Search

AbtractsApplying spatial transcriptomics (ST) to explore a vast amount of formalin-fixed paraffin-embedded (FFPE) archival cancer tissues has been highly challenging due to several critical technical issues. In this work, we optimised ST protocols to generate unprecedented spatial gene expression data for FFPE skin cancer. Skin is among the most challenging tissue types for ST due to its fibrous structure and a high risk of RNAse contamination. We evaluated tissues collected from ten years to two years ago, spanning a range of tissue qualities and complexity. Technical replicates and multiple patient samples were assessed. Further, we integrated gene expression profiles with pathological information, revealing a new layer of molecular information. Such integration is powerful in cancer research and clinical applications. The data allowed us to detect the spatial expression of non-coding RNAs. Together, this work provides important technical perspectives to enable the applications of ST on archived cancer tissues.

Here, we present a rare case of a patient who developed multiple primary melanomas within the boundaries of two nevi depigmentosa. The melanomas were excised, and as a preventive measure, the remainder of the nevi depigmentosa were removed. We performed whole-exome sequencing on excised tissue from the nevus depigmentosus, adjacent normal skin, and saliva to explain this intriguing phenomenon. We also performed a GeneTrails Comprehensive Solid Tumor Panel analysis on one of the melanoma tissues. Genetic analysis revealed germline MC1R V92M and TYR R402Q polymorphisms and a MET E168D germline mutation that may have increased the risk of melanoma development. This genetic predisposition, combined with a patient-reported history of substantial sun exposure and sunburns, which were more severe within the boundaries of the nevi depigmentosa due to the lack of photoprotective melanin, produced numerous somatic mutations in the melanocytes of the nevi depigmentosa. Fitting with this paradigm for melanoma development in chronically sun-damaged skin, the patient's melanomas harbored somatic mutations in CDKN2A (splice site), NF1, and ATRX and had a tumor mutation burden in the 90-95th percentile for melanoma.

Melanoma may arise within a pre-existing nevus, but commonly forms in the skin adjacent to nevi. We have performed global DNA methylation profiling using the Illumina EPIC array (>800K loci) of 32 dermoscopically ('globular' vs 'non-globular' pattern) and histopathologically classified nevi, together with matching adjacent perilesional skin, and discovered that methylation patterns can accurately decompose the proportion of cell types present in the tissue. In this study, we sought to assess whether there are differences in the DNA methylation profiles of histologically normal skin samples that surround nevi, independent of cellular composition. Among the 739,270 CpG loci included in the analysis, there were a total of 8,200 CpG loci identified as significantly differentially methylated (Q < 0.05) in ‘globular skin’ vs ‘non-globular skin’ adjusted for age, sex, and the estimated relative proportions of epithelial cells, fibroblasts, and melanocytes in each sample. After imposing a more stringent threshold (M value of ≥0.5) a total 816 CpG loci were found to be DM, with 696 loci identified as significantly hypermethylated, and 120 loci significantly hypomethylated (Q < 0.05 and ΔM > 0.5) in ‘globular’ vs ‘non-globular’ skin. Mapping of the DM (Q < 0.05 and |ΔM| < 0.5) CpG loci to their respective genes revealed that eight of the top 20 DM genes, by proportion of differentially methylated loci, were positioned in proximity to suggest regulation of members of the HOX gene family. We next assessed the differential gene expression of the eight DM HOX genes in the perilesional skin as well as the matching adjacent nevus. After adjusting for age, sex, and sample cellular composition, none of the assessed HOX genes demonstrated statistically significant (P ≤ 0.05) differential expression in the nevi, however, ‘non-globular skin’ demonstrated a statistically significant (P < 0.05) increase in the expression of five of the eight HOX genes relative to ‘globular skin’. Importantly, this increased gene expression corresponds to the methylation status of the DM loci. HOX genes are known to be involved in limb and axial development as well as the positioning of dermal fibroblasts. Moreover, melanocytes from different anatomic positions have distinct transcriptional profiles and the anatomical position of melanocytes plays a key role in determining whether genetic alterations will subsequently drive melanoma formation. This positioning of melanoma subtypes (cutaneous vs acral) was recently shown to be related to interaction of specific HOX genes with site-specific oncogenic alterations. The distinct HOX gene family members we have revealed, differ to those related to melanoma development and positioning, as such we propose that these may contribute to the positioning of acquired melanocytic nevi, and in combination with common somatic alterations in BRAF, may contribute to nevus formation. Citation Format: Meghan E. Muse, Helmut Schaider, Harald Oey, H. Peter Soyer, Brock C. Christensen, Mitchell S. Stark. Distinct HOX gene family DNA methylation profiles in histologically normal skin dependent on dermoscopic pattern of adjacent nevi [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6000.

Ultraviolet radiation-induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV-induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome-wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

Early detection of skin pathologies with current clinical diagnostic tools is challenging, particularly when there are no visible colour changes or morphological cues present on the skin. In this study, we present a terahertz (THz) imaging technology based on a narrow band quantum cascade laser (QCL) at 2.8 THz for human skin pathology detection with diffraction limited spatial resolution. THz imaging was conducted for three different groups of unstained human skin samples (benign naevus, dysplastic naevus, and melanoma) and compared to the corresponding traditional histopathologic stained images. The minimum thickness of dehydrated human skin that can provide THz contrast was determined to be 50 µm, which is approximately one half-wavelength of the THz wave used. The THz images from different types of 50 µm-thick skin samples were well correlated with the histological findings. The per-sample locations of pathology vs healthy skin can be separated from the density distribution of the corresponding pixels in the THz amplitude–phase map. The possible THz contrast mechanisms relating to the origin of image contrast in addition to water content were analyzed from these dehydrated samples. Our findings suggest that THz imaging could provide a feasible imaging modality for skin cancer detection that is beyond the visible.

Large-giant congenital melanocytic nevi have been well characterized clinically, yet questions remain about the heterogenous phenotypes observed. Martins da Silva et al. (2018) highlight the genotypic diversity between "classic" and "spilus-like" congenital melanocytic nevi by analyzing multiple biopsy sites and matching satellite nevi. This study provides evidence for alternative modes of development beyond the well-established NRAS mutation paradigm.

Acquired melanocytic nevi grow and persist in a stable form into adulthood. Using genome-wide methylation profiling, we evaluated 32 histopathologically and dermoscopically characterized nevi to identify the key epigenetic regulatory mechanisms involved in nevogenesis. Benign (69% globular and 31% nonspecific dermoscopic pattern) and dysplastic (95% reticular/nonspecific dermoscopic pattern) nevi were dissimilar, with only two shared differentially methylated loci. Benign nevi showed an increase in both genome-scale methylation and methylation of Alu/LINE-1 retrotransposable elements, a marker of genomic stability, as well as global methylation. In contrast, dysplastic nevi showed evidence for genomic instability through the hypomethylation of Alu/LINE-1 (Alu: P = 0.00019; LINE-1: P = 0.000035). Using dermoscopic classifications, reticular/nonspecific patterned nevi had 59,572 5'-C-phosphate-G-3' differentially methylated loci (Q0.05), whereas globular nevi had no significant differentially methylated loci. In reticular/nonspecific patterned nevi, the tumor suppressor PTEN had the greatest proportion of hypermethylated 5'-C-phosphate-G-3' loci in its promoter region than all other assayed gene promoters. The relative activity of reticular/nonspecific nevi was evidenced by 50,720 hypomethylated loci being enriched for accessible chromatin and 8,852 hypermethylated loci strongly enriched, for example, marks of active gene promoters, which suggests that gain of DNA methylation observed in these nevus types plays a role in gene regulation.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten–driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Benign naevi are closely linked to melanoma, as risk factors, simulators, or sites of melanoma formation. There is a heavy genetic overlap between the two lesions, a shared environmental influence of ultraviolet radiation, and many similar cellular features, yet naevi remain locally situated while melanomas spread from their primary site and may progress systemically to distal organs. Untangling the overlapping contributors and predictors of naevi and melanoma is an ongoing area of research and should eventually lead to more personalized prevention and treatment strategies, through the development of melanoma risk stratification tools and early detection of evolving melanomas. This will be achieved through a range of complementary strategies: risk-adjusted primary prevention counseling; the use of lesion imaging technologies such as sequential 3D total body photography and consumer-performed lesion imaging; artificial intelligence deep phenotyping and clinical assistance; a better understanding of genetic drivers of malignancy, risk variants, clinical genetics, and polygenic effects; and the interplay between genetics, phenotype and the environment.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

IMPORTANCE: Use of prognostic gene expression profile (GEP) testing in cutaneous melanoma (CM) is rising despite a lack of endorsement as standard of care. OBJECTIVE: To develop guidelines within the national Melanoma Prevention Working Group (MPWG) on integration of GEP testing into the management of patients with CM, including (1) review of published data using GEP tests, (2) definition of acceptable performance criteria, (3) current recommendations for use of GEP testing in clinical practice, and (4) considerations for future studies. EVIDENCE REVIEW: The MPWG members and other international melanoma specialists participated in 2 online surveys and then convened a summit meeting. Published data and meeting abstracts from 2015 to 2019 were reviewed. FINDINGS: The MPWG members are optimistic about the future use of prognostic GEP testing to improve risk stratification and enhance clinical decision-making but acknowledge that current utility is limited by test performance in patients with stage I disease. Published studies of GEP testing have not evaluated results in the context of all relevant clinicopathologic factors or as predictors of regional nodal metastasis to replace sentinel lymph node biopsy (SLNB). The performance of GEP tests has generally been reported for small groups of patients representing particular tumor stages or in aggregate form, such that stage-specific performance cannot be ascertained, and without survival outcomes compared with data from the American Joint Committee on Cancer 8th edition melanoma staging system international database. There are significant challenges to performing clinical trials incorporating GEP testing with SLNB and adjuvant therapy. The MPWG members favor conducting retrospective studies that evaluate multiple GEP testing platforms on fully annotated archived samples before embarking on costly prospective studies and recommend avoiding routine use of GEP testing to direct patient management until prospective studies support their clinical utility. CONCLUSIONS AND RELEVANCE: More evidence is needed to support using GEP testing to inform recommendations regarding SLNB, intensity of follow-up or imaging surveillance, and postoperative adjuvant therapy. The MPWG recommends further research to assess the validity and clinical applicability of existing and emerging GEP tests. Decisions on performing GEP testing and patient management based on these results should only be made in the context of discussion of testing limitations with the patient or within a multidisciplinary group.

Objectives The major cause of mucosal squamous cell carcinomas of the head and neck (HNSCCs) has been attributed to human papillomavirus (HPV) infection. Here we investigate if microRNA expression in HNSCC can be used as a prognostic tool with or without HPV status. Materials and Methods We performed a discovery miRNA microarray (miRBase v.21) profiling of 52 tonsillar SCCs with TaqMan real-time PCR validation of 228 HNSCCs. Patients had a histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx or larynx. Logistic regression models were used to estimate the magnitude of the effect of association with clinical factors and miRNAs associated with HPV status. For recurrence and survival analysis, we used unadjusted and multivariable adjusted Cox proportional hazard regression models. Results Seventeen miRNAs were significantly associated with better prognosis in the discovery phase and were validated in the extended dataset. The best fitting model (AUC = 0.92) for HPV status included age, smoking, and miRNAs: miR-15b, miR-20b, miR-29a, miR-29c, miR-142, miR-146a and miR-205. Using Cox regression model for recurrence, miR-29a was associated with 49% increased risk of recurrence while miR-30e and miR-342 were associated with decreased risk of recurrence with HRs 0.92 (95% CI 0.85–0.99) and 0.84 (95% CI 0.73–0.98), respectively. Our best fitting model for survival included age, gender, alcohol consumption, N stage, recurrence, HPV status, together with miRNAs-20b, 29a, and 342. Conclusion miRNAs show potential to serve as usual biomarkers to predict the clinical course of patients with mucosal HNSCC.

1-5% of individuals with melanoma have a strong family history with a causal gene identified in ~30% of those families, most commonly CDKN2A. Genetic testing and counselling for CDKN2A improves sun-protective and surveillance behaviour without negative psychological sequelae. Given these advantages, dermatologists need to recognise those with a sufficient a priori risk to warrant the offer of genetic testing.

Lung cancer is currently the leading cause of cancer death in both developing and developed countries. Given that lung cancer has poor prognosis in later stages, it is essential to achieve an early diagnosis to maximize patients’ overall survival. Non-small cell lung cancer (NSCLC) is the most common form of primary lung cancer in both smokers and non-smokers. The current standard screening method, low‐dose computed tomography (LDCT), is the only radiological method that demonstrates to have mortality benefits across multiple large randomized clinical trials (RCT). However, these RCTs also found LDCT to have a significant false positive rate that results in unnecessary invasive biopsies being performed. Due to the lack of both sensitive and specific screening methods for the early detection of lung cancer, there is an urgent need for alternative minimally or non-invasive biomarkers that may provide diagnostic, and/or prognostic information. This has led to the identification of circulating biomarkers that can be readily detectable in blood and have been extensively studied as prognosis markers. Circulating microRNA (miRNA) in particular has been investigated for these purposes as an augmentation to LDCT, or as direct diagnosis of lung cancer. There is, however, a lack of consensus across the studies on which miRNAs are the most clinically useful. Besides miRNA, other potential circulating biomarkers include circulating tumor cells (CTCs), circulating tumor DNA (ctDNAs) and non-coding RNAs (ncRNAs). In this review, we provide the current outlook of several of these biomarkers for the early diagnosis of NSCLC.

Melanocytic naevi are common melanocytic proliferations that may simulate the appearance of cutaneous melanoma. Naevi commonly harbour somatic mutations implicated in melanomagenesis but in most cases lack the necessary genomic alterations required for melanoma development. While the mitogen-activated protein kinase pathway and ultraviolet radiation strongly contribute to naevogenesis, the somatic mutational landscape of dermoscopic naevus subsets distinguishes some of the molecular hallmarks of naevi in relation to melanoma. We herein discuss the classification of naevi and theories of naevogenesis and review the current literature on the somatic alterations in naevi and melanoma. This review focusses on the clinical-dermoscopic-pathological and genomic correlation of naevi that shapes the current understanding of naevi.

Precise detection of early melanomas is essential as the stage of disease guides treatment options. One growing field that may facilitate the advancement of early melanoma detection, is achieved through profiling serum extracellular vesicles (EVs) using sensitive nanotechnology. As a proof of principle, using a detection platform that combines a microfluidic device and surface-enhanced Raman spectroscopy (SERS), the expression profiles of 4 protein biomarkers in serum EVs (termed as “EV SERS signatures”) derived from 20 early stage melanoma patients (including in situ melanoma) and 21 healthy participants are multiplexed. Significantly higher signal intensities of selected protein biomarkers are observed in serum EVs from melanoma patients compared with healthy participants, with mean fold-changes ranging from 3.7 to 4.2. It is demonstrated that the EV SERS signatures can accurately separate melanoma patients and healthy individuals, with an area under the curve of 0.95. Thus, with further development, this ultra-sensitive detection platform, combined with the panel of melanoma-associated biomarkers, has the ability to differentiate early stage melanoma patients from healthy participants.

Acquired naevi can have unique dermoscopic patterns that correspond to distinct microanatomical growth patterns. Previous studies on acquired naevi stratified according to dermoscopic pattern focused on the frequency of somatic BRAF mutations, whereas NRAS mutations remained to be elucidated. To investigate the BRAF and NRAS mutation prevalence and activation of the mitogen-activated protein kinase (MAPK) pathway in distinct dermoscopic subtypes of acquired naevi. Common mutations present in BRAF and NRAS were assessed in 40 globular, reticular and peripheral rim of globules (PG) subtypes of acquired naevi from 27 participants (19 male, 8 female; mean age 46·7 years) selected from 1261 eligible volunteers. Mutations were determined using the highly sensitive and quantitative QX200 droplet digital™ polymerase chain reaction (ddPCR) system. The BRAF V600E (c.1799T>A or c.1799_1800delTGinsA) and BRAF V600K mutations were detected in 85% (n = 34/40) of naevi. All BRAF wild-type naevi (15%; n = 6/40) harboured an NRAS codon 12/13 or 61 mutation. BRAF mutations were present in 92% (n = 12/13) of globular and 100% (n = 12/12) of PG naevi, whereas reticular naevi were 67% (n = 10/15) BRAF- and 33% (n = 5/15) NRAS-mutant (P = 0·037). We discovered that 100% of the assessed acquired naevi had either a BRAF or NRAS mutation. Using sensitive techniques capable of single-cell mutation detection, it is likely that all acquired naevi will be mutated for BRAF or NRAS. Because both of these mutations are prevalent in distinct dermoscopic naevus subsets, our study supports the role of the MAPK pathway in the development of benign melanocytic proliferations, indicating that additional genomic events besides somatic mutations in BRAF or NRAS are required for melanoma development.

Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.

Melanoma is the most dangerous form of skin cancer: around 15% of cases worldwide are fatal, but with early detection, survival rates are better than 95%. Having many large moles or having red hair increases a person's risk of developing melanoma; having certain versions of the MC1R gene, called R alleles, the main cause of red hair, also increases melanoma risk. This study, from Australia, aimed to find out if having both those risk factors increased melanoma risk more than the two risk levels simply added together. 611 people with melanoma and 656 who had never had melanoma donated a DNA sample, and researchers recorded the number of large moles, their skin and eye colour, and hair colour at age 21. The authors found that people with red hair and 20+ large moles were 10 times more likely to have melanoma than people with 0-4 moles and dark brown hair. Looking specifically at the types of the MC1R gene that people carried, people with 20+ moles and MC1R R/R genotype were 25 times more likely to have melanoma than people with 0-4 moles and MC1R wildtype/wildtype (consensus) genotype. While the R/R genotype, with or without the red hair phenotype, and a high mole count are both strong melanoma risk factors on their own, when combined they result in an extremely high-risk profile for melanoma. The absolute lifetime risk to age 75 of getting melanoma in Australia is 23.3% for men and 19.3% for women who have 20+ moles and MC1R R/R genotype, compared to just 0.8% for men and 0.7% for women with 0-4 moles and MC1R wildtype/wildtype genotype. This is higher than the risk threshold for screening in other cancers, so the authors suggest that people with many moles and red hair should be regularly checked for melanoma by a doctor, and that people with many moles but non-red hair should also be checked to determine if they have an MC1R R/R genotype.

A high total body naevus count is the highest risk factor for melanoma; the phenotype of red hair colour, freckling and pale skin that burns easily, produced by MC1R R alleles, also predisposes to melanoma.To determine whether the known melanoma risk factors of high naevus count and red hair or MC1R R alleles act synergistically to increase melanoma risk.The Brisbane Naevus Morphology Study involved 1267 participants from volunteers presenting at a melanoma unit, dermatology outpatient clinic, private dermatology clinics, the Brisbane Longitudinal Twin Study and the QSkin Study. We examined pigmentation characteristics, total body naevus ≥ 5 mm count, and MC1R, ASIP and CDKN2A genotype in participants with and without a personal history of melanoma, living in Queensland, Australia, which is an area of high ultraviolet radiation.Cases were older than controls (median 57 vs. 33 years). Compared with individuals with dark brown hair and zero to four naevi, individuals with red hair and ≥ 20 naevi had a melanoma odds ratio of 10·0 (95% confidence interval 4·2-24·3). Individuals with MC1R R/R genotype and ≥ 20 naevi (≥ 5 mm diameter) had a melanoma odds ratio of 25·1 (95% confidence interval 8·4-82·7) compared with wild-type (WT)/WT individuals with zero to four naevi. The highest risk group is Australian men with the MC1R R/R genotype and ≥ 20 moles, who have an absolute risk of melanoma to age 75 years of 23·3%, compared with 0·8% for men with the WT/WT genotype and zero to four naevi.Patients who live in areas of high ultraviolet radiation, and have many large naevi and the red hair colour phenotype, particularly those with the MC1R R/R genotype, have a high risk of melanoma above the threshold recommended for screening in other cancers. Therefore, they should undergo intensive physician-led surveillance. What's already known about this topic? A high number of acquired melanocytic naevi, the red hair phenotype and MC1R R alleles all independently increase melanoma risk. Women with atypical naevi have an increasing melanoma risk gradient from darker hair to lighter hair. Women with many naevi have an increasing melanoma risk gradient from those with no elements of the red hair phenotype, to those with freckles but not red hair, to those with red hair. What does this study add? In Queensland, Australia, people with ≥ 20 naevi (≥ 5 mm diameter) and MC1R R/R genotype have a 25-fold increased melanoma risk, relative to people with zero to four naevi and the MC1R WT/WT genotype. In Queensland, individuals with ≥ 20 naevi and the MC1R R/R genotype have an absolute melanoma risk to age 75 years of 23·3% for men and 19·3% for women. This effect is independent of CDKN2A genotype. Further research is required to determine the effect of areas of lower ultraviolet radiation, as this study took place in the Queensland, Australia, which is an area of high ultraviolet radiation. MC1R R/r genotype is associated with increased total body naevus count but this is not the case for R/R. What is the translational message? Patients with many large naevi and the red hair colour phenotype, particularly those with an MC1R R/R genotype, have an unusually high risk of melanoma. In a high ultraviolet environment, this risk exceeds the threshold recommended for screening in other cancers, and such individuals should undergo intensive, regular, physician-led surveillance. Patients with many large naevi but with non-red colour hair may benefit further from clinical MC1R genotyping.

Amelanotic/hypomelanotic melanoma is a clinicopathologic subtype with absent or minimal melanin. This study assessed previously reported coding variants in albinism genes (TYR, OCA2, TYRP1, SLC45A2, SLC24A5, LRMDA) and common intronic, regulatory variants of OCA2 in individuals with amelanotic/hypomelanotic melanoma, pigmented melanoma cases and controls. Exome sequencing was available for 28 individuals with amelanotic/hypomelanotic melanoma and 303 individuals with pigmented melanoma, which were compared to whole exome data from 1144 Australian controls. Microarray genotyping was available for a further 17 amelanotic/hypomelanotic melanoma, 86 pigmented melanoma, 147 melanoma cases (pigmentation unknown) and 652 unaffected controls. Rare deleterious variants in TYR/OCA1 were more common in amelanotic/hypomelanotic melanoma cases than pigmented melanoma cases (set mixed model association tests P = 0.0088). The OCA2 hypomorphic allele p.V443I was more common in melanoma cases (1.8%) than controls (1.0%, X2 P = 0.02), and more so in amelanotic/hypomelanotic melanoma (4.4%, X2 P = 0.007). No amelanotic/hypomelanotic melanoma cases carried an eye and skin darkening haplotype of OCA2 (including rs7174027), present in 7.1% of pigmented melanoma cases (P = 0.0005) and 9.4% controls. Variants in TYR and OCA2 may play a role in amelanotic/hypomelanotic melanoma susceptibility. We suggest that somatic loss of function at these loci could contribute to the loss of tumor pigmentation, consistent with this we found a higher rate of somatic mutation in TYR/OCA2 in amelanotic/hypomelanotic melanoma vs pigmented melanoma samples (28.6% vs 3.0%; P = 0.021) from The Cancer Genome Atlas Skin Cutaneous Melanoma collection.

We recently performed whole exome sequencing of 30 melanocytic nevi which surprisingly revealed numerous regions of copy number loss. It was found that common reticular nevi (mostly with a junctional component) have a high incidence of copy number aberrations (CNA). Importantly, concurrent with the loss of tumor suppressor genes (e.g. CDKN2A, TP53, and NF1) was the loss of potent oncogenes (e.g. NRAS, MITF, and MDM2). We postulated that the balanced nature of these CNAs confers protection from transformation, thus keeping the lesion in the benign state. Along with copy-number loss of these well-known genes, further interrogation of these data revealed a loss on chr8q24.3 which was frequently observed in 7/30 (23%) nevi. In the smallest region of overlap, the block of proliferation 1 (BOP1) gene was identified as a candidate for functional validation. BOP1 is part of the trimeric “PeBoW” complex, essential for ribosome biogenesis and cell cycle progression. In melanoma cell lines (MM96L, HT144, and MM253), we used siRNA mediated gene targeting to achieve differing BOP1 expression levels (high, low, absent), which resulted in altered cell states. For example, high BOP1 expression caused an increase in proliferation and colony formation, whereas low BOP1 expression conferred reduced proliferation, colony formation, and a senescent like phenotype. Interestingly, low BOP1 expression also resulted in increased migration and invasive capacity. BOP1 immunofluorescence assessment of benign nevi revealed limited expression whereas in early invasive melanomas, BOP1 expression was clearly evident in proliferative melanoma nests. These data support the role of BOP1 playing a role in melanoma progression by contributing to phenotype switching. Citation Format: Mitchell S. Stark, Sabrina Hammerlindl, Lisa Tom, Kasturee Jagirdar, Jean-Marie Tan, Helmut Schaider, Richard A. Sturm, Blake O'Brien, Michael Walsh, Angus Collins, H.Peter Soyer. BOP1 expression contributes to the proliferative/invasive phenotype in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5861.

The melanoma transformation rate of an individual nevus is very low despite the detection of oncogenic BRAF or NRAS mutations in 100% of nevi. Acquired melanocytic nevi do, however, mimic melanoma, and approximately 30% of all melanomas arise within pre-existing nevi. Using whole-exome sequencing of 30 matched nevi, adjacent normal skin, and saliva we sought to identify the underlying genetic mechanisms for nevus development. All nevi were clinically, dermoscopically, and histopathologically documented. In addition to identifying somatic mutations, we found mutational signatures relating to UVR mirroring those found in cutaneous melanoma. In nevi we frequently observed the presence of the UVR mutation signature compared with adjacent normal skin (97% vs. 10%, respectively). Copy number aberration analysis showed that for nevi with copy number loss of tumor suppressor genes, this loss was balanced by loss of potent oncogenes. Moreover, reticular and nonspecific patterned nevi showed an increased (P0.0001) number of copy number aberrations compared with globular nevi. The mutation signature data generated in this study confirms that UVR strongly contributes to nevogenesis. Copy number changes reflect at a genomic level the dermoscopic differences of acquired melanocytic nevi. Finally, we propose that the balanced loss of tumor suppressor genes and oncogenes is a protective mechanism of acquired melanocytic nevi.

The overall 5-year survival for melanoma is 91%. However, if distant metastasis occurs (stage IV), cure rates are, Highlights • A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%). • In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases. • The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.

// Mitchell S. Stark 1,2 , Vanessa F. Bonazzi 3 , Glen M. Boyle 1 , Jane M. Palmer 1 , Judith Symmons 1 , Catherine M. Lanagan 1 , Christopher W. Schmidt 1 , Adrian C. Herington 2 , Robert Ballotti 3 , Pamela M. Pollock 2 and Nicholas K. Hayward 1 1 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 2 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia 3 Inserm U1065, Centre Mediterraneen de Medecine Moleculaire, Equipe 1, Biologie et pathologies des melanocytes, Nice, France Correspondence to: Mitchell S. Stark, email: // Keywords : miRNA, microRNA, miR-514a, BRAFi, NF1 Received : March 16, 2015 Accepted : April 07, 2015 Published : April 23, 2015 Abstract To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1 , CDK2 , MC1R , and NF1 , previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E -targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies. Mitchell S. Stark

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh-frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR-150-5p, miR-142-3p and miR-142-5p. Co-analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR-150-5p and the miR-142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor genePTPRJin 19% of cases. NoBRAFmutations were detected, but activatingRASmutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.MDM2amplifications (24%) andTP53mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen inBRAFwild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.AUTHOR SUMMARYMelanoma, an aggressive cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, which most often occurs in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Clinical features of canine melanoma resemble those of human melanoma, particularly the less common sun-shielded human subtypes. However, whereas the genomic basis of diverse human melanoma subtypes is well understood, canine melanoma genomics remain poorly defined. Similarly, although diverse new treatments for human melanoma based on a biologic disease understanding have recently shown dramatic improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain universally poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving the lives of pet dogs while also helping to establish canine melanoma as a comparative model system for informing human melanoma biology and treatment. In order to better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such asRAS,MDM2, andTP53as well mutational patterns that share important similarities with human melanoma subtypes. We have also found a new putative cancer gene,PTPRJ, frequently mutated in canine melanoma. These data will guide additional biologic and therapeutic studies in canine melanoma while framing the utility of comparative studies of canine and human cancers more broadly.

Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma.

Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases1, while rare variants in CDK4, BRCA2, BAP1, and the promoter of TERT, have also been linked to the disease2-5. Here we set out to identify novel high-penetrance susceptibility genes in unexplained cases by sequencing 184 melanoma patients from 105 pedigrees recruited in the United Kingdom, the Netherlands, and Australia that were negative for variants in known predisposition genes. We identify families where melanoma co-segregates with loss-of-function variants in the protection of telomeres 1 (POT1) gene, a proportion of members presenting with an early age of onset and multiple primaries. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding, leading to increased telomere length. Thus, POT1 variants predispose to melanoma formation via a direct effect on telomeres.

A hallmark of cancer is genomic instability that is considered to provide the adaptive capacity of cancers to thrive under conditions in which the normal precursors would not survive. Recent genomic analysis has revealed a very high degree of genomic instability in melanomas, although the mechanism by which this instability arises is not known. Here we report that a high proportion (68%) of melanoma cell lines are either partially (40%) or severely (28%) compromised for the G2 phase decatenation checkpoint that normally functions to ensure that the sister chromatids are able to separate correctly during mitosis. The consequence of this loss of checkpoint function is a severely reduced ability to partition the replicated genome in mitosis and thereby increase genomic instability. We also demonstrate that decatenation is dependent on both TopoIIα and β isoforms. The high incidence of decatenation checkpoint defect is likely to be a major contributor to the high level of genomic instability found in melanomas.

Purpose To determine if a circulating microRNA (miRNA) panel could be used to distinguish between uveal melanoma and uveal nevi. Methods We report on a multicenter, cross-sectional study conducted between June 2012 and September 2015. The follow-up time was approximately 3 to 5 years. Blood was drawn from participants presenting with a uveal nevus (n = 10), localized uveal melanoma (n = 50), or metastatic uveal melanoma (n = 5). Levels of 17 miRNAs were measured in blood samples of study participants using a sensitive real-time PCR system. Results A panel of six miRNAs (miR-16, miR-145, miR-146a, miR-204, miR-211, and miR-363-3p) showed significant differences between participants with uveal nevi compared with patients with localized and metastatic uveal melanoma. Importantly, miR-211 was able to accurately distinguish metastatic disease from localized uveal melanoma (P < 0.0001; area under the curve = 0.96). When the six-miRNA panel was evaluated as a group it had the ability to identify uveal melanoma when four or more miRNAs (93% sensitivity and 100% specificity) reached or exceeded their cut-point. Conclusions This miRNA panel, in tandem with clinical findings, may be suited to confirm benign lesions. In addition, due to the panel's high precision in identifying malignancy, it has the potential to augment melanoma detection in subsequent clinical follow-up of lesions with atypical clinical features. Translational Relevance Uveal nevi mimic the appearance of uveal melanoma and their transformation potential cannot be definitively determined without a biopsy. This panel is most relevant at the nevus stage and in lesions with uncertain malignant potential as a companion diagnostic tool to assist in clinical decision-making.

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation "hotspot" at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.