Genetic testing for familial melanoma.

While the average lifetime risk of melanoma worldwide is approximately 3%, those with inherited high-penetrance mutations face an increased lifetime risk of 52-84%. In countries of low melanoma incidence, such as in Southern Europe, familial melanoma genetic testing may be warranted when there are two first degree relatives with a melanoma diagnosis. Testing criteria for high incidence countries such as USA, or with very-high incidence, such as Australia and New Zealand, would require a threshold of 3 to 4 affected family members. A mutation in the most common gene associated with familial melanoma, CDKN2A, is identified in approximately 10-40% of those meeting testing criteria. However, the use of multi-gene panels covering additional less common risk genes can significantly increase the diagnostic yield. Currently, genetic testing for familial melanoma is typically conducted by qualified genetic counsellors, however with increasing demand on testing services and high incidence rate in certain countries, a mainstream model should be considered. With appropriate training, dermatologists are well placed to identify high risk individuals and offer melanoma genetic test in dermatology clinics. Genetic testing should be given in conjunction with pre- and post-test consultation. Informed patient consent should cover possible results, the limitations and implications of testing including inconclusive results, and potential for genetic discrimination. Previous studies reporting on participant outcomes of genetic testing for familial melanoma have found significant improvements in both sun protective behavior and screening frequency in mutation carriers.

Standardized Computer-Assisted Analysis of 5-hmC Immunoreactivity in Dysplastic Nevi and Superficial Spreading Melanomas.

5-Hydroxymethylcytosine (5-hmC) is an important intermediate of DNA demethylation. Hypomethylation of DNA is frequent in cancer, resulting in deregulation of 5-hmC levels in melanoma. However, the interpretation of the intensity and distribution of 5-hmC immunoreactivity is not very standardized, which makes its interpretation difficult. In this study, 5-hmC-stained histological slides of superficial spreading melanomas (SSM) and dysplastic compound nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. Receiver operating characteristic/area under the curve (ROCAUC) and t-tests were performed. A p-value of <0.05 was used for statistical significance, and a ROCAUC score of >0.8 was considered a "good" result. In total, 92 5-hmC-stained specimens were analyzed, including 42 SSM (45.7%) and 50 DN (54.3%). The mean of 5-hmC-positive cells/mm2 for the epidermis and dermo-epidermal junction and the entire lesion differed significantly between DN and SSM (p = 0.002 and p = 0.006, respectively) and showed a trend towards higher immunoreactivity in the dermal component (p = 0.069). The ROCAUC of 5-hmC-positive cells of the epidermis and dermo-epidermal junction was 0.79, for the dermis 0.74, and for the entire lesion 0.76. These results show that the assessment of the epidermal with junctional expression of 5-hmC is slightly superior to dermal immunoreactivity in distinguishing between DN and SSM.

Transcriptome Analysis Identifies Oncogenic Tissue Remodeling during Progression from Common Nevi to Early Melanoma.

Early detection and treatment of melanoma, the most aggressive skin cancer, improves the median 5-year survival rate of patients from 25% to 99%. Melanoma development involves a stepwise process during which genetic changes drive histologic alterations within nevi and surrounding tissue. Herein, a comprehensive analysis of publicly available gene expression data sets of melanoma, common or congenital nevi (CN), and dysplastic nevi (DN), assessed molecular and genetic pathways leading to early melanoma. The results demonstrate several pathways reflective of ongoing local structural tissue remodeling activity likely involved during the transition from benign to early-stage melanoma. These processes include the gene expression of cancer-associated fibroblasts, collagens, extracellular matrix, and integrins, which assist early melanoma development and the immune surveillance that plays a substantial role at this early stage. Furthermore, genes up-regulated in DN were also overexpressed in melanoma tissue, supporting the notion that DN may serve as a transitional phase toward oncogenesis. CN collected from healthy individuals exhibited different gene signatures compared with histologically benign nevi tissue located adjacent to melanoma (adjacent nevi). Finally, the expression profile of microdissected adjacent nevi tissue was more similar to melanoma compared with CN, revealing the melanoma influence on this annexed tissue.

Biology and genetics of acquired and congenital melanocytic naevi.

Acquired and congenital melanocytic naevi are common benign neoplasms. Understanding their biology and genetics will help clinicians and pathologists correctly diagnose melanocytic tumours, and generate insights into naevus aetiology and melanomagenesis. Genomic data from published studies analysing acquired and congenital melanocytic naevi, including oncogenic driver mutations, common melanoma associated mutations, copy number aberrations, somatic mutation signature patterns, methylation profile, and single nucleotide polymorphisms, were reviewed. Correlation of genomic changes to dermoscopic features, particular anatomic sites and total body naevus counts, was also performed. This review also highlights current scientific theories and evidence concerning naevi growth arrest. Acquired and congenital melanocytic naevi show simple genomes, typically characterised by mutually exclusive single oncogenic driver mutations in either BRAF or NRAS genes. Genomic differences exist between acquired and congenital naevi, common and dysplastic naevi, and by dermoscopic features. Acquired naevi show a higher rate of BRAF hotspot mutations and a lower rate of NRAS hotspot mutations compared to congenital naevi. Dysplastic naevi show upregulation of follicular keratinocyte-related genes compared to common naevi. Anatomical locations and DNA signatures of naevi implicates ultraviolet radiation and non-ultraviolet radiation pathways in naevogenesis. DNA driver point mutations in acquired and congenital melanocytic naevi have been well characterised. Future research is required to better understand transcriptional and epigenetic changes in naevi, as well as those regulating naevus growth arrest and cell environment signalling.

Dysplastic nevus part II: Dysplastic nevi: Molecular/genetic profiles and management.

Dermatologists frequently see patients with clinically atypical nevi and dermatopathologists interpret histologically dysplastic nevi on a near-daily basis, but there is great variability in the definition and management of such lesions. This part of the CME review focuses on information published since the previous comprehensive review (2012), with emphasis on molecular and genetic attributes of histologically dysplastic nevi and clinical management.

Dysplastic melanocytic nevus: Are molecular findings the key to the diagnosis?

The primary differential diagnosis of melanoma is dysplastic nevus. Until now, the final diagnosis is based on histological findings. With modern techniques, pathologists receive very early melanocytic lesions, which do not fit all malignant criteria. In those cases, even the concurrence between specialists and intraobserver agreement is not good. A molecular test could be developed to improve the accuracy of melanocytic lesions diagnosis and help in challenging lesions. The objective of this study is to provide a literary review looking for molecular markers that characterize dysplastic nevi and could help surgical pathologists differentiate them from melanoma. Articles from PubMed presenting case series of dysplastic nevi and melanoma genomic analyses were considered. The search was conducted in PubMed looking for papers written in English, published in the ten years preceding April 2020. This review confirmed the absence of a pathognomonic molecular marker of dysplastic nevi. This is a heterogeneous group of lesions with an uncertain risk to become a melanoma. The molecular heterogeneity of dysplastic nevi, the variation of histological diagnostic criteria among services, and the diverse molecular techniques applied are challenging features that might hamper definitive diagnoses. However, currently, there appears to be limited value for molecular testing in the diagnosis of dysplastic nevi.

Dysplastic nevi and melanoma: microRNAs tell a divergent story.

BACKGROUND: dysplastic nevi (DN) share some clinical and histological features with melanoma and have been considered intermediate lesions toward malignant transformation. However, scientific evidence of DN representing melanoma precursors is still incomplete, and many observations pointed toward their being a distinct biological entity. The current definition of DN is also confusing and the practical consequence of this uncertainty is the excessive excision of DN with severe atypia. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and whose global expression can classify normal and pathological tissues. OBJECTIVES: given these considerations, we decided to perform a small RNA profiling study in a group of DN and invasive melanomas obtained from the same patient, to assess tumor evolution according to the global microRNA expression. METHODS: we performed a small-RNA sequencing of 6 DN, 2 congenital nevi and 4 cutaneous melanomas obtained from 4 subjects and evaluated the global miRNA expression correlation between samples. RESULTS AND CONCLUSIONS: the hierarchical clustering and principal component analyses of global miRNA expression, independently grouped together DN and their matching congenital nevi and showed a divergence of DN miRNA profile from melanoma. Our study suggests that DN have a peculiar and different miRNA expression profile compared to melanomas developed in the same patient, thus supporting the hypothesis that DN are distinct biological entities and not melanoma precursors.

Dysplastic Nevi: Morphology and Molecular and the Controversies In-between.

Dysplastic nevi are distinctive melanocytic lesions in the larger group of atypical nevi. They often are multiple and sporadic with genetic features intermediate between common acquired nevi and melanoma. Dysplastic nevi may be multiple, familial, and seen in patients with familial melanoma syndrome. Although their behavior is benign, they rarely represent a precursor to melanoma. If clinically suspicious, dysplastic nevi should be removed for adequate histopathologic examination and to exclude possibility of melanoma. Partial sampling should be avoided because reliable separation from melanoma requires visualization of the entire lesion to allow for examination of architectural histopathologic features and avoid sampling error.

Neurofibromatosis type 1 and melanoma of the iris arising from a dysplastic nevus: A rare yet casual association?

PURPOSE: We investigated the molecular causes of an unusual pigmented and ulcerated iris lesion detected in a patient diagnosed with neurofibromatosis type 1 (NF1). CASE REPORT: A 52-year-old man was referred to our clinic with a non-traumatic ulcer in his left eye. Hyphema reabsorption disclosed a pigmented iris mass, thus ultrasound biomicroscopy and anterior segment fluorescein angiography were performed to investigate for the presence of a malignant lesion. Upon angiography, the lesion appeared highly vascularized but prevented posterior iris examination. Therefore, a gonioscopy was executed revealing extension of the lesion into the peripheral iris. Histopathology of the excisional iris biopsy revealed iris melanoma over a dysplastic nevus. NF1 is an autosomal dominant disorder characterized by pigmented cutaneous lesions, multiple skin tumors, and spinal and cranial nerve tumors. Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 92% of cutaneous melanomas occur in patients with dysplastic nevus syndrome. Skin melanomas have been found in 0.1%-5.4% of NF1 patients. In literature, only 18 reports of uveal melanoma have been documented in association with NF1, including three cases of iris melanoma. RESULTS: NF1 gene testing identified a causative mutation in the germline but no loss of the wild-type allele in the iris melanoma. CONCLUSIONS: Occurrence of both diseases in one patient is extremely rare, but the common origin of Schwann cells and melanoblasts suggests a non-casual association. Therefore, we propose that NF1 patients should be screened for nevi, both cutaneous and uveal, for better patients' management.

BRAF fusion Spitz neoplasms; clinical morphological, and genomic findings in six cases.

BACKGROUND: Fusions involving the BRAF gene are responsible for 5% of Spitz neoplasms. To better characterize them, we report the clinical, morphological, and genomic findings of six BRAF fusion Spitz tumors. METHODS: The morphological, clinical, and molecular findings of six BRAF fusion Spitz neoplasms assessed by next generation sequencing (NGS) were compared to a control set of Spitz without BRAF fusions. RESULTS: BRAF fusion Spitz tumors had frequent predominance of epithelioid morphology (4/6 cases), frequent high-grade nuclear atypia and pleomorphism (5/6 cases), and a frequent desmoplastic base (3/6 cases). Five of six cases were diagnosed as atypical Spitz tumor and one as Spitz nevus. All cases had uneventful clinical follow-up. There were five different fusion partners, with CLIP2 being the most frequent. Secondary pathogenic mutations were frequent and chromosomal copy number changes were seen in three of six cases by an NGS platform. CONCLUSIONS: BRAF fusions Spitz usually have epithelioid morphology, high-grade nuclear atypia, and desmoplasia. Chromosomal copy number changes are not infrequent. While our cases had uneventful follow-up, a meta-analysis of the literature suggests that among the fusion subtypes associated with Spitz tumors, they are among the subgroups more likely to develop distant metastasis.

PRAME expression in melanocytic proliferations with intermediate histopathologic or spitzoid features.

BACKGROUND: PRAME (PReferentially expressed Antigen in MElanoma) has shown utility in distinguishing melanoma from benign melanocytic lesions, but knowledge of its expression pattern in intermediate melanocytic and spitzoid proliferations is limited. METHODS: Immunohistochemical expression of PRAME was examined in 112 melanocytic proliferations with intermediate histopathologic or spitzoid features. RESULTS: Any intensity of nuclear PRAME staining in at least 60% of lesional melanocytes was determined as the best threshold for diffuse staining in this cohort. Nearly all non-spitzoid melanomas (23/24; 95.8%) demonstrated diffuse PRAME expression. PRAME was completely negative in 95.6% (43/45) of mitotically-active nevi, traumatized nevi, nevi with persistent/recurrent features, and dysplastic nevi. Most Spitz nevi (15/20) and atypical Spitz tumors (10/13) entirely lacked PRAME expression. One Spitz nevus, one atypical Spitz tumor, and one spitzoid melanoma (1/2) demonstrated diffuse PRAME expression. CONCLUSIONS: Although diffuse PRAME expression is generally limited to malignant melanoma, benign Spitz nevi and atypical Spitz tumors can infrequently express diffuse PRAME. PRAME immunohistochemistry can be useful in the evaluation of atypical melanocytic proliferations with intermediate histopathologic features but should be interpreted with caution in the setting of spitzoid neoplasms.

Development of esophageal squamous cell cancer in patients with FAMMM syndrome: Two clinical reports.

Familial atypical multiple mole melanoma (FAMMM) syndrome is a hereditary syndrome characterized by multiple dysplastic nevi and melanoma. Patients with FAMMM may have a heterozygous, inactivating, pathogenic germline variant in the CDKN2A gene, especially the NM_000077.4: c.225_243del19 (p.p75fs) variant, also known as p16-Leiden variant. Patients with this variant are at high risk for developing melanomas and pancreatic cancer due to somatic inactivation of the wild-type CDKN2A allele. The combination of an inactivating germline CDKN2A mutation and somatic inactivation of the wild-type CDKN2A allele in the same cell results in tumor formation. It has been suggested that carriers of a germline CDKN2A mutation are also at increased risk for several other cancer types, including esophageal cancer. Here, we describe two unrelated patients with the p16-Leiden variant who developed esophageal squamous cell cancer. Evidence of loss of the wild-type CDKN2A allele was obtained in the tumor tissue of both patients indicating biallelic inactivation of p16 in the tumor cells. These results suggest that these patients developed esophageal squamous cell cancer in the context of FAMMM syndrome.

Analysis of the CDKN2A Gene in FAMMM Syndrome Families Reveals Early Age of Onset for Additional Syndromic Cancers.

Familial atypical multiple mole melanoma (FAMMM) syndrome is a hereditary cancer syndrome that results from mutations in several genes, including the CDKN2A gene. In addition to melanoma, certain other malignancies such as pancreatic cancer are known to occur more frequently in family members who carry the mutation. However, as these families have been followed over time, additional cancers have been observed in both carriers and noncarriers. We sought to determine whether these additional cancers occur at higher frequencies in carriers than noncarriers. We performed survival analyses using 10 FAMMM syndrome families (N = 1,085 individuals) as well as a mixed effects Cox regression, with age at last visit to the clinic or age at cancer diagnosis as our time variable. This analysis was done separately for the known FAMMM-related cancers and "other" cancer groups. The survival curves showed a significant age effect with carriers having a younger age at cancer onset than noncarriers for FAMMM-related cancers (as expected) as well as for newly associated cancers. The Cox regression reflected what was seen in the survival curves, with all models being highly significant (P = 7.15E-20 and P = 5.00E-13 for the FAMMM-related and other cancers, respectively). These analyses support the hypothesis that CDKN2A mutation carriers in FAMMM syndrome families have increased risk for early onset of several cancer types beyond the known cancers. Therefore, these individuals should be screened for additional cancers, and mutation screening should be extended to more than first-degree relatives of an index carrier patient. SIGNIFICANCE: This study shows that carriers of mutations in the CDKN2A gene in FAMMM syndrome are at increased risk for early onset of several cancer types beyond the known cancers.

Dysplastic/Clark naevus in the era of molecular pathology.

Dysplastic naevus has been a controversial entity since its first description by Clark in 1978. Despite a recent paradigm shift from the initially proposed notion that dysplastic naevus is a precursor to melanoma, its management has been increasingly more aggressive in the last decade. The latter is due to an unresolved uncertainty regarding its biological nature which necessitates further clarification. Recent molecular genetics, epigenetic and transcriptomic discoveries have revealed that a subset of dysplastic naevi exhibits a genomic profile which is intermediate between that of benign naevus and melanoma. This group of lesions often shows somatic mutations in non-V600E BRAF, NRAS and TERT and hemizygous deletion of CDKN2A gene as well as upregulation of genes involved in proliferation, cell adhesion and migration, and epidermal and follicular keratinocyte-related genes. These new genomic insights suggest that a proportion of dysplastic naevi have a greater propensity to evolve to melanoma; however, the clinical and histopathological features of this proposed intermediate category are still to be elucidated by further research.

CDKN2A germline mutations are not associated with poor survival in an Italian cohort of melanoma patients.

BACKGROUND: Cyclin dependent kinase inhibitor 2A gene (CDKN2A) germline mutations have recently been associated with poor survival in patients with melanoma. Despite the high mutation rate in our cohort (up to 10% in patients with apparently sporadic melanoma), information on the impact of CDKN2A on survival in this cohort is lacking. OBJECTIVE: To investigate whether poor survival associated with CDKN2A germline mutations was confirmed in a high mutation-prevalence cohort of Italian patients with melanoma undergoing a mutation-based follow-up. METHODS: A total of 1239 patients with cutaneous melanoma were tested for CDKN2A mutational status and then assigned to a follow-up scheme according not only to family history but also to CDKN2A mutational status, as follow-up intervals were more frequent for CDKN2A germline mutation-positive (MUT+) patients. From this cohort, we selected 106 MUT+ patients (with familial melanoma or apparently sporadic melanoma) and 199 CDKN2A germline mutation-negative (MUT-) patients with sporadic melanoma who were matched by age and sex and had a similar tumor stage distribution. RESULTS: We found no difference in overall survival (hazard ratio, 0.85; 95% confidence interval, 0.48-1.52; P = .592,) or melanoma-specific survival (hazard ratio, 0.86; 95% confidence interval, 0.38-1.95; P = .718,) between MUT+ and MUT- patients. MUT+ patients were more likely to develop multiple melanomas and to undergo surgical excision of dysplastic nevi than were MUT- patients. LIMITATIONS: Retrospective study. CONCLUSION: CDKN2A mutations were not associated with survival in our cohort.

Risks of Melanoma and Other Cancers in Melanoma-Prone Families over 4 Decades.

Since 1976, melanoma-prone families have been followed at the National Cancer Institute to identify etiologic factors for melanoma. We compared risks of melanoma and other cancers in 1,226 members of 56 families followed for up to 4 decades with population rates in the Surveillance, Epidemiology, and End Results program. All families were tested for mutations in CDKN2A and CDK4; 29 were mutation-positive and 27 mutation-negative. We compared rates of invasive melanomas, both first and second, by family mutation status, with Surveillance, Epidemiology, and End Results program. Comparing three calendar periods of the study, risk of first primary melanoma decreased slightly. Risks of melanoma after first examination, however, were approximately one-third the risks prior to the first examination in both mutation-positive and mutation-negative families. Among patients with melanoma, risk of a second melanoma was increased 10-fold in all families; risk was somewhat higher in mutation-positive families. Risks of other second cancers were increased only for pancreatic cancer after melanoma in mutation-positive families. Over 4 decades, prospective risk of melanoma has decreased substantially in both mutation-positive and mutation-negative families, when melanoma has greatly increased in the general population. TRIAL REGISTRATION: NCI 02-C-0211, ClinicalTrials.gov ID NCT00040352.

Trajectories of premalignancy during the journey from melanocyte to melanoma.

A stepwise progression from melanocytic precursors to cutaneous melanoma is a well-established model, based on decades of careful observation and morphological analysis. The steps identified are benign melanocytic naevus, dysplastic naevus, 'radial growth phase' melanoma (including melanoma in situ) and 'vertical growth phase' melanoma (also termed tumourigenic melanoma). Recent genomic data have refined the understanding of the steps of melanoma development and their relationship to one another. These data support the existence of dysplastic naevi as distinct lesions; suggest the importance of clonal dynamics in the precursor steps of melanoma; and confirm the carcinogenic role of ultraviolet radiation throughout early melanoma development and progression. In this review, the steps of melanoma development and progression are summarised and discussed in the context of recent genomic studies. This new understanding of melanoma pathogenesis that has been facilitated through careful correlation of morphological and molecular features will allow the identification and development of robust biomarkers to assist in more accurate diagnosis and prognostication of melanocytic tumours.

Improvement of Genetic Testing for Cutaneous Melanoma in Countries With Low to Moderate Incidence: The Rule of 2 vs the Rule of 3.

Importance: Genetic testing for melanoma-prone mutation in France, a country with low to moderate incidence of melanoma, is proposed in cases with 2 invasive cutaneous melanomas and/or related cancers in the same patient, or in first- or second-degree relatives (rule of 2). In preclinical studies, these rules led to disclosure of mutation(s) in more than 10% of these families, the threshold widely accepted to justify genetic testing for cancers. Objective: To reconsider these criteria in a general population testing of patients. Design, Setting, and Participants: This was a retrospective study, performed from 2004 to 2015 at Angers and Lyons University Hospitals, of a cohort of 1032 patients who underwent genetic testing. Main Outcomes and Measures: Frequency of mutation in high (CDKN2A, CDK4, and BAP1) and intermediate (MITF) susceptibility genes; statistical effect of histologic subtype, age, dysplastic nevi syndrome, and associated cancers on mutation rate; and evaluation of cases with anamnestic uncertainty. Results: The mutation rate was 67 of 1032 patients (6.5%). Their mean (SD) age was 54.5 (14.2) years [range, 18-89 years], and 543 (52.6%) were men. It increased to 38 of 408 patients (9.3%) when applying a rule of 3 (those with ≥3 primary melanomas or genetically related cancers) (P = .68) and to 27 of 150 patients (18.0%) with a rule of 4 (4 primary melanomas or related cancer) (P < .001). The impact of age at first melanoma was observed only in those younger than 40 years, with a rate of 32 of 263 (12.1%) (P = .12) for the rule of 2 and 22 of 121 (18.2%) (P = .001) for the rule of 3. Use of the rule of 2 in patients younger than 40 years reduced the number of missed CDKN2A-mutated-families when applying the rule of 3 from 14 of 43 to 7 of 43. Anamnestic uncertainty, found in 88 families (8.5%), if excluded, would have led us to withdraw of only 21 cases (23.8%), and only 1 mutation would have been missed. Conclusions and Relevance: We propose using the rule of 3 to recommend genetic testing in France and countries with low to moderate incidence of melanoma, except in families and patients with a first melanoma occurrence before age 40 years in whom the rule of 2 could be maintained.

Familial Melanoma Associated with Li-Fraumeni Syndrome and Atypical Mole Syndrome: Total-body Digital Photography, Dermoscopy and Confocal Microscopy.

Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disorder caused by a mutation in the p53 gene. Melanoma is considered to be a rare, controversial component of LFS. The aim of this study is to describe the utility of systematic screening for melanoma in patients with LFS and atypical mole syndrome. Two 28-year-old identical twin sisters with LFS and atypical moles were monitored by physical examination, total-body digital photography and dermoscopy be-tween 2006 and 2014. A total of 117, predominantly dark-brown, reticular naevi were identified on case 1 and 105 on case 2. Excisions were performed during the evaluation period of 1 in-situ melanoma and 3 basal cell carcinomas in case 1, and 1 in-situ melanoma and 1 early invasive melanoma in case 2. The remaining melanocytic lesions in both patients were stable during follow-up. The 3 melanomas were new atypical lesions detected with total-body photography and dermoscopy. In conclusion, monitoring LFS patients with total-body photography and dermoscopy may be useful to detect early melanoma.