AbstractBackground: 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.

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.

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

Supplementary Figures S1-3, Tables S1-4. Supplemental Figure S1. Racial differences in breast tumor methylation at candidate CpG probes when race is self-reported or defined by AIMs markers. Supplemental Figure S2. Methylation detected at KCNK4 and DSC2 by the GoldenGate Cancer Panel I array or quantitative methylation-specific PCR. Supplemental Figure S3. Kaplan-Meier plots showing disease-specific survival in AAs and non-AAs according to tumor methylation level for the top probes varying by race. Cases were divided into three methylation groups (high, intermediate, low) based on beta values in the combined dataset, and Kaplan-Meier plots were generated within AAs and non-AAs. Log-rank p-values are given for differences according to methylation level within each racial group. Supplemental Table S1. Reported genomic variants overlapping CpG probes and their frequencies by ancestry Supplemental Table S2. CpGs significantly differentially methylated by race in HR+ or HR- breast tumors Supplemental Table S3. Correlation between 450K CpG methylation and gene expression in TCGA breast tumors for genes showing differential methylation by race in CBCS Supplemental Table S4. Comparison of methylation in lymphoblastoid cell lines from African Americans and Caucasian Americans

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.

Supplementary Table 6. Significant pathways identified by Ingenuity Pathway Analysis for Reverse Phase Protein Array (RPPA) results of RPMI 8322

Supplemental Table 1. Correlation between BMI-associated methylation sites (overall and among ER-positive tumors) and gene expression in breast tumors from The Cancer Genome Atlas (TCGA)

Supplementary Table 2. Staining protocols for antibodies utilized for immunohistochemistry throughout the study

Supplementary Figure 2. Ingenuity Pathway Analysis Results from RPPA assay and effects of ITK activity on specific cellular proteins by westerns and RPPA results.

Legends for Supplementary Figures 1 and 2

Supplementary Tables 3 and 4. Supplementary Table 3. ITK protein expression levels in cell lines by single fluorescent immunocytochemistry Supplementary Table 4. Statistical analysis of shRNA and BI 10N effects on the proliferation and motility rates of melanoma cell lines

Supplementary Table 5. BI 10N kinase selectivity at ATP concentrations of approximate Km for ATP and 1 mM ATP

Differential methylation plays an important role in melanoma development and is associated with survival, progression and response to treatment. However, the mechanisms by which methylation promotes melanoma development are poorly understood. The traditional explanation of selective advantage provided by differential methylation postulates that hypermethylation of regulatory 5’-cytosine-phosphate-guanine-3’ dinucleotides (CpGs) downregulates the expression of tumor suppressor genes and therefore promotes tumorigenesis. We believe that other (not necessarily alternative) explanations of the selective advantages of methylation are also possible. Here, we hypothesize that melanoma cells use methylation to shut down transcription of nonessential genes – those not required for cell survival and proliferation. Suppression of nonessential genes allows tumor cells to be more efficient in terms of energy and resource usage, providing them with a selective advantage over the tumor cells that transcribe and subsequently translate genes they do not need. We named the hypothesis the Rule Out (RO) hypothesis. The RO hypothesis predicts higher methylation of CpGs located in regulatory regions (CpG islands) of nonessential genes. It also predicts the higher methylation of regulatory CpGs linked to nonessential genes in melanomas compared to nevi and lower expression of nonessential genes in malignant (derived from melanoma) versus normal (derived from nonaffected skin) melanocytes. The analyses conducted using in-house and publicly available data found that all predictions derived from the RO hypothesis hold, providing observational support for the hypothesis.

Supplementary Figure 1. IHC staining for co-localization of immune markers with cells expressing ITK and determining the presence of ITK in immune cell subtypes infiltrating the tumor and the effect of ITK ablation on the cell cycle of the melanoma cells

Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns, and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low methylation, intermediate methylation, or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP). CIMP melanomas occurred as early as tumor stage 1b and, compared with low-methylation melanomas, were associated with age at diagnosis ≥65 years, lentigo maligna melanoma histologic subtype, presence of ulceration, higher American Joint Committee on Cancer stage and tumor stage, and lower tumor-infiltrating lymphocyte grade (all P < 0.05). Patients with CIMP melanomas had worse melanoma-specific survival (hazard ratio = 11.84; confidence interval = 4.65‒30.20) than those with low-methylation melanomas, adjusted for age, sex, American Joint Committee on Cancer stage, and tumor-infiltrating lymphocyte grade. Genes hypermethylated in CIMP compared with those in low-methylation melanomas included PTEN, VDR, PD-L1, TET2, and gene sets related to development/differentiation, the extracellular matrix, and immunity. CIMP melanomas exhibited hypermethylation of genes important in melanoma progression and tumor immunity, and although present in some early melanomas, CIMP was associated with worse survival independent of known prognostic factors.

Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns, and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low methylation, intermediate methylation, or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP). CIMP melanomas occurred as early as tumor stage 1b and, compared with low-methylation melanomas, were associated with age at diagnosis >= 65 years, lentigo maligna melanoma histologic subtype, presence of ulceration, higher American Joint Committee on Cancer stage and tumor stage, and lower tumor-infiltrating lymphocyte grade (all P < 0.05). Patients with CIMP melanomas had worse melanoma-specific survival (hazard ratio = 11.84; confidence interval = 4.65.30.20) than those with low-methylation melanomas, adjusted for age, sex, American Joint Committee on Cancer stage, and tumor-infiltrating lymphocyte grade. Genes hypermethylated in CIMP compared with those in lowmethylation melanomas included PTEN, VDR, PD-L1, TET2, and gene sets related to development/differentiation, the extracellular matrix, and immunity. CIMP melanomas exhibited hypermethylation of genes important in melanoma progression and tumor immunity, and although present in some early melanomas, CIMP was associated with worse survival independent of known prognostic factors.

Background: Genome-wide association studies have reported that genetic variation at ANRIL (CDKN2B-AS1) is associated with risk of several chronic diseases including coronary artery disease, coronary artery calcification, myocardial infarction, and type 2 diabetes mellitus. ANRIL is located at the CDKN2A/B locus, which encodes multiple melanoma tumor suppressors. We investigated the association of these variants with melanoma prognostic characteristics. Methods: The Genes, Environment, and Melanoma Study enrolled 3,285 European origin participants with incident invasive primary melanoma. For each of ten disease-associated SNPs at or near ANRIL, we used linear and logistic regression modeling to estimate, respectively, the per allele mean changes in log of Breslow thickness and ORs for presence of ulceration and tumor-infiltrating lymphocytes (TIL). We also assessed effect modification by tumor NRAS/ BRAF mutational status. Results: Rs518394, rs10965215, and rs564398 passed false discovery and were each associated (P <= 0.005) with TILs, although only rs564398 was independently associated (P = 0.0005) with TILs. Stratified by NRAS/BRAF mutational status, rs564398*A was significantly positively associated with TILs among NRAS/BRAF mutant, but not wild-type, cases. We did not find SNP associations with Breslow thickness or ulceration. Conclusions: ANRIL rs564398 was associated with TIL presence in primary melanomas, and this association may be limited to NRAS/BRAF-mutant cases. Impact: Pathways related to ANRIL variants warrant exploration in relationship to TILs in melanoma, especially given the impact of TILs on immunotherapy and survival.

Using a genome-wide association study of familial melanoma pedigrees (excluding CDKN2A+ pedigrees) and genetically matched controls, Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar identified three single nucleotide polymorphisms (SNPs) in close proximity and high linkage disequilibrium in the 10q25.1 region (rs17119434, rs17119461, and rs17119490) associated with melanoma (Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar). These SNPs had low minor allele frequencies of 0.005 among controls utilized by Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar, making detection of associations via traditional case–control methods challenging. We sought to confirm the relationship between these SNPs and melanoma utilizing the population-based Genes, Environment, and Melanoma (GEM) Study, designed to detect associations of rare genetic variants with melanoma (Begg et al., 2006Begg C.B. Hummer A.J. Mujumdar U. Armstrong B.K. Kricker A. Marrett L.D. et al.A design for cancer case-control studies using only incident cases: experience with the GEM study of melanoma.Int J Epidemiol. 2006; 35: 756-764Crossref PubMed Scopus (62) Google Scholar). The GEM Study is an international population-based case–control study of melanoma in which controls are those diagnosed with an invasive single primary melanoma (SPM) and cases are those diagnosed with multiple primary melanoma (MPM) ascertained between 1998 and 2003 in Australia, Canada, Italy, and the United States (Begg et al., 2006Begg C.B. Hummer A.J. Mujumdar U. Armstrong B.K. Kricker A. Marrett L.D. et al.A design for cancer case-control studies using only incident cases: experience with the GEM study of melanoma.Int J Epidemiol. 2006; 35: 756-764Crossref PubMed Scopus (62) Google Scholar, Millikan et al., 2006Millikan R.C. Hummer A. Begg C. Player J. de Cotret A.R. Winkel S. et al.Polymorphisms in nucleotide excision repair genes and risk of multiple primary melanoma: the Genes Environment and Melanoma Study.Carcinogenesis. 2006; 27: 610-618Crossref PubMed Scopus (86) Google Scholar). Per GEM protocol, in situ melanomas were considered to be incident melanomas if patients had prior invasive melanomas, in view of the careful surveillance that such patients would have received. The institutional review board at each participating recruitment site approved the study. Participants provided written informed consent. Patient characteristics were collected from phone interviews and self-completed questionnaires. DNA was collected from buccal brushes (Begg et al., 2005Begg C.B. Orlow I. Hummer A.J. Armstrong B.K. Kricker A. Marrett L.D. et al.Lifetime risk of melanoma in CDKN2A mutation carriers in a population-based sample.J Natl Cancer Inst. 2005; 97: 1507-1515Crossref PubMed Scopus (168) Google Scholar). SNPs were genotyped using the MassArray iPLEX platform (Agena Bioscience, San Diego, CA) with quality-control measures described previously (Orlow et al., 2016Orlow I. Reiner A.S. Thomas N.E. Roy P. Kanetsky P.A. Luo L. et al.Vitamin D receptor polymorphisms and survival in patients with cutaneous melanoma: a population-based study.Carcinogenesis. 2016; 37: 30-38Crossref PubMed Scopus (44) Google Scholar). The tumor characteristics were obtained from the diagnostic pathology reports or centralized pathology review as described previously (Kricker et al., 2013Kricker A. Armstrong B.K. Goumas C. Thomas N.E. From L. Busam K. et al.Survival for patients with single and multiple primary melanomas: the Genes, Environment, and Melanoma Study.JAMA Dermatol. 2013; 149: 921-927Crossref PubMed Scopus (27) Google Scholar, Taylor et al., 2015Taylor N.J. Busam K.J. From L. Groben P.A. Anton-Culver H. Cust A.E. et al.Inherited variation at MC1R and histological characteristics of primary melanoma.PLoS One. 2015; 10: e0119920Google Scholar). Logistic regression models estimated the odds ratios (ORs) and 95% confidence intervals (CIs) for each SNP adjusted for study features (age, sex, and study center) and an age by sex interaction. Participants with SPM who developed MPM during the ascertainment period (n = 96) were included as both cases and controls. All tests were two-sided with P < 0.05 considered significant. All data were analyzed using Stata, version 15 (StataCorp, College Station, TX). The demographics and tumor characteristics of the 2,458 controls and 1,205 cases in GEM are in Supplementary Table S1 online, excluding 12 participants not of European descent. The SNPs were in high linkage disequilibrium with each other: D′ = 0.92 for rs17119434 and rs17119461, 0.95 for rs17119434 and rs17119490, and 1.00 for rs17119461 and rs17119490. Minor allele frequencies were between 0.012 and 0.013 for cases and 0.008 and 0.009 for controls, and the genotype frequency of homozygous minor allele carriage was zero for all three SNPs. The associations of these SNPs with MPM compared to SPM are in Table 1, and reported ORs reflect the comparison of heterozygous versus homozygous major allele genotypes. SNPs rs17119461 and rs17119490 were significantly associated with MPM (P < 0.05), and rs17119434 approached significance (P < 0.08). rs17119461 had the strongest independent association with MPM (OR = 1.77, 95% CI = 1.06–2.97).Table 1Associations of genotypes from the 10q25.1 chromosomal region with multiple primary melanoma (n = 1,205) compared with single primary melanoma (n = 2458) patients in the Genes, Environment, and Melanoma Study1Limited to participants of European origin.SNP (hg19)A/aGenotype Frequency, n (%)MAFSingle Primary Melanoma (n = 2,458)Multiple Primary Melanoma (n = 1,205)Aa Versus AA, OR (95% CI)2We used logistic regression models to estimate the ORs and 95% CIs adjusted for study features (age at diagnosis [continuous], sex, and study center) and an age by sex interaction. The genotype frequency of homozygous minor allele carriage was zero for all three SNPs, and the ORs reflect the comparison of heterozygous versus homozygous major allele genotypes.P-ValueControlsCasesMissingAAAaMissingAAAars17119434 (107,505,161)A/G0.0090.01373 (3.0)2344 (95.4)41 (1.7)21 (1.7)1154 (95.8)30 (2.5)1.59 (0.94–2.67)0.08rs17119461 (107,516,352)T/C0.0090.01364 (2.0)2353 (95.7)41 (1.7)18 (1.5)1156 (95.9)31 (2.6)1.77 (1.06–2.97)0.03rs17119490 (107,522,927)G/A0.0080.01284 (3.4)2334 (95.0)40 (1.6)28 (2.3)1148 (95.3)29 (2.4)1.70 (1.00–2.88)0.05Bold type indicates the SNP with the strongest association.Abbreviations: A, major allele; a, minor allele; CI, confidence interval; hg19, human genome reference version 19; MAF, minor allele frequency; OR, odds ratio; SNP, single nucleotide polymorphism.1 Limited to participants of European origin.2 We used logistic regression models to estimate the ORs and 95% CIs adjusted for study features (age at diagnosis [continuous], sex, and study center) and an age by sex interaction. The genotype frequency of homozygous minor allele carriage was zero for all three SNPs, and the ORs reflect the comparison of heterozygous versus homozygous major allele genotypes. Open table in a new tab Bold type indicates the SNP with the strongest association. Abbreviations: A, major allele; a, minor allele; CI, confidence interval; hg19, human genome reference version 19; MAF, minor allele frequency; OR, odds ratio; SNP, single nucleotide polymorphism. To our knowledge, we provide the first confirmation of associations between SNPs in the 10q25.1 region and melanoma occurrence. The ORs (1.6–1.8) for MPM versus SPM were lower in GEM than the ORs (6.8–8.4) for familial melanoma cases versus genetically matched controls in Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar. As previously found for CDKN2A mutations, melanoma risk variants in the general population can have a lower relative risk of melanoma than in a high-risk population (Begg et al., 2005Begg C.B. Orlow I. Hummer A.J. Armstrong B.K. Kricker A. Marrett L.D. et al.Lifetime risk of melanoma in CDKN2A mutation carriers in a population-based sample.J Natl Cancer Inst. 2005; 97: 1507-1515Crossref PubMed Scopus (168) Google Scholar). Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar proposed a common ancestor to explain the high risk related to the 10q25.1 SNPs among their familial melanoma cases. A more plausible explanation, perhaps, is that the Teerlink et al., 2012Teerlink C. Farnham J. Allen-Brady K. Camp N.J. Thomas A. Leachman S. et al.A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.Hum Genet. 2012; 131: 77-85Crossref PubMed Scopus (23) Google Scholar estimate is simply an overestimate, a common feature of many initial epidemiologic discoveries (Xiao and Boehnke, 2009Xiao R. Boehnke M. Quantifying and correcting for the winner's curse in genetic association studies.Genet Epidemiol. 2009; 33: 453-462Crossref PubMed Scopus (131) Google Scholar). An advantage of the GEM study is that low-frequency genetic variants are more likely to be observable in SPMs than normal controls (Begg et al., 2005Begg C.B. Orlow I. Hummer A.J. Armstrong B.K. Kricker A. Marrett L.D. et al.Lifetime risk of melanoma in CDKN2A mutation carriers in a population-based sample.J Natl Cancer Inst. 2005; 97: 1507-1515Crossref PubMed Scopus (168) Google Scholar). Further, the ORs found in the GEM study are more likely to represent the impact of these SNPs in the general population than the ORs found for multiple case families. The 10q25.1 gene region lacks genes known to be associated with malignancy. A pseudogene, YWHAZP5, is the closest at 65 kb away. SORCS3 and SORCS1 genes, both involved with vacuolar protein production, fall within 1 Mb in either direction of the SNPs. Thus, the mechanism for these SNP associations with melanoma risk remains unknown. Notably, rs17119461 and rs17119490 were found to be nominally associated with pancreatic cancer, which shares genetic risk with familial melanoma (Wu et al., 2014Wu L. Goldstein A.M. Yu K. Yang X.R. Rabe K.G. Arslan A.A. et al.Variants associated with susceptibility to pancreatic cancer and melanoma do not reciprocally affect risk.Cancer Epidemiol Biomarkers Prev. 2014; 23: 1121-1124Crossref PubMed Scopus (14) Google Scholar). Some melanoma genetic testing panels for patients meeting specific criteria include intermediate risk variants, such as MITF c.952 G>A that have a low minor allele frequency (∼0.0015) (Delaunay et al., 2017Delaunay J. Martin L. Bressac-de Paillerets B. Duru G. Ingster O. Thomas L. Improvement of genetic testing for cutaneous melanoma in countries with low to moderate incidence: the rule of 2 vs the rule of 3.JAMA Dermatol. 2017; 153: 1122-1129Crossref PubMed Scopus (6) Google Scholar). Thus, if validated in additional studies, rs17119461 may be a potential candidate for genetic testing in populations at high risk for melanoma. Further, additional studies investigating the mechanism for the 10q25.1 SNP associations with melanoma risk are warranted. KJB has received minor royalties from editing a textbook with Elsevier. The remaining authors state no conflict of interest. This work was supported by the National Cancer Institute (P01CA206980 to NET and MB, R01CA112243 to NET, U01CA83180 and R01CA112524 to MB, R01CA098438 to CBB, R03CA125829 and R03CA173806 to IO, P30CA016086 (to Henry Shelton Earp), P30CA014089 (to SBG), and P30CA008748 (to Craig B. Thompson); National Institute of Environmental Health Sciences (P30ES010126 to James A. Swenberg). AEC was supported by Career Development Fellowships from the National Health and Medical Research Council is (1147843) and Cancer Institute of New South Wales (15/CDF/1-14). GEM Study Group: Coordinating Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA: Marianne Berwick (principal investigator [PI], currently at the University of New Mexico, Albuquerque, NM, USA), Colin Begg (co-PI), Irene Orlow (co-investigator), Klaus J. Busam (dermatopathologist), Pampa Roy (senior laboratory technician), Siok Leong (research assistant), Sergio Corrales-Guerrero (senior research technician), Keimya Sadeghi (senior laboratory technician), Anne Reiner (biostatistician). University of New Mexico, Albuquerque, NM, USA: Marianne Berwick (PI), Li Luo (biostatistician), Tawny W. Boyce (data manager). Study centers: The University of Sydney and The Cancer Council New South Wales, Sydney, Australia: Anne E. Cust (PI), Bruce K. Armstrong (former PI), Anne Kricker (former co-PI); Menzies Institute for Medical Research University of Tasmania, Hobart, Australia: Alison Venn (current PI), Terence Dwyer (PI, currently at University of Oxford, Oxford, UK), Paul Tucker (dermatopathologist); British Columbia Cancer Research Centre, Vancouver, Canada: Richard P. Gallagher (PI), Agnes Lai, Research Coordinator, Cancer Care Ontario, Toronto, Canada: Loraine D. Marrett (PI), Lynn From (dermatopathologist); CPO, Center for Cancer Prevention, Torino, Italy: Roberto Zanetti, M.D (PI), Stefano Rosso (co-PI); University of California, Irvine, CA, USA: Hoda Anton-Culver (PI); University of Michigan, Ann Arbor, MI, USA: University of Michigan, Ann Arbor, MI, USA: Stephen B. Gruber (PI, currently at University of Southern California, Los Angeles, CA, USA), Shu-Chen Huang (co-investigator, joint at University of Southern California–University of Michigan); University of North Carolina, Chapel Hill, NC, USA: Nancy E. Thomas (PI), Kathleen Conway (co-investigator), David W. Ollila (co-Investigator), Pamela A. Groben (dermatopathologist), Sharon N. Edmiston (research analyst), Honglin Hao (laboratory specialist), Eloise Parrish (laboratory specialist), Jill S. Frank (Research Assistant), David C. Gibbs (Research Assistant, Emory University, Atlanta, GA, USA); University of Pennsylvania, Philadelphia, PA, USA: Timothy R. Rebbeck (former PI), Peter A. Kanetsky (PI, currently at H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA); UV data consultants: Julia Lee Taylor and Sasha Madronich, National Centre for Atmospheric Research, Boulder, CO, USA. Download .pdf (.04 MB) Help with pdf files Supplementary Table S1

Early diagnosis improves melanoma survival, yet the histopathological diagnosis of cutaneous primary melanoma can be challenging, even for expert dermatopathologists. Analysis of epigenetic alterations, such as DNA methylation, that occur in melanoma can aid in its early diagnosis. Using a genome-wide methylation screening, we assessed CpG methylation in a diverse set of 89 primary invasive melanomas, 73 nevi, and 41 melanocytic proliferations of uncertain malignant potential, classified based on interobserver review by dermatopathologists. Melanomas and nevi were split into training and validation sets. Predictive modeling in the training set using ElasticNet identified a 40-CpG classifier distinguishing 60 melanomas from 48 nevi. High diagnostic accuracy (area under the receiver operator characteristic curve = 0.996, sensitivity = 96.6%, and specificity = 100.0%) was independently confirmed in the validation set (29 melanomas, 25 nevi) and other published sample sets. The 40-CpG melanoma classifier included homeobox transcription factors and genes with roles in stem cell pluripotency or the nervous system. Application of the 40-CpG melanoma classifier to the diagnostically uncertain samples assigned melanoma or nevus status, potentially offering a diagnostic tool to assist dermatopathologists. In summary, the robust, accurate 40-CpG melanoma classifier offers a promising assay for improving primary melanoma diagnosis.

BRAF and NRAS mutations arise early in melanoma development, but their associations with low-penetrance melanoma susceptibility loci remain unknown. In the Genes, Environment and Melanoma Study, 1,223 European-origin participants had their incident invasive primary melanomas screened for BRAF/NRAS mutations and germline DNA genotyped for 47 single-nucleotide polymorphisms identified as low-penetrant melanoma-risk variants. We used multinomial logistic regression to simultaneously examine each single-nucleotide polymorphism's relationship to BRAF V600E, BRAF V600K, BRAF other, and NRAS+ relative to BRAF-/NRAS- melanoma adjusted for study features. IRF4 rs12203592*T was associated with BRAF V600E (odds ratio [OR] = 0.59, 95% confidence interval [CI] = 0.43-0.79) and V600K (OR = 0.65, 95% CI = 0.41-1.03), but not BRAF other or NRAS+ melanoma. A global test of etiologic heterogeneity (Pglobal = 0.001) passed false discovery (Pglobal = 0.0026). PLA2G6 rs132985*T was associated with BRAF V600E (OR = 1.32, 95% CI = 1.05-1.67) and BRAF other (OR = 1.82, 95% CI = 1.11-2.98), but not BRAF V600K or NRAS+ melanoma. The test for etiologic heterogeneity (Pglobal) was 0.005. The IRF4 rs12203592 associations were slightly attenuated after adjustment for melanoma-risk phenotypes. The PLA2G6 rs132985 associations were independent of phenotypes. IRF4 and PLA2G6 inherited genotypes may influence melanoma BRAF/NRAS subtype development.