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

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

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

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 For patients with sentinel lymph node (SLN)-positive cutaneous melanoma, the Second Multicenter Selective Lymphadenectomy trial demonstrated equivalent disease-specific survival (DSS) with active surveillance using nodal ultrasound versus completion lymph node dissection (CLND). Adoption and outcomes of active surveillance in clinical practice and in adjuvant therapy recipients are unknown. Methods In a retrospective cohort of SLN-positive adults treated at 21 institutions in Australia, Europe, and the United States from June 2017 to November 2019, the authors evaluated the impact of active surveillance and adjuvant therapy on all-site recurrence-free survival (RFS), isolated nodal RFS, distant metastasis-free survival (DMFS), and DSS using Kaplan-Meier curves and Cox proportional hazard models. Results Among 6347 SLN biopsies, 1154 (18%) were positive and had initial negative distant staging. In total, 965 patients (84%) received active surveillance, 189 (16%) underwent CLND. Four hundred thirty-nine patients received adjuvant therapy (surveillance, 38%; CLND, 39%), with the majority (83%) receiving anti-PD-1 immunotherapy. After a median follow-up of 11 months, 220 patients developed recurrent disease (surveillance, 19%; CLND, 22%), and 24 died of melanoma (surveillance, 2%; CLND, 4%). Sixty-eight patients had an isolated nodal recurrence (surveillance, 6%; CLND, 4%). In patients who received adjuvant treatment without undergoing prior CLND, all isolated nodal recurrences were resectable. On risk-adjusted multivariable analyses, CLND was associated with improved isolated nodal RFS (hazard ratio [HR], 0.36; 95% CI, 0.15-0.88), but not all-site RFS (HR, 0.68; 95% CI, 0.45-1.02). Adjuvant therapy improved all-site RFS (HR, 0.52; 95% CI, 0.47-0.57). DSS and DMFS did not differ by nodal management or adjuvant treatment. Conclusions Active surveillance has been adopted for most SLN-positive patients. At initial assessment, real-world outcomes align with randomized trial findings, including in adjuvant therapy recipients. Lay Summary For patients with melanoma of the skin and microscopic spread to lymph nodes, monitoring with ultrasound is an alternative to surgically removing the remaining lymph nodes. The authors studied adoption and real-world outcomes of ultrasound monitoring in over 1000 patients treated at 21 centers worldwide, finding that most patients now have ultrasounds instead of surgery. Although slightly more patients have cancer return in the lymph nodes with this strategy, typically, it can be removed with delayed surgery. Compared with up-front surgery, ultrasound monitoring results in the same overall risk of melanoma coming back at any location or of dying from melanoma.

e21014 Background: The ECHO-301/KEYNOTE-252 trial failed to show clinical benefit of adding epacadostat to pembrolizumab in PD-1 inhibitor-naïve MM. We reasoned that the expression of other TMEs and/or LAT1 by melanoma cells plays a more important role. Methods: Melanoma tissues from stage III/IV pts were stained for the 4 TMEs (TPH1, TPH2, TDO2, IDO1) and LAT1 by single-color immunohistochemistry. Tissues were scored for the status of tumor-infiltrating lymphocytes (TILs) and the expression of 5 proteins separately in melanoma cells and TILs (if present). Association between protein expression, TIL status, and melanoma-specific overall survival was performed. Results: Tissues from 87 pts (stage IV, n = 25) were available. Expression of all 4 TMEs and LAT1 was significantly higher in melanoma cells compared to TILs ( p< 0.001); TPH1, TPH2, and TDO2 expression in melanoma cells was significantly higher than IDO1 (p < 0.001). Lower TPH1 expression in melanoma cells was associated with presence of TILs. Multivariate analysis using a Cox proportional hazards model that included expression of the 5 proteins in melanoma cells showed that high IDO1 was a favorable and high LAT1 expression was an adverse prognostic factor. Conclusions: Expression of TMEs and LAT1 by melanoma cells may have more important role in metastatic melanoma by depleting an essential amino acid from the tumor microenvironment. Pharmacologic targeting of TPH1/TPH2 (e.g. telotristat) may be more clinically relevant in MM as opposed to IDO1 inhibition.

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

Background: Little is known about the prognostic significance of somatically mutated genes in metastatic melanoma (MM). We have employed a combined clinical and bioinformatics approach on tumor samples from cutaneous melanoma (SKCM) as part of The Cancer Genome Atlas project (TCGA) to identify mutated genes with potential clinical relevance. Methods: After limiting our DNA sequencing analysis to MM samples (n = 356) and to the CANCER CENSUS gene list, we filtered out mutations with low functional significance (snpEFF). We performed Cox analysis on 53 genes that were mutated in ≥3% of samples, and had ≥50% difference in incidence of mutations in deceased subjects versus alive subjects. Results: Four genes were potentially prognostic [RAC1, FGFR1, CARD11, CIITA; false discovery rate (FDR) < 0.2]. We identified 18 additional genes (e.g., SPEN, PDGFRB, GNAS, MAP2K1, EGFR, TSC2) that were less likely to have prognostic value (FDR < 0.4). Most somatic mutations in these 22 genes were infrequent (< 10%), associated with high somatic mutation burden, and were evenly distributed across all exons, except for RAC1 and MAP2K1. Mutations in only 9 of these 22 genes were also identified by RNA sequencing in >75% of the samples that exhibited corresponding DNA mutations. The low frequency, UV signature type and RNA expression of the 22 genes in MM samples were confirmed in a separate multi-institution validation cohort (n = 413). An underpowered analysis within a subset of this validation cohort with available patient follow-up (n = 224) showed that somatic mutations in SPEN and RAC1 reached borderline prognostic significance [log-rank favorable (p = 0.09) and adverse (p = 0.07), respectively]. Somatic mutations in SPEN, and to a lesser extent RAC1, were not associated with definite gene copy number or RNA expression alterations. High (>2+) nuclear plus cytoplasmic expression intensity for SPEN was associated with longer melanoma-specific overall survival (OS) compared to lower (≤ 2+) nuclear intensity (p = 0.048). We conclude that expressed somatic mutations in infrequently mutated genes beyond the well-characterized ones (e.g., BRAF, RAS, CDKN2A, PTEN, TP53), such as RAC1 and SPEN, may have prognostic significance in MM.

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.

Telomerase reverse transcriptase (TERT) promoter mutations are commonly found in malignant melanomas but rare in melanocytic nevi. To assess its potential diagnostic utility for the distinction of melanoma from nevus, we determined the TERT promoter mutation status of 86 primary melanomas, 72 melanocytic nevi, and 40 diagnostically problematic melanocytic proliferations. Of the 86 melanomas, 67 (77.9%) were TERT-positive, defined as harboring a hotspot TERT promoter mutation at positions -124C>T, -124_125CC>TT, -138_139CC>TT, or -146C>T. Of the 72 nevi, only 1 (1.4%) was TERT-positive. Of the 40 diagnostically uncertain melanocytic proliferations, 2 (5.0%) were TERT-positive. TERT positivity as a test for melanoma versus nevus had an accuracy of 87.3% [95% confidence interval (CI), 81.1-92.1], a sensitivity of 77.9% (95% CI, 68.9-85.4), a specificity of 98.6% (95% CI, 95.8-100), a positive predictive value of 98.5% (95% CI, 95.6-100), and a negative predictive value of 78.9% (95% CI, 72.6-85.4). Our results indicate that hotspot TERT promoter mutation status may be a useful ancillary parameter for the diagnosis of melanoma. In particular, the high specificity of these mutations for melanoma indicates the presence of a TERT promoter mutation in a melanocytic neoplasm associated with diagnostic controversy, or uncertainty should increase concern for a melanoma.

Tumor mitotic rate (TMR) is a known prognostic variable in thin melanoma patients. Its significance in stage II melanoma patients is yet to be demonstrated. Retrospective analysis of a prospective melanoma database from 9/1997 to 7/2015 was performed. All stage II melanoma, with documented TMR, and six months of follow-up were included. We evaluated the association of clinicopathologic variables, TMR, as a continuous and categorical variable with recurrence-free survival (RFS) and overall survival (OS) using Cox proportional hazards modeling. We used a statistical model, X-tile, to develop optimal categorizations of TMR. A total of 265 patient characteristics are included in this study. Recurrences occurred in 82 (30.9%) patients, including 5 local, 41 regional, and 36 distant patients. In multivariate model, ulceration, Breslow, and continuous TMR were associated with worse RFS\OS. Continuous TMR demonstrated worse RFS (hazards ratio [HR] 1.02 (1.00–1.05)) and OS (HR 1.02 (1.00–1.04)), whereas dichotomized TMR (≥1 vs <1) was not significant. TMR >10.4 mitoses/mm2 has a 5-year RFS\OS of 27.2 and 44.3 per cent, respectively, compared with 57.4 and 71.4 per cent, respectively, for TMR <3.2 mitoses/mm2. Continuous TMR predicts incidence of recurrence in stage II melanoma. We propose a new categorization method developed by statistical modeling for optimal stratification that may guide surveillance for this disparate patient population.

Associations of MC1R with BRAF mutations in melanoma have been inconsistent between studies. We sought to determine for 1,227 participants in the international population-based Genes, Environment, and Melanoma (GEM) study whether MC1R and phenotypes were associated with melanoma BRAF/NRAS subtypes. We used logistic regression adjusted by age, sex, and study design features and examined effect modifications. BRAF(+) were associated with younger age, blond/light brown hair, increased nevi, and less freckling, and NRAS(+) with older age relative to the wild type (BRAF(-)/NRAS(-)) melanomas (all P < 0.05). Comparing specific BRAF subtypes to the wild type, BRAF V600E was associated with younger age, blond/light brown hair, and increased nevi and V600K with increased nevi and less freckling (all P < 0.05). MC1R was positively associated with BRAF V600E cases but only among individuals with darker phototypes or darker hair (P-interaction < 0.05) but inversely associated with BRAF V600K (P-trend = 0.006) with no significant effect modification by phenotypes. These results support distinct etiologies for BRAF V600E, BRAF V600K, NRAS(+), and wild-type melanomas. MC1R's associations with BRAF V600E cases limited to individuals with darker phenotypes indicate that MC1R genotypes specifically provide information about BRAF V600E melanoma risk in those not considered high risk based on phenotype. Our results also suggest that melanin pathways deserve further study in BRAF V600E melanomagenesis.

BACKGROUND: Patients with stage II melanoma have a considerable risk for recurrence. Current guidelines are imprecise as to optimal follow-up. We hypothesized that by examining recurrence patterns, we could help to better inform guidelines.STUDY DESIGN: We queried IRB-approved melanoma databases of Thomas Jefferson University and University of North Carolina, identifying 581 patients with stage II melanoma between 1996 and 2015 with at least 1 year of follow-up. Data included location of first recurrence and how recurrence was detected (ie patient symptom, physician examination, or routine surveillance imaging). Cox regression with backward elimination was used for multivariable analysis.RESULTS: One hundred and seventy-one patients had a recurrence (29.4%), the incidence increased considerably by stage sub-group. Significant predictors of recurrence included male sex (p = 0.003), ulceration (p = 0.03), and stage (p < 0.001). On multivariable analysis, male sex and stage continued to be significant (p < 0.01). For overall survival, regression, ulceration, stage, and age were significant predictors of survival. Stage, regression, and age remained significant by multivariable analysis. Patient symptoms were the most frequent mode of detection (40%), followed by physician examination (30%) and surveillance imaging (26%) dthis did not differ significantly by stage. Regional nodes were the most common site of recurrence (30%), followed by lung (27%) and in-transit (18%).CONCLUSIONS: The majority of recurrences in stage II melanoma are detected by patients and their physicians and rarely by routine imaging. As such, clinical follow-up and patient education are critical factors in detection of recurrence. With the prevalence of regional nodal recurrences, ultrasound might prove to be an important strategy in early recurrence detection. (C) 2017 by the American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Abstract Purpose: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation. Experimental Design: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined. Results: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0–G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Ptennull/BrafV600E) melanomas. Conclusions: We conclude that ITK, formerly considered an immune cell–specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Ptennull/BrafV600E mouse melanoma model supports this possibility. Clin Cancer Res; 21(9); 2167–76. ©2015 AACR.