David Lorenzo Duffy

Germline genetic variation can influence the number of nevi on the skin. However, nevi can be classified in many ways (e.g., by shape, size or evolution); little is known of how genetics influences the density of different nevus morphologies on the body. Here, we explore how germline genetics influences the density of flat, raised and atypical nevi, and identify which classification correlates most with genetic risk of melanoma. We estimate heritability and perform genome‐wide association analysis of flat, raised, and atypical nevus count within the Brisbane Twin Nevus Study ( N = 3862). We compare the effect estimates of genetic loci associated with each morphology. We also assess how well each morphology correlates with the genetic risk of melanoma using a polygenic risk score of melanoma. Heritability estimates revealed both unique and shared genetic effects between morphologies. We identified two loci near IRF4 and MC1R showing opposing effects on flat and raised nevus counts. Variation in raised nevus count showed the strongest correlation with melanoma risk using polygenic risk scores. Although some genetic effects are shared between nevus morphologies, they appear to be genetically distinct phenotypes. Given the opposing effects of IRF4 and MC1R on flat and raised nevus counts, future studies of nevus count should consider, where possible, analysing each nevus morphology separately as well as combined.

Background: Approximately 2–20% of cutaneous melanomas (CMs) are diagnosed as amelanotic/hypopigmented melanoma (AHM) and represent a challenge for early diagnosis. Objectives: To investigate loss-of-function mutations in key pigmentation genes in matched germline and AHM, as well as pigmented melanoma (PM), tumour DNA samples. Methods: Analysis of clinical and histopathological characteristics – together with whole-exome sequencing data of 34 fresh frozen primary CMs, graded according to the amount of pigmentation present – was performed. Together with germline and somatic variant analysis, 30 samples had previously been analysed for copy number aberration (CNA) changes. This study focused on germline and somatic variants in the coding region of 16 genes known to be associated with albinism/hypopigmentation or variation in human pigmentation in all samples. Chromosomal regions encompassing these 16 genes were examined for DNA copy loss or gain. Results: The finding that red hair-related MC1R and TYR R402Q loss-of-activity gene variant alleles and genotypes are associated with AHM was confirmed. Germline AHM-related gene variants were enriched in 70% (n = 7/10) of patients with AHM vs. 8% (n = 2/24) of those with PM. This surprisingly high frequency of rare germline variants in people with AHM constitutes the ‘first hit’ and confirms that those with AHM are more likely to be albinism allele carriers than individuals with PM. Next, in CNA analysis of each tumour sample, 50% (n = 4/8) of AHM samples with a pigmentation gene variant had loss of heterozygosity (LOH) in the region containing the corresponding gene and 25% (n = 2/8) had LOH in chromosomal regions of two AHM-related genes. Conclusions: This study proposes that the likely molecular mechanism for the development of amelanogenesis in AHM is carriage of an albinism/hypopigmentation allele followed by LOH of the corresponding gene in the tumour.

Nodular melanoma (NM) is a challenge to diagnose early due to its rapid growth and more atypical clinical presentation, making it the largest contributor to melanoma mortality. Our study aimed to perform a rare variant allele analysis of whole exome sequenced NM and non-NM patients (minor allele frequency ≤1% Non- Finnish European) for a set of 500 candidate genes potentially implicated in melanoma. This study recruited 131 NM and 194 non-NM participants from South-east Queensland and NM patients from Victoria to perform a comparative analysis of possible genetic differences or similarities between the two melanoma cohorts. Phenotypic analysis revealed that a majority of patients diagnosed with NM were older males with a higher frequency of fair skin and red hair than is seen in the general population. The distribution of common melanoma polygenic risk scores (PRS) was similar in NM and non-NM patients, with over 28% in the highest quantile of scores. There was also a similar frequency of carriage of familial/high penetrant melanoma gene and loss of function (LOF) variants. We identified 39 genes by filtering 500 candidate genes based on the greatest frequency in NM compared to non-NM cases. The genes with rare variant alleles of greatest frequency in NM included PTCH1, ARID2 and GHR. Rare variants in the SMO gene that interacts with PTCH1 as ligand and receptor were also identified, providing evidence that the Hedgehog pathway may contribute to NM risk. There was a cumulative effect in carrying multiple rare variants in the NM associated genes. A 14.8 fold increased ratio for NM compared to non-NM was seen when 2 rare variants alleles of the 39 genes were carried by a patient. This study highlights the importance of considering frequency of rare variant alleles to identify those at risk of NM in addition to known high penetrance genes.

Population-wide screening for melanoma is not cost-effective, but genetic characterization could facilitate risk stratification and targeted screening. Common Melanocortin-1 receptor (MC1R) red hair colour (RHC) variants and Microphthalmia-associated transcription factor (MITF) E318K separately confer moderate melanoma susceptibility, but their interactive effects are relatively unexplored. To evaluate whether MC1R genotypes differentially affect melanoma risk in MITF E318K⁺ vs. E318K^– individuals. Melanoma status (affected or unaffected) and genotype data (MC1R and MITF E318K) were collated from research cohorts (five Australian and two European). In addition, RHC genotypes from E318K⁺ individuals with and without melanoma were extracted from databases (The Cancer Genome Atlas and Medical Genome Research Bank, respectively). χ² and logistic regression were used to evaluate RHC allele and genotype frequencies within E318K^+/– cohorts depending on melanoma status. Replication analysis was conducted on 200 000 general-population exomes (UK Biobank). The cohort comprised 1165 MITF E318K^– and 322 E318K⁺ individuals. In E318K^– cases MC1R R and r alleles increased melanoma risk relative to wild type (wt), P < 0.001 for both. Similarly, each MC1R RHC genotype (R/R, R/r, R/wt, r/r and r/wt) increased melanoma risk relative to wt/wt (P < 0.001 for all). In E318K⁺ cases, R alleles increased melanoma risk relative to the wt allele [odds ratio (OR) 2.04 (95% confidence interval 1.67–2.49); P = 0.01], while the r allele risk was comparable with the wt allele [OR 0.78 (0.54–1.14) vs. 1.00, respectively]. E318K⁺ cases with the r/r genotype had a lower but not significant melanoma risk relative to wt/wt [OR 0.52 (0.20–1.38)]. Within the E318K⁺ cohort, R genotypes (R/R, R/r and R/wt) conferred a significantly higher risk compared with non-R genotypes (r/r, r/wt and wt/wt) (P < 0.001). UK Biobank data supported our findings that r did not increase melanoma risk in E318K⁺ individuals. RHC alleles/genotypes modify melanoma risk differently in MITF E318K^– and E318K⁺ individuals. Specifically, although all RHC alleles increase risk relative to wt in E318K^– individuals, only MC1R R increases melanoma risk in E318K⁺ individuals. Importantly, in the E318K⁺ cohort the MC1R r allele risk is comparable with wt. These findings could inform counselling and management for MITF E318K⁺ individuals.

Melanoma incidence rates are high among individuals with fair skin and multiple naevi. Established prognostic factors are tumour specific, and less is known about prognostic host factors. A total of 556 stage I to stage IV melanoma patients from Germany with phenotypic and disease-specific data were analysed; 64 of these patients died of melanoma after a median follow-up time of 8 years. Germline DNA was assessed by the HumanCoreExome BeadChip and data of 356,384 common polymorphisms distributed over all 23 chromosomes were used for a genome-wide analysis. A suggestive genome-wide significant association of the intronic allele rs7551288*A with diminished melanoma-specific survival was detected (p = 2 × 10⁻⁶). The frequency of rs7551288*A was 0.43 and was not associated with melanoma risk, hair and eye colour, tanning and total naevus count. Cox regression multivariate analyses revealed a 5.31-fold increased risk of melanoma-specific death for patients with the rs7551288 A/A genotype, independent of tumour thickness, ulceration and stage of disease at diagnoses. The variant rs7551288 belongs to the DHCR24 gene, which encodes Seladin-1, an enzyme involved in the biosynthesis of cholesterol. Further investigations are needed to confirm this genetic variant as a novel prognostic biomarker and to explore whether specific treatment strategies for melanoma patients might be derived from it.

A GWAS including 192,986 European and 1636 Asian participants identifies 50 novel discrete associations with eye color.

Visceral hemangiosarcoma (HSA) is one of the more frequent cancers in dogs and has a high metastatic rate and poor prognosis, as clinical signs only become apparent in advanced stages of tumor development. In order to improve early and differential diagnostic capabilities and hence, prognosis for dogs with HSA, two types of biomarker are needed: a point-of-care diagnostic biomarker and a prognostic biomarker—preferentially based on samples obtained with minimally invasive methods. In this study, we applied a lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS/MS) workflow through discovery and validation phases to discover serum glycoprotein biomarker candidates for canine HSA. By this approach, we found that Datura stramonium (DSA), wheat germ agglutinin (WGA), Sambucus nigra (SNA), and Pisum sativum (PSA) lectins captured the highest number of validated candidate glycoproteins. Secondly, we independently validated serum LeMBA-MS/MS results by demonstrating the in situ relationship of lectin-binding with tumor cells. Using lectin-histochemistry and immunohistochemistry (IHC) for key proteins on tissues with HSA and semi-quantitation of the signals, we demonstrate that a combination of DSA histochemistry and IHC for complement C7 greatly increases the prospect of a more specific diagnosis of canine HSA.

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.