BACKGROUND:Recent genome-wide association studies (GWAS) have identified new susceptibility loci for melanoma, but their associations with multiple primary melanoma (MPM) are unclear. METHODS:We investigated the associations of 69 SNPs in 39 GWAS-identified loci with odds of MPM relative to single primary melanoma in the international, population-based Genes, Environment, and Melanoma study. Per-minor-allele ORs and 95% confidence intervals (CI) for individuals with MPM "cases" (n = 1,205) relative to single primary melanoma "controls" (n = 2,458) were estimated using multivariable logistic regression, and polygenic risk scores (PRS) were calculated and weighted based on a 2020 GWAS meta-analysis (57 of the 68 independent GWAS SNPs available). RESULTS:Thirteen SNPs in 11 gene regions (PARP1, CYP1B1/RMDN3, TERT, RAPGEF5, TYRP1, MTAP, CDKN2A/CDKN2B, KLF4, TYR, SOX6, and ASIP) were statistically significantly associated (P < 0.05) with MPM adjusting for age, sex, age-by-sex interaction, and study center. The highest versus lowest PRS quintile was associated with a 2.81-fold higher odds of MPM (95% CI, 2.10-3.78; P = 7.5 × 10-13); this association was attenuated but remained statistically significant after excluding SNPs individually associated with MPM (OR = 1.75, 95% CI, 1.32-2.31). CONCLUSIONS:Inherited genetic variants spanning 11 gene regions were independently associated with MPM. Nonsignificant SNPs were associated with MPM when aggregated into a PRS, indicating that their cumulative effect may influence MPM risk despite lacking individual statistical significance in our study population. IMPACT:Our findings provide additional evidence that these loci are associated with melanoma risk and estimate the magnitude of their genetic effect on subsequent (multiple) primary melanoma risk.

To investigate the landscape of the activated kinome in MM, we applied a proteomic approach that interrogates the activation status of more than half of the entire kinome (∼300), termed multiplexed kinase inhibitor bead affinity chromatography coupled with mass spectrometry (MIB/MS) on 45 MM patient (pt) tumors (BRAFV600, n=25; NRASQ61, n=5, NF1 loss-of-function, n=4; Triple wild-type n=11) in conjunction with whole exome sequencing and RNA sequencing (RNAseq). We performed exploratory analyses (Wilcoxon rank sum test for each kinase) on the untreated tumors to identify differentially activated kinases in BRAFV600-mutant MM vs. all others. While no activated kinases were differentially expressed in BRAFV600-mutant MM after adjustment for multiple testing, AKT1, PDGFRA, and MAP3K1 kinases exhibited higher MIB binding (i.e., more activated) in BRAFV600E-mutant pts (unadjusted p-value < 0.05). Spearman’s rank correlation tests between the activated kinase and its gene expression by RNAseq and adjusted for multiple testing did not identify a significant correlation for any kinase, suggesting a complex mechanism of kinase activation other than sole gene expression. K-means clustering analysis to classify melanomas into 4 subtypes using a 6-gene discriminant expression signature showed that BRAFV600-mutant tumors were more frequently classified as melanocytic (9/24, MLANA/MITF/SOX10high, AXLlow). In contrast, triple wild-type tumors were more frequently classified as transitory (5/10, MITF/SOX10/ETV4high, AXLlow). We then performed similar exploratory analyses on the baseline (untreated) tumors from the subset of the 20 BRAFV600E-mutant pts who received D+T as part of a prospective clinical trial (NCT01726738; median follow-up 30.5 months, range 2.0-116.1 months) to identify differentially activated kinases that would predict shorter (<12 months) vs. longer progression-free survival. Again, while no kinases survived multiple comparison testing, the bromodomain protein TRIM28, known to regulate melanoma plasticity, exhibited a more consistent loss of MIB binding in BRAFV600E-mutant pts who progressed shorter than in pts who progressed longer than 12 months on D+T (unadjusted p-value < 0.05). 7 baseline-progression tumor pairs were evaluated for MIB/MS, and 6 were for RNAseq. Although diverse resistance mechanisms were identified, as previously described, we also identified melanoma subtype switch in 4/6 pt tumors and a consistent loss of MIB binding of the non-receptor tyrosine kinase PTK6 in response to D+T, an off-target of D and vemurafenib but not encorafenib. Our in vitro findings revealed that off-target PTK6 inhibition by D could activate beta-catenin signaling and contribute to the development of resistance. Steve P. Angus, Naim U. Rashid, Andrew J. Walther, David L. Corcoran, Steven D. Rhodes, Frances A. Collichio, C. Paige Jones, Mikaela J. Bauer, Joel S. Parker, Noah Sciaky, 1Alisha R. Coffey, Samantha M. Bevill, Brian T. Golitz, v Timothy J. Stulhmiller, Nancy E. Thomas, David W. Ollila, Norman E. Sharpless, Carrie B. Lee, Gary L. Johnson, Stergios J. Moschos. Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in BRAFV600-mutant metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2051.

PURPOSE:Patients with stage II and resected stage III melanomas have variable clinical outcomes, providing evidence of underlying biological differences in tumors and/or the patients themselves, beyond stage. The approval of adjuvant immunotherapy for stage IIB/C and resected stage III/IV disease (and adjuvant targeted therapy for resected stage III disease) has created a pressing need to develop biomarkers to accurately distinguish patients at low risk versus high risk for recurrence and death from melanoma. miRNAs are promising biomarkers because of their stability in tissues and fluids and their demonstrated functional and prognostic roles in melanoma. We hypothesized that miRNA expression could be integrated into prognostic models that would classify 5-year survival outcomes more accurately than clinical factors alone. EXPERIMENTAL DESIGN:Using a NanoString miRNA Expression Assay, we analyzed 715 primary melanomas from patients with stage II or stage III disease within the InterMEL consortium and examined associations between miRNA expression and melanoma-specific death. RESULTS:When integrated into clinical prognostic models for 5-year melanoma-specific survival, miRNA signatures improved the area under the receiver operating characteristic curve for patients in stage II from 0.71 for a "clinical factors-only" model to 0.81 for a "clinical plus miRNA" model in an independent test set, an improvement of 0.10 with a 95% confidence interval (0.03-0.19). The improvement was more modest for patients in stage III who were included in the analysis. CONCLUSIONS:Incorporating miRNA expression in primary melanomas may enhance the accuracy of clinical prognostic models and potentially aid in the selection of patients with melanoma for adjuvant treatment and clinical trials.

Sex differences in melanoma are prominent, with female having a significant survival advantage. However, it is unclear why we see this survival advantage. Here, we investigate the relationship between sex, clinicopathologic variables, and melanoma specific survival in 1753 single primary melanomas from patients in the GEM (Genes, Environment, and Melanoma) study. Using Cox proportional hazard models and formal mediation analysis, the effect of sex on survival is explained largely by differences in the clinicopathologic features of tumors at diagnosis. Specifically, we find evidence that 86.5% of the effect of sex on melanoma survival is mediated by differences in age at diagnosis, Breslow thickness, ulceration, mitoses, and site (hazard ratio [HR] = 1.85, P < .001). This analysis indicates that the female survival advantage in melanoma is not primarily due to a direct effect of sex (HR = 1.19, P = .42) but is largely a result of an indirect effect of sex mediated by clinicopathologic features.

BACKGROUND:Hidradenitis suppurativa (HS) is a common, chronic and debilitating inflammatory disease that most commonly affects intertriginous skin. Despite its high heritability, the genetic underpinnings of HS remain poorly understood. OBJECTIVES:To identify genetic signals associated with HS, determine genetic relationships with other diseases and investigate potential molecular genetic mechanisms. METHODS:We performed a genome-wide association meta-analysis of six studies, totalling 4540 patients with HS and > 1 million control participants, and identified genetic correlations with other common diseases. We integrated the HS data with expression quantitative trait loci from 10 trait-relevant tissues, epigenomic and transcriptomic data from human scalp, differential expression data from HS lesions vs. adjacent skin and mesenchymal Hi-C chromatin looping data. To identify functional noncoding variants, we performed transcriptional reporter assays for signals near KLF5 and SOX9. RESULTS:We identified 11 significant HS signals across 7 loci: 4 corresponded to previously reported associations, 4 represented novel signals within known loci and 3 were signals in newly implicated loci. We identified significant genetic correlations between HS and other inflammatory conditions, particularly inflammatory bowel disease, rheumatoid arthritis, type 2 diabetes mellitus and asthma. We prioritized candidate genes for the 11 signals. The risk allele at KLF5 exhibited 10-fold greater transcriptional activity than the nonrisk allele, while risk alleles at SOX9 showed significantly reduced transcriptional activity. CONCLUSIONS:Our results provide insights into potential genetic mechanisms underlying HS and suggest potential therapeutic targets for this challenging condition.

Linear IgA bullous dermatosis (LABD) is an acquired autoimmune subepidermal blistering skin disease characterized by circulating and tissue-bound IgA autoantibodies that recognize epitopes within the hemidesmosomal protein BP180, including its NC16A domain. Histologically, LABD has long been defined by neutrophil infiltration and dermal-epidermal separation. However, the pathogenic roles of anti-NC16A IgA and neutrophils in LABD, as well as their interactions, have not been thoroughly studied. We show that passive transfer of patient-derived anti-NC16A IgA induce clinical and histologic LABD pathology in humanized NC16A mice that are reconstituted locally or systemically with human neutrophils. The lesional skin of mice exhibits significantly elevated levels of the neutrophil chemoattractants CXCL-1 and CXCL-2. Furthermore, we show significantly increased levels of the neutrophil chemoattractant IL-8 in blister fluids of patients with LABD. This study provides direct evidence that anti-NC16A IgA in patients with LABD are pathogenic and interact with neutrophils to mediate tissue injury and subepidermal blister formation. This study further corroborates the importance of neutrophil-mediated tissue injury in LABD disease physiology and establishes a clinically relevant in vivo model system that can be used to systematically dissect the immunopathogenesis of LABD.

Ideally, detection of somatic mutations in a tumor is accomplished using a patient-matched sample of normal cells as the benchmark. In this way somatic mutations can be distinguished from rare germline mutations. In large retrospective studies, archival tissue collection can pose challenges in obtaining samples of normal DNA. In this article we propose a protocol that improves somatic mutation analysis in the absence of a matched normal sample. The method was motivated by the InterMEL study, a large-scale epidemiologic investigation involving multiomic, multi-institutional genomic profiling of 1000 primary melanoma samples. The key insight for accomplishing improved mutation calling is the fact that germline mutations should produce a variant allele frequency (VAF) of around 50%. While a similar VAF of 50% would also be expected for somatic mutations in pure tumor samples, typically the tumor purity is much less than 50%, resulting in a considerably lower VAF. Making use of a technique that can simultaneously estimate both tumor purity and VAF from tumor-only samples we have developed a method for better distinguishing somatic versus germline variants. Based on 137 melanomas from the InterMEL Study with matched normal tissue to provide a gold standard we show that the conventional pipeline using a panel of (unmatched) normal samples has a false positive rate of 15.6% and a false negative rate of 3.5%. Our new technique improves these error rates to 6.4% and 2.1%, respectively.

PURPOSE Patients with stage II and III cutaneous primary melanoma vary considerably in their risk of melanoma-related death. We explore the ability of methylation profiling to distinguish primary melanoma methylation classes and their associations with clinicopathologic characteristics and survival. MATERIALS AND METHODS InterMEL is a retrospective case-control study that assembled primary cutaneous melanomas from American Joint Committee on Cancer (AJCC) 8th edition stage II and III patients diagnosed between 1998 and 2015 in the United States and Australia. Cases are patients who died of melanoma within 5 years from original diagnosis. Controls survived longer than 5 years without evidence of melanoma recurrence or relapse. Methylation classes, distinguished by consensus clustering of 850K methylation data, were evaluated for their clinicopathologic characteristics, 5-year survival status, and differentially methylated gene sets. RESULTS Among 422 InterMEL melanomas, consensus clustering revealed three primary melanoma methylation classes (MethylClasses): a CpG island methylator phenotype (CIMP) class, an intermediate methylation (IM) class, and a low methylation (LM) class. CIMP and IM were associated with higher AJCC stage (both P = .002), Breslow thickness (CIMP P = .002; IM P = .006), and mitotic index (both P < .001) compared with LM, while IM had higher N stage than CIMP ( P = .01) and LM ( P = .007). CIMP and IM had a 2-fold higher likelihood of 5-year death from melanoma than LM (CIMP odds ratio [OR], 2.16 [95% CI, 1.18 to 3.96]; IM OR, 2.00 [95% CI, 1.12 to 3.58]) in a multivariable model adjusted for age, sex, log Breslow thickness, ulceration, mitotic index, and N stage. Despite more extensive CpG island hypermethylation in CIMP, CIMP and IM shared similar patterns of differential methylation and gene set enrichment compared with LM. CONCLUSION Melanoma MethylClasses may provide clinical value in predicting 5-year death from melanoma among patients with primary melanoma independent of other clinicopathologic factors.

Objective:To evaluate the relation between solar elastosis and tumor mutation burden (TMB) in a large clinically annotated cohort of stage II and III melanoma patients. Methods:Primary cutaneous melanomas from 469 AJCC (8th edition) stage II and III patients with clinical annotation including outcome at 5 years of diagnosis were histopathologically evaluated for solar elastosis. Next-generation sequencing assay MSK-IMPACT™ was employed to determine TMB. Analysis by Fisher's exact test, chi-square, and Kruskal-Wallis were performed, as well as uni- and multivariate logistic regression. Results:Tumors stratified by low and high TMB showed marked and statistically significant differences in presence and extent of associated solar elastosis. Lower risk patient stage (II versus III by AJCC 8th edition) as well as better 5-year melanomaspecific survival (as binary variable of controls-survivors versus cases-dead of disease at 5 years of diagnosis) were associated with severe solar elastosis. On univariate and multivariate logistic regression models, severe solar elastosis predicted significantly decreased odds of dying of melanoma within 5 years of diagnosis (OR 0.60, 95 % CI 0.39-0.89; and OR 0.42, 95 % CI 0.20-0.83, respectively; both p<0.05). Conclusion:The association of solar elastosis to TMB and 5-year melanoma specific survival points to its potential as a biomarker of clinical relevance that can be assessed by routine histopathology.

The multiplexed immunofluorescence (mIF) platform enables biomarker discovery through the simultaneous detection of multiple markers on a single tissue slide, offering detailed insights into intratumor heterogeneity and the tumor-immune microenvironment at spatially resolved single cell resolution. However, current mIF image analyses are labor-intensive, requiring specialized pathology expertise which limits their scalability and clinical application. To address this challenge, we developed CellGate, a deep-learning (DL) computational pipeline that provides streamlined, end-to-end whole-slide mIF image analysis including nuclei detection, cell segmentation, cell classification, and combined immuno-phenotyping across stacked images. The model was trained on over 750,000 single cell images from 34 melanomas in a retrospective cohort of patients using whole tissue sections stained for CD3, CD8, CD68, CK-SOX10, PD-1, PD-L1, and FOXP3 with manual gating and extensive pathology review. When tested on new whole mIF slides, the model demonstrated high precision-recall AUC. Further validation on whole-slide mIF images of 9 primary melanomas from an independent cohort confirmed that CellGate can reproduce expert pathology analysis with high accuracy. We show that spatial immuno-phenotyping results using CellGate provide deep insights into the immune cell topography and differences in T cell functional states and interactions with tumor cells in patients with distinct histopathology and clinical characteristics. This pipeline offers a fully automated and parallelizable computing process with substantially improved consistency for cell type classification across images, potentially enabling high throughput whole-slide mIF tissue image analysis for large-scale clinical and research applications.

Automated region of interest detection in histopathological image analysis is a challenging and important topic with tremendous potential impact on clinical practice. The deep learning methods used in computational pathology may help us to reduce costs and increase the speed and accuracy of cancer diagnosis. We started with the UNC Melanocytic Tumor Dataset cohort which contains 160 hematoxylin and eosin whole slide images of primary melanoma (86) and nevi (74). We randomly assigned 80% (134) as a training set and built an in-house deep learning method to allow for classification, at the slide level, of nevi and melanoma. The proposed method performed well on the other 20% (26) test dataset; the accuracy of the slide classification task was 92.3% and our model also performed well in terms of predicting the region of interest annotated by the pathologists, showing excellent performance of our model on melanocytic skin tumors. Even though we tested the experiments on a skin tumor dataset, our work could also be extended to other medical image detection problems to benefit the clinical evaluation and diagnosis of different tumors.

Table S3. Association of haplotypes with multiple primary melanoma in the GEM study

Table S1. Demographic and phenotypic characteristics among muliple and single primary melanoma patients in the GEM study

Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled alpha-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov, NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n = 68). Melanoma cell-specific TPH1 and LAT1 expressions were significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cutpoint survival analysis of pretreatment 'high' vs. 'low' C11-AMT SUVmax of the hottest tumor lesion per patient revealed that the 'low' C11-AMT SUVmax was associated with longer progression-free survival in our clinical trial (n = 26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

Supplementary Table 1. Clinical and histologic characteristics of cutaneous melanocytic nevi, primary melanomas, and melanoma metastases and their relationship to ITK protein levels

Table S5. Genotype and haplotype associations with multiple primary melanoma stratified by phenotypic index and back nevi in the GEM study

Linear IgA bullous dermatosis (LABD) is a skin autoimmune disease characterized by the linear deposition of IgA autoantibodies at the basement membrane zone (BMZ), subepidermal blisters, and neutrophil infiltration. These IgA autoantibodies specifically recognize the hemidesmosomal component BP180 (also called type XVII collagen) and its processed 120 kDa and 97 kDa extracellular regions. The main epitopes recognized by LABD IgA autoantibodies are in the NC16A domain of BP180. Since there is a lack of immune cross-reactivity between mouse and human BP180 in the NC16A domain, we generated a humanized mouse strain expressing human BP180 NC16A domain (termed hNC16A mice). hNC16A mice, when reconstituted with purified human neutrophils and injected with purified anti-NC16A IgA from LABD patients' sera, developed subepidermal blisters 48 h post IgA injection. The lesional skin showed IgA deposition at the BMZ, extensive neutrophilic infiltration, and significantly increased levels of neutrophil elastase (NE) and MMP-9.In vitro, anti-NC16A IgA in the presence of recombinant NC16A, activates human neutrophils to release NE and MMP-9. Blockade of NE but not MMP-9 significantly reduced the disease severity in the LABD mouse model. These findings suggest that NE plays a pivotal role in anti-NC16A IgA-induced LABD.

Importance:Hidradenitis suppurativa (HS) is a common and severely morbid chronic inflammatory skin disease that is reported to be highly heritable. However, the genetic understanding of HS is insufficient, and limited genome-wide association studies (GWASs) have been performed for HS, which have not identified significant risk loci. Objective:To identify genetic variants associated with HS and to shed light on the underlying genes and genetic mechanisms. Design, Setting, and Participants:This genetic association study recruited 753 patients with HS in the HS Program for Research and Care Excellence (HS ProCARE) at the University of North Carolina Department of Dermatology from August 2018 to July 2021. A GWAS was performed for 720 patients (after quality control) with controls from the Add Health study and then meta-analyzed with 2 large biobanks, UK Biobank (247 cases) and FinnGen (673 cases). Variants at 3 loci were tested for replication in the BioVU biobank (290 cases). Data analysis was performed from September 2021 to December 2022. Main Outcomes and Measures:Main outcome measures are loci identified, with association of P < 1 × 10-8 considered significant. Results:A total of 753 patients were recruited, with 720 included in the analysis. Mean (SD) age at symptom onset was 20.3 (10.57) years and at enrollment was 35.3 (13.52) years; 360 (50.0%) patients were Black, and 575 (79.7%) were female. In a meta-analysis of the 4 studies, 2 HS-associated loci were identified and replicated, with lead variants rs10512572 (P = 2.3 × 10-11) and rs17090189 (P = 2.1 × 10-8) near the SOX9 and KLF5 genes, respectively. Variants at these loci are located in enhancer regulatory elements detected in skin tissue. Conclusions and Relevance:In this genetic association study, common variants associated with HS located near the SOX9 and KLF5 genes were associated with risk of HS. These or other nearby genes may be associated with genetic risk of disease and the development of clinical features, such as cysts, comedones, and inflammatory tunnels, that are unique to HS. New insights into disease pathogenesis related to these genes may help predict disease progression and novel treatment approaches in the future.

Introduction We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. Methods Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation- profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). Results Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p< 0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). Conclusion Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids coextracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.