Melanoma risk and melanocyte biology

Funding text This work was supported by the Ligue Contre le Cancer, INCa, ITMO Cancer, Fondation ARC (PGA), and is under the program “Investissements d’Avenir” launched by the French Government and implemented by ANR Labex CelTisPhyBio (ANR-11-LA-BX-0038 and ANR-10-IDEX-0001-02 PSL). This work was also supported by a grant from the Icelandic Research Fund (grant number 184861-051 to ES). BBdP is nationwide coordinator of melanoma oncogenetics for INCA. Publisher Copyright: © 2020 Acta Dermato-Venereologica.Cutaneous melanoma arises from melanocytes following genetic, epigenetic and allogenetic (i.e. other than epi/genetic) modifications. An estimated 10% of cutaneous melanoma cases are due to inherited variants or de novo mutations in approximately 20 genes, found using linkage, next-generation sequencing and association studies. Based on these studies, 3 classes of predisposing melanoma genes have been defined based on the frequency of the variants in the general population and lifetime risk of developing a melanoma: (i) ultra-rare variants with a high risk, (ii) rare with a moderate risk, and (iii) frequent variants with a low risk. Most of the proteins encoded by these genes have been shown to be involved in melanoma initiation, including proliferation and senescence bypass. This paper reviews the role(s) of these genes in the transformation of melanocytes into melanoma. It also describes their function in the establishment and renewal of melanocytes and the biology of pigment cells, if known.Peer reviewe

Apport du séquençage d’exomes constitutionnels dans l’identification de nouveaux gènes de prédisposition aux cancers, sarcomes et mélanomes pédiatriques.

The aim of this thesis was to identify by sequencing of exomes new predisposing genes in 2 rare pathologies whose etiology is not well known.The first project involved a family with 3 cases of sarcomas, without mutation of TP53 gene. We identified a pathogenic mutation of the CDKN2A gene, in the 3 cases. Since CDKN2A gene is the major melanoma predisposing gene, we have searched in 3 different collections, sarcoma cases link to CDKN2A mutations. In total, we have identified 8 independent sarcomas cases, carrying a germline mutation of the CDKN2A gene and showed a loss of heterozygosity at the site of the constitutional mutation of CDKN2A in 5/7 cases, thus proving a complete loss of function. Since sarcomas are rare in carriers of CDKN2A mutations, we looked for potential modifying rare variants, by sequencing constitutional exomes of the 8 index cases, and identified 3 variants of the PDGFRA gene. Modeling studies have shown that 2 of these variants could have an impact on the structure of the extracellular domain of the PDGFRA protein. We have thus demonstrated that the CDKN2A gene can predispose to sarcomas and identified PDGFRA as a potential modifier gene. In the second project, we worked on childhood melanomas, of sporadic appearance. Our working hypothesis was that these melanomas occur as a result of a de novo genetic accident. We analyzed constitutional exomes of 41 trios (affected child and his 2 healthy parents). Our bioinformatics analyzes identified the existence of de novo post-zygotic mutations of genes involved in neural crest development or cancer, in 5 cases. More complete analyzes of tumor exomes are underway, as well as functional studies of genes and their mutations, in a chick embryo model.This work improves the understanding of molecular and cellular mechanisms that are deregulated in these diseases, thus opening up new therapeutic perspectives.Le but de ce travail de thèse a été d’identifier par séquençage d’exomes, de nouveaux gènes de prédisposition dans 2 pathologies rares dont l’étiologie est peu connue. Le 1er projet a porté sur une famille à 3 cas de sarcomes, sans mutation du gène TP53. Nous avons identifié une mutation pathogénique du gène CDKN2A, dans les 3 cas. Le gène CDKN2A étant le gène majeur de prédisposition au mélanome, nous avons recherché parmi 3 collections différentes des cas de sarcomes associés à une mutation du gène CDKN2A. Nous avons identifié 8 cas de sarcomes indépendants, porteurs d’une mutation du gène CDKN2A et montré une perte d’hétérozygotie au niveau du site de la mutation constitutionnelle CDKN2A dans 5/7 cas, montrant ainsi une perte de fonction complète. Les sarcomes étant rares chez les porteurs de mutations CDKN2A, nous avons recherché des variants rares modificateurs potentiels, par séquençages d’exomes constitutionnels des 8 cas index, et identifié 3 variants du gène PDGFRA. Des études de modélisation ont montré que 2 de ces variants, pourraient avoir un impact sur la structure du domaine extracellulaire de la protéine PDGFRA. Nous avons ainsi démontré que le gène CDKN2A peut prédisposer aux sarcomes et identifié PDGFRA comme gène modificateur potentiel.Dans le second projet nous avons travaillé sur les mélanomes de l’enfant, d’apparence sporadique. Notre hypothèse de travail était que ces mélanomes surviennent suite à un accident génétique de novo. Nous avons analysé des exomes constitutionnels de 41 trios (enfant atteint et ses 2 parents sains). Nos analyses bio-informatiques ont montré l’existence de mutations de novo post-zygotiques de gènes impliqués dans le développement de la crête neurale ou le cancer, dans 5 cas. Les analyses plus complètes des exomes tumoraux sont en cours, ainsi que des études fonctionnelles des gènes et de leurs mutations, dans un modèle d’embryon de poulet. Ce travail permettra d’accroitre la compréhension des mécanismes moléculaires et cellulaires dérégulés dans ces maladies, ouvrant ainsi, de nouvelles perspectives thérapeutiques

Constitutional Exome Sequencing contribution for identification of new cancer predisposing genes, sarcoma and pediatric melanoma.

Le but de ce travail de thèse a été d’identifier par séquençage d’exomes, de nouveaux gènes de prédisposition dans 2 pathologies rares dont l’étiologie est peu connue. Le 1er projet a porté sur une famille à 3 cas de sarcomes, sans mutation du gène TP53. Nous avons identifié une mutation pathogénique du gène CDKN2A, dans les 3 cas. Le gène CDKN2A étant le gène majeur de prédisposition au mélanome, nous avons recherché parmi 3 collections différentes des cas de sarcomes associés à une mutation du gène CDKN2A. Nous avons identifié 8 cas de sarcomes indépendants, porteurs d’une mutation du gène CDKN2A et montré une perte d’hétérozygotie au niveau du site de la mutation constitutionnelle CDKN2A dans 5/7 cas, montrant ainsi une perte de fonction complète. Les sarcomes étant rares chez les porteurs de mutations CDKN2A, nous avons recherché des variants rares modificateurs potentiels, par séquençages d’exomes constitutionnels des 8 cas index, et identifié 3 variants du gène PDGFRA. Des études de modélisation ont montré que 2 de ces variants, pourraient avoir un impact sur la structure du domaine extracellulaire de la protéine PDGFRA. Nous avons ainsi démontré que le gène CDKN2A peut prédisposer aux sarcomes et identifié PDGFRA comme gène modificateur potentiel.Dans le second projet nous avons travaillé sur les mélanomes de l’enfant, d’apparence sporadique. Notre hypothèse de travail était que ces mélanomes surviennent suite à un accident génétique de novo. Nous avons analysé des exomes constitutionnels de 41 trios (enfant atteint et ses 2 parents sains). Nos analyses bio-informatiques ont montré l’existence de mutations de novo post-zygotiques de gènes impliqués dans le développement de la crête neurale ou le cancer, dans 5 cas. Les analyses plus complètes des exomes tumoraux sont en cours, ainsi que des études fonctionnelles des gènes et de leurs mutations, dans un modèle d’embryon de poulet. Ce travail permettra d’accroitre la compréhension des mécanismes moléculaires et cellulaires dérégulés dans ces maladies, ouvrant ainsi, de nouvelles perspectives thérapeutiques.The aim of this thesis was to identify by sequencing of exomes new predisposing genes in 2 rare pathologies whose etiology is not well known.The first project involved a family with 3 cases of sarcomas, without mutation of TP53 gene. We identified a pathogenic mutation of the CDKN2A gene, in the 3 cases. Since CDKN2A gene is the major melanoma predisposing gene, we have searched in 3 different collections, sarcoma cases link to CDKN2A mutations. In total, we have identified 8 independent sarcomas cases, carrying a germline mutation of the CDKN2A gene and showed a loss of heterozygosity at the site of the constitutional mutation of CDKN2A in 5/7 cases, thus proving a complete loss of function. Since sarcomas are rare in carriers of CDKN2A mutations, we looked for potential modifying rare variants, by sequencing constitutional exomes of the 8 index cases, and identified 3 variants of the PDGFRA gene. Modeling studies have shown that 2 of these variants could have an impact on the structure of the extracellular domain of the PDGFRA protein. We have thus demonstrated that the CDKN2A gene can predispose to sarcomas and identified PDGFRA as a potential modifier gene. In the second project, we worked on childhood melanomas, of sporadic appearance. Our working hypothesis was that these melanomas occur as a result of a de novo genetic accident. We analyzed constitutional exomes of 41 trios (affected child and his 2 healthy parents). Our bioinformatics analyzes identified the existence of de novo post-zygotic mutations of genes involved in neural crest development or cancer, in 5 cases. More complete analyzes of tumor exomes are underway, as well as functional studies of genes and their mutations, in a chick embryo model.This work improves the understanding of molecular and cellular mechanisms that are deregulated in these diseases, thus opening up new therapeutic perspectives

Atypical BRAF and NRAS Mutations in Mucosal Melanoma

Primary mucosal melanomas represent a minority of melanomas, but have a significantly worse prognosis than cutaneous melanomas. A better characterization of the molecular pathogenesis of this melanoma subtype could help us understand the risk factors associated with the development of mucosal melanomas and highlight therapeutic targets. Because the Mitogen-Activated Protein Kinase (MAPK) pathway plays such a significant role in melanoma development, we explore v-raf murine sarcoma viral oncogene homolog B (BRAF) and neuroblastoma RAS viral oncogene homolog (NRAS) mutations in mucosal melanoma and compare them to the mutation profiles in cutaneous melanoma and other tumors with BRAF and NRAS mutations. We show that in addition to being less frequent, BRAF and NRAS mutations are different in mucosal melanoma compared to cutaneous melanomas. Strikingly, the BRAF and NRAS mutation profiles in mucosal melanoma are closer to those found in cancers such as lung cancer, suggesting that mutations in mucosal melanoma could be linked to some genotoxic agents that remain to be identified. We also show that the atypical BRAF and NRAS mutations found in mucosal melanomas have particular effects on protein activities, which could be essential for the transformation of mucosal melanocytes

The PI3K/mTOR Pathway Is Targeted by Rare Germline Variants in Patients with Both Melanoma and Renal Cell Carcinoma

Background: Malignant melanoma and RCC have different embryonic origins, no common lifestyle risk factors but intriguingly share biological properties such as immune regulation and radioresistance. An excess risk of malignant melanoma is observed in RCC patients and vice versa. This bidirectional association is poorly understood, and hypothetic genetic co-susceptibility remains largely unexplored. Results: We hereby provide a clinical and genetic description of a series of 125 cases affected by both malignant melanoma and RCC. Clinical germline mutation testing identified a pathogenic variant in a melanoma and/or RCC predisposing gene in 17/125 cases (13.6%). This included mutually exclusive variants in MITF (p.E318K locus, N = 9 cases), BAP1 (N = 3), CDKN2A (N = 2), FLCN (N = 2), and PTEN (N = 1). A subset of 46 early-onset cases, without underlying germline variation, was whole-exome sequenced. In this series, thirteen genes were significantly enriched in mostly exclusive rare variants predicted to be deleterious, compared to 19,751 controls of similar ancestry. The observed variation mainly consisted of novel or low-frequency variants (<0.01%) within genes displaying strong evolutionary mutational constraints along the PI3K/mTOR pathway, including PIK3CD, NFRKB, EP300, MTOR, and related epigenetic modifier SETD2. The screening of independently processed germline exomes from The Cancer Genome Atlas confirmed an association with melanoma and RCC but not with cancers of established differing etiology such as lung cancers. Conclusions: Our study highlights that an exome-wide case-control enrichment approach may better characterize the rare variant-based missing heritability of multiple primary cancers. In our series, the co-occurrence of malignant melanoma and RCC was associated with germline variation in the PI3K/mTOR signaling cascade, with potential relevance for early diagnostic and clinical management

A Melanoma-Tailored Next-Generation Sequencing Panel Coupled with a Comprehensive Analysis to Improve Routine Melanoma Genotyping

International audienceBackground: Tumor molecular deciphering is crucial in clinical management. Pan-cancer next-generation sequencing panels have moved towards exhaustive molecular characterization. However, because of treatment resistance and the growing emergence of pharmacological targets, tumor-specific customized panels are needed to guide therapeutic strategies.Objective: The objective of this study was to present such a customized next-generation sequencing panel in melanoma.Methods: Melanoma patients with somatic molecular profiling performed as part of routine care were included. High-throughput sequencing was performed with a melanoma tailored next-generation sequencing panel of 64 genes involved in molecular classification, prognosis, theranostic, and therapeutic resistance. Single nucleotide variants and copy number variations were screened, and a comprehensive molecular analysis identified clinically relevant alterations.Results: Four hundred and twenty-one melanoma cases were analyzed (before any treatment initiation for 94.8% of patients). After bioinformatic prioritization, we uncovered 561 single nucleotide variants, 164 copy number variations, and four splice-site mutations. At least one alteration was detected in 368 (87.4%) lesions, with BRAF, NRAS, CDKN2A, CCND1, and MET as the most frequently altered genes. Among patients with BRAFV600 mutated melanoma, 44.5% (77 of 173) harbored at least one concurrent alteration driving potential resistance to mitogen-activated protein kinase inhibitors. In patients with RAS hotspot mutated lesions and in patients with neither BRAFV600 nor RAS hotspot mutations, alterations constituting potential pharmacological targets were found in 56.9% (66 of 116) and 47.7% (63 of 132) of cases, respectively.Conclusions: Our tailored next-generation sequencing assay coupled with a comprehensive analysis may improve therapeutic management in a significant number of patients with melanoma. Updating such a panel and implementing multi-omic approaches will further enhance patients' clinical management

The PI3K/mTOR pathway Is targeted by rare germline variants in patients with both melanoma and renal cell carcinoma

International audienceBackground: Malignant melanoma and RCC have different embryonic origins, no common lifestyle risk factors but intriguingly share biological properties such as immune regulation and radioresistance. An excess risk of malignant melanoma is observed in RCC patients and vice versa. This bidirectional association is poorly understood, and hypothetic genetic co-susceptibility remains largely unexplored. Results: We hereby provide a clinical and genetic description of a series of 125 cases affected by both malignant melanoma and RCC. Clinical germline mutation testing identified a pathogenic variant in a melanoma and/or RCC predisposing gene in 17/125 cases (13.6%). This included mutually exclusive variants in MITF (p.E318K locus, N = 9 cases), BAP1 (N = 3), CDKN2A (N = 2), FLCN (N = 2), and PTEN (N = 1). A subset of 46 early-onset cases, without underlying germline variation, was whole-exome sequenced. In this series, thirteen genes were significantly enriched in mostly exclusive rare variants predicted to be deleterious, compared to 19,751 controls of similar ancestry. The observed variation mainly consisted of novel or low-frequency variants (<0.01%) within genes displaying strong evolutionary mutational constraints along the PI3K/mTOR pathway, including PIK3CD, NFRKB, EP300, MTOR, and related epigenetic modifier SETD2. The screening of independently processed germline exomes from The Cancer Genome Atlas confirmed an association with melanoma and RCC but not with cancers of established differing etiology such as lung cancers. Conclusions: Our study highlights that an exome-wide case-control enrichment approach may better characterize the rare variant-based missing heritability of multiple primary cancers. In our series, the co-occurrence of malignant melanoma and RCC was associated with germline variation in the PI3K/mTOR signaling cascade, with potential relevance for early diagnostic and clinical management

Recurrent PAK2 rearrangements in poroma with folliculo‐sebaceous differentiation

International audienceAims: Poroma is a benign adnexal neoplasm with differentiation towards the upper portion of the sweat gland apparatus. In 2019, Sekine et al. demonstrated recurrent YAP1::MAML2 and YAP1::NUTM1 fusion in poroma and porocarcinoma. Follicular, sebaceous and/or apocrine differentiation has been reported in rare cases of poroma and whether these tumours constitute a variant of poroma or represent a distinctive tumour is a matter to debate. Herein we describe the clinical, immunophenotypic, and molecular features of 13 cases of poroma with folliculo-sebaceous differentiation.Methods and results: Most of the tumours were located on the head and neck region (n = 7), and on the thigh (n = 3). All presented were adults with a slight male predilection. The median tumour size was 10 mm (range: 4-25). Microscopically, lesions displayed features of poroma with nodules of monotonous basophilic cells associated with a second population of larger eosinophilic cells. In all cases, ducts and scattered sebocytes were identified. Infundibular cysts were present in 10 cases. In two cases high mitotic activity was noted, and in three cases cytologic atypia and areas of necrosis were identified. Whole transcriptome RNA sequencing demonstrated in-frame fusion transcripts involving RNF13::PAK2 (n = 4), EPHB3::PAK2 (n = 2), DLG1::PAK2 (n = 2), LRIG1::PAK2 (n = 1), ATP1B3::PAK2 (n = 1), TM9SF4::PAK2 (n = 1), and CTNNA1::PAK2 (n = 1). Moreover, fluorescence in situ hybridisation (FISH) analysis revealed PAK2 rearrangement in an additional case. No YAP1::MAML2 or YAP1::NUTM1 fusion was detected.Conclusion: Recurrent fusions involving the PAK2 gene in all analysed poroma with folliculo-sebaceous differentiation in this study confirms that this neoplasm represents a separate tumour entity distinct from YAP1::MAML2 or YAP1::NUTM1 rearranged poromas