associated with familial melanoma. Next-generation sequencing studies focused on the analysis of this region may allow the identification of new melanoma susceptibility genes or variants. Finally, in the last two studies, we focused on melanoma prognosis. We determined that IRF4 rs12203592 T functional variant, associated with a low melanogenesis (and low nevus count) and immune tolerance, correlates with a worse melanoma survival. Moreover, inherited functional variants of the lymphocyte receptor CD5, associated with more immune reactivity, correlated with better melanoma outcome. To conclude, we have established the genetic bases of melanoma susceptibility in our population and refined genetic counseling, knowing the mutation prevalence of each gene and adapting secondary prevention measures in melanoma and other tumors according to the genetic testing results. Genome-wide linkage analysis in melanoma-prone families from Spain has allowed the identification of a new locus at 11q involved in familial melanoma. We have identified new genes modulating melanoma outcome based on the study of candidate genes involved in melanoma susceptibility, nevi count, and immune regulation.[cat] El melanoma és el més agressiu dels càncers de pell comuns. És el tumor amb una major heretabilitat. La susceptibilitat genètica depèn de variants d’alt, mig o baix risc, o la seva combinació. Hi ha una gran correlació entre factors genètics associats al risc i al pronòstic del melanoma. Els objectius d’aquesta tesi han estat: 1. Caracteritzar gens coneguts en pacients amb alt risc a desenvolupar melanoma per millorar el consell genètic. 2. Identificar nous loci implicats en la susceptibilitat a melanoma mitjançant anàlisi de lligament massiu. 3. Estudiar el paper de gens candidats en el pronòstic del melanoma En els primers tres estudis, es va avaluar la prevalença de mutacions a CDKN2A, POT1, promotor de TERT i la variant p.Glu318Lys a MITF en pacients amb alt risc a desenvolupar melanoma. També es van estudiar les característiques de les famílies i portadors d’aquestes variants. Els resultats observats mostren que els portadors de mutació a CDKN2A, a banda de les mesures de fotoprotecció i seguiment dermatològic, haurien d’evitar el tabac i es poden incloure en programes de detecció precoç de càncer de pàncrees, pulmó o mama. Els portadors de la variant p.Glu318Lys a MITF haurien de tenir accés ràpid a la consulta dermatològica pel risc a desenvolupar melanoma de ràpid creixement. POT1 s’hauria d’incloure en el test genètic. En el quart, hem identificat un nou locus a 11q associat a la susceptibilitat del melanoma familiar. Finalment, en els dos últims treballs, hem observat que la variant funcional d’IRF4 rs12203592 T, associada a un recompte de nevus baix i immunotolerància, correlaciona amb un pitjor pronòstic. A més, variants funcionals de CD5 associades amb una major reactivitat immunològica correlacionen amb un millor pronòstic. En conclusió, hem establert les bases genètiques de la susceptibilitat del melanoma en la nostra població i millorat el consell genètic. Estudis de lligament massiu han permès la identificació d’un nou locus associat al melanoma familiar. Per acabar, hem identificat nous gens que modulen el pronòstic del melanoma basant-nos en l’estudi de candidats relacionats amb la susceptibilitat a melanoma, el recompte de nevus i la regulació del sistema immune

Role of CPI-17 in restoring skin homoeostasis in cutaneous field of cancerization: effects of topical application of a film-forming medical device containing photolyase and UV filters

Cutaneous field of cancerization (CFC) is caused in part by the carcinogenic effect of the cyclobutane pyrimidine dimers CPD and 6‐4 photoproducts (6‐4PPs). Photoreactivation is carried out by photolyases which specifically recognize and repair both photoproducts. The study evaluates the molecular effects of topical application of a film‐forming medical device containing photolyase and UV filters on the precancerous field in AK from seven patients. Skin improvement after treatment was confirmed in all patients by histopathological and molecular assessment. A gene set analysis showed that skin recovery was associated with biological processes involved in tissue homoeostasis and cell maintenance. The CFC response was associated with over‐expression of the CPI‐17 gene, and a dependence on the initial expression level was observed (P = 0.001). Low CPI‐17 levels were directly associated with pro‐inflammatory genes such as TNF (P = 0.012) and IL‐1B (P = 0.07). Our results suggest a role for CPI‐17 in restoring skin homoeostasis in CFC lesions

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

Prevalence of MITF p.E318K in patients with melanoma independent of the presence of CDKN2A causative mutations

Importance The main high-penetrance melanoma susceptibility gene is CDKN2A, encoding p16INK4A and p14ARF. The gene MITF variant p.E318K also predisposes to melanoma and renal cell carcinoma. To date, the prevalence of MITF p.E318K and its clinical and phenotypical implications has not been previously assessed in a single cohort of Spanish patients with melanoma or in p16INK4A mutation carriers.Objectives To evaluate the prevalence of MITF p.E318K in Spanish patients with melanoma and assess the association with clinical and phenotypic features.Design, Setting, and Participants A hospital-based, case-control study was conducted at the Melanoma Unit of Hospital Clinic of Barcelona, with MITF p.E318K genotyped in all patients using TaqMan probes. We included 531 patients: 271 patients with multiple primary melanoma (MPM) without mutations affecting p16INK4A (wild-type p16INK4A); 191 probands from melanoma-prone families with a single melanoma diagnosis and without mutations affecting p16INK4A, and 69 probands from different families carrying CDKN2A mutations affecting p16INK4A. A population-based series of 499 age- and sex-matched cancer-free individuals from the Spanish National Bank of DNA were included as controls. Patients were recruited between January 1, 1992, and June 30, 2014; data analysis was conducted from September 1 to November 30, 2014.Main Outcomes and Measures The genetic results of the MITF p.E318K variant were correlated with clinical and phenotypic features.Results Among the 531 patients, the prevalence of the MITF p.E318K variant was calculated among the different subsets of patients included and was 1.9% (9 of 462) in all melanoma patients with wild-type p16INK4A, 2.6% (7 of 271) in those with MPM, and 2.9% (2 of 69) in the probands of families with p16INK4A mutations. With results reported as odds ratio (95% CI), the MITF p.E318K was associated with an increased melanoma risk (3.3 [1.43-7.43]; P 200 nevi) (8.4 [2.14-33.19]; P < .01). Two fast-growing melanomas were detected among 2 MITF p.E318K carriers during dermatologic digital follow-up.Conclusions and Relevance In addition to melanoma risk, MITF p.E318K is associated with a high nevi count and could play a role in fast-growing melanomas. Testing for MITF p.E318K should not exclude patients with known mutations in p16INK4A. Strict dermatologic surveillance, periodic self-examination, and renal cell carcinoma surveillance should be encouraged in this context

Association between confocal morphologic classification and clinical phenotypes of multiple primary and familial melanomas

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

Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas.

BACKGROUND: BRAF and NRAS mutation detection is crucial for advanced melanoma treatment. Our aim was to evaluate how different characteristics from formalin-fixed paraffin-embedded (FFPE) samples, age of the block or DNA concentration could influence the success of BRAF and NRAS mutational screening. METHODS: DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis). BRAF and NRAS were sequenced by Sanger sequencing. RESULTS: Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them. BRAF was mutated in 55% (29/53) and NRAS in 11% (5/45) of the primary melanomas sequenced. DNA concentration correlated with the tumor area used for DNA extraction (mm2) (adj p-value<0.01, r=0.73). The age of the block did not affect sequencing success. In 60% of samples kept for more than 10 years, both BRAF and NRAS were successfully sequenced. CONCLUSIONS: Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival

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

Melanocortin-1 receptor (MC1R) genotypes do not correlate with size in two cohorts of medium-to-giant congenital melanocytic nevi

Congenital melanocytic nevi (CMN) are cutaneous malformations whose prevalence is inversely correlated with projected adult size. CMN are caused by somatic mutations, but epidemiological studies suggest that germline genetic factors may influence CMN development. In CMN patients from the U.K., genetic variants in MC1R, such as p.V92M and loss-of-function variants, have been previously associated with larger CMN. We analyzed the association of MC1R variants with CMN characteristics in two distinct cohorts of medium-to-giant CMN patients from Spain (N = 113) and from France, Norway, Canada, and the United States (N = 53), similar at the clinical and phenotypical level except for the number of nevi per patient. We found that the p.V92M or loss-of-function MC1R variants either alone or in combination did not correlate with CMN size, in contrast to the U.K. CMN patients. An additional case-control analysis with 259 unaffected Spanish individuals showed a higher frequency of MC1R compound heterozygous or homozygous variant genotypes in Spanish CMN patients compared to the control population (15.9% vs. 9.3%; p = .075). Altogether, this study suggests that MC1R variants are not associated with CMN size in these non-UK cohorts. Additional studies are required to define the potential role of MC1R as a risk factor in CMN development.© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Lethal congenital contracture syndrome 11: A case report and literature review

Lethal congenital contracture syndrome 11 (LCCS11) is caused by homozygous or compound heterozygous variants in the GLDN gene on chromosome 15q21. GLDN encodes gliomedin, a protein required for the formation of the nodes of Ranvier and development of the human peripheral nervous system. We report a fetus with ultrasound alterations detected at 28 weeks of gestation. The fetus exhibited hydrops, short long bones, fixed limb joints, absent fetal movements, and polyhydramnios. The pregnancy was terminated and postmortem studies confirmed the prenatal findings: distal arthrogryposis, fetal growth restriction, pulmonary hypoplasia, and retrognathia. The fetus had a normal chromosomal microarray analysis. Exome sequencing revealed two novel compound heterozygous variants in the GLDN associated with LCCS11. This manuscript reports this case and performs a literature review of all published LCCS11 cases

A Common Variant in the MC1R Gene (p.V92M) is associated with Alzheimer's Disease Risk.

Despite the recent identification of some novel risk genes for Alzheimer's disease (AD), the genetic etiology of late-onset Alzheimer's disease (LOAD) remains largely unknown. The inclusion of these novel risk genes to the risk attributable to the APOE gene accounts for roughly half of the total genetic variance in LOAD. The evidence indicates that undiscovered genetic factors may contribute to AD susceptibility. In the present study, we sequenced the MC1R gene in 525 Spanish LOAD patients and in 160 controls. We observed that a common MC1R variant p.V92M (rs2228479), not related to pigmentation traits, was present in 72 (14%) patients and 15 (9%) controls and confers increased risk of developing LOAD (OR: 1.99, 95% CI: 1.08-3.64, p = 0.026), especially in those patients whose genetic risk could not be explained by APOE genotype. This association remains and even increased in the subset of 69 patients with typical AD cerebrospinal fluid profile (OR: 3.40 95% CI: 1.40-8.27, p = 0.007). We did not find an association between p.V92M and age of onset of AD. Further studies are necessary to elucidate the role of MC1R in brain cells through the different MC1R pathways