Richard A. Sturm

We evaluated an inexpensive portable hand-held spectrophotometer for skin colour measurement and classification. Under standardised conditions, skin reflectance and colorimetric data were collected from 40 participants of diverse ancestral backgrounds at three anatomical sites: forehead (FH), right posterior forearm (FA), and right upper inner arm (RUA). Demographic and ancestral data, Fitzpatrick Skin Phototype classification, standardised iPhone 13 images, and visual and device-based skin colour matches were also obtained. Participants spanned the five-point EHSCS scale; visually matched Pantone SkinTone colours numbered 25 for FH, 33 for FA, and 28 for RUA. ITA values derived from colorimetric data were used to generate site-specific classifications using the five-point EHSCS, seven-point ITA scale incorporating Del Bino categories, and ten-point MST categories defined by Ulrich or Lipnick. This versatile spectrophotometer-based approach supports affordable, reproducible, race-, ethnicity-, and ancestry-independent skin colour measurement and classification across multiple scales for dermatologists and other users.

Background: Naevi are considered benign analogues for melanoma; indeed, they share many clinical and molecular features, including oncogenic activation, and the transient adoption of senescence-like phenotypes. We previously determined dynamic mechanisms for maintenance of benign naevi (showing conventional features under histopathology) via DNA methylation, which was not shared by dysplastic naevi. Objectives: To determine gene sets enriched in actively growing naevi by evaluating 32 differently patterned naevi using whole-transcriptome sequencing. Methods: Matched specimens (naevi and adjacent perilesional skin) characterized by histopathology and dermoscopy underwent differential expression analysis to identify novel genes related to naevus growth. Cell culture-based phenotypic assays and immunohistochemistry were performed to determine the function and cell-specific localization in the context of melanocytic lesions. Results: We focused on an apoptosis-related gene, HRK, a Bcl2 family member, which has been found to be downregulated in therapy-induced senescent melanoma cells. Whole-transcriptome profiling confirmed that increased HRK expression was significantly correlated with dysplastic naevi (R2 = 0.86; P = 1.55 × 10–17) and in naevi featuring a peripheral rim of globules (R2 = 0.67; P = 1.54 × 10–8). Immunohistochemistry confirmed that Harakiri (HRK; also known as DP5) was lowly expressed in benign (conventional) naevi compared with dysplastic naevi, with a marked increase in thin invasive T1a melanoma tissues. Next, small interfering RNA knockdown of HRK in melanoma cell lines resulted in up to twofold BAX (proapoptotic) expression and significant increases in sub G0/G1, indicative of apoptotic cells. Decreased HRK expression led to significantly reduced proliferative capacity, wound healing, colony formation and an increase in senescent cells, indicating that HRK activity promotes escape from senescence. Conclusions: We show that HRK expression is distinctly regulated between different subtypes of naevi and melanoma, with supporting evidence that this differential expression contributes to the regulation of melanocytic proliferation.

Background: The origins of cutaneous melanoma are often traced to visible precursor lesions (e.g. melanocytic naevi), but approximately two-thirds of melanomas arise from clinically healthy skin with no detectable precursor lesion. The BRAF V600E mutation drives melanoma and naevus formation, but eruptive naevi syndromes and the disproportionate role early-life ultraviolet exposure plays in melanoma risk suggest the presence of a hidden field of oncogene-mutant melanocytes in clinically healthy skin, which may represent an earlier step in tumorigenesis. Objectives: To determine whether BRAF V600E-mutant melanocytes were commonly present in nonlesional skin from adults at high risk of melanoma. Methods: We examined BRAF V600E mutations in 97 histologically and clinically healthy, nonlesional skin samples from an Australian cohort of individuals at high risk of melanoma. The skin selected was adjacent to a naevus or a prior melanoma site; photodamaged skin proximal (approximately 5 cm) from melanoma excision; photoprotected skin distant from lesions; and low-risk neonatal foreskin-derived melanoblasts. We used immunohistochemistry to locate BRAF V600E-mutant cells in histopathology sections; droplet digital polymerase chain reaction to determine the fractional abundance of BRAF V600E in whole skin; and single-cell RNA sequencing to confirm cells as melanocytes and detect their transcriptional programmes. Results: We identified BRAF V600E-mutant melanocytes in skin surrounding naevi and primary melanomas, even years after tumour excision. Fields of BRAF V600E-mutant melanocytes were commonly found in the skin of patients at high risk for melanoma and were up to 20-fold denser and 50% more frequent in tumour-adjacent skin. Field cells exhibited a gene expression profile characteristic of BRAF V600E-induced growth arrest, consistent with dormant yet genetically primed cells. Conclusions: There is a reservoir of oncogene-harbouring melanocytes in normal skin. Our findings challenge the prevailing notion that melanocyte expression of BRAF V600E is inherently tumorigenic. The presence of such fields suggests that the scope of melanoma detection and prevention efforts might be extended beyond visible lesions, to encompass potentially precancerous fields of driver-mutant cells. An author video to accompany this article is available online.

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.

An apical component of the cell cycle checkpoint and DNA damage repair response is the ataxia-telangiectasia mutated (ATM) Ser/Thr protein kinase. A variant of ATM, Ser49Cys (rs1800054; minor allele frequency = 0.011), has been associated with an elevated risk of melanoma development; however, the functional consequence of this variant is not defined. ATM-dependent signalling in response to DNA damage has been assessed in a panel of patient-derived lymphoblastoid lines and primary human melanocytic cell strains heterozygous for the ATM Ser49Cys variant allele. The ATM Ser49Cys allele appears functional for acute p53-dependent signalling in response to DNA damage. Expression of the variant allele did reduce the efficacy of oncogene expression in inducing senescence. These findings demonstrate that the ATM 146C>G Ser49Cys allele has little discernible effect on the acute response to DNA damage but has reduced function observed in the chronic response to oncogene over-expression. Analysis of melanoma, naevus and skin colour genomics and GWAS analyses have demonstrated no association of this variant with any of these outcomes. The modest loss of function detected suggest that the variant may act as a modifier of other variants of ATM/p53-dependent signalling.

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.