[Show abstract][Hide abstract] ABSTRACT: Thirteen common susceptibility loci have been reproducibly associated with cutaneous malignant melanoma (CMM). We report the results of an international 2-stage meta-analysis of CMM genome-wide association studies (GWAS). This meta-analysis combines 11 GWAS (5 previously unpublished) and a further three stage 2 data sets, totaling 15,990 CMM cases and 26,409 controls. Five loci not previously associated with CMM risk reached genome-wide significance (P < 5 × 10(-8)), as did 2 previously reported but unreplicated loci and all 13 established loci. Newly associated SNPs fall within putative melanocyte regulatory elements, and bioinformatic and expression quantitative trait locus (eQTL) data highlight candidate genes in the associated regions, including one involved in telomere biology.
[Show abstract][Hide abstract] ABSTRACT: Telomere length has been associated with risk of many cancers, but results are inconsistent. Seven single nucleotide polymorphisms
(SNPs) previously associated with mean leukocyte telomere length were either genotyped or well-imputed in 11108 case patients
and 13933 control patients from Europe, Israel, the United States and Australia, four of the seven SNPs reached a P value under .05 (two-sided). A genetic score that predicts telomere length, derived from these seven SNPs, is strongly associated
(P = 8.92x10-9, two-sided) with melanoma risk. This demonstrates that the previously observed association between longer telomere length
and increased melanoma risk is not attributable to confounding via shared environmental effects (such as ultraviolet exposure)
or reverse causality. We provide the first proof that multiple germline genetic determinants of telomere length influence
JNCI Journal of the National Cancer Institute 10/2014; 106(10). DOI:10.1093/jnci/dju267 · 12.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysplastic naevi are melanocytic lesions that represent an intermediate stage between common naevus and melanoma. Histopathological distinction of dysplastic naevus from melanoma can be challenging and there is a requirement for molecular diagnostic markers. In this study we examined promoter CpG island methylation of a selected panel of genes, identified in a genome-wide methylation screen, across a spectrum of 405 melanocytic neoplasms. Promoter methylation analysis in common naevi, dysplastic naevi, primary melanomas and metastatic melanomas demonstrated progressive epigenetic deregulation. Dysplastic naevi were affected by promoter methylation of genes that are frequently methylated in melanoma but not in common naevi. We assessed the diagnostic value of the methylation status of five genes in distinguishing primary melanoma from dysplastic naevus. In particular CLDN11 promoter methylation was specific for melanoma as it occurred in 50% of primary melanomas but in only 3% of dysplastic naevi. A diagnostic algorithm that incorporates methylation of the CLDN11, CDH11, PPP1R3C, MAPK13 and GNMT genes was validated in an independent sample set and helped distinguish melanoma from dysplastic naevus (AUC 0.81). Melanoma-specific methylation of these genes supports the utility as epigenetic biomarkers and could point to their significance in melanoma development.Journal of Investigative Dermatology accepted article preview online, 07 July 2014; doi:10.1038/jid.2014.270.
[Show abstract][Hide abstract] ABSTRACT: A critical first step in the metastatic progression of cutaneous melanoma, invasive growth into the dermal compartment, would ideally be studied in the proper three-dimensional tissue microenvironment. In this study, we compared the growth and behavior of four melanoma cell lines originating from primary and metastatic human cutaneous melanomas (AN, RU, M14, and WK) in in-vitro human skin equivalents (HSEs) generated with four different dermal matrices: human fibroblast-seeded rat tail collagen, human fibroblast-derived matrix (FDM), noncellular human de-epidermized dermis (DED), and a novel fully cellular human DED with an intact pre-existent basement membrane. Melanoma cells showed proliferation in all HSEs, indicating that the microenvironment formed in all HSEs studied here allows the growth of melanoma cells in concert with epidermal keratinocytes for multiple weeks in vitro. Melanoma cells did not affect epidermal proliferation and terminal differentiation. Growth of melanoma cells in the dermal compartment, as a measure of invasive potential, differs markedly between the four types of in-vitro human melanoma models. Notably, the growth of melanoma cells in the dermal matrix was observed in all HSEs cultured with cell lines originating from metastatic melanoma, except for cDED-based HSEs, and the growth of melanoma cells of nonmetastatic origin was observed in the dermal compartment of FDM-based HSEs. Our results show that the type of dermal equivalent and the presence of an intact basement membrane should be taken into consideration when studying melanoma invasion using in-vitro HSEs.
Melanoma Research 06/2014; 24(4). DOI:10.1097/CMR.0000000000000079 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, their depletion abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas compared to nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-β-galactosidase activity, interleukins and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Topical imiquimod cream is increasingly applied in the treatment of lentigo maligna (LM), in particular for large lesions where surgery may lead to disfiguring scars. Published studies suggest that more frequent and prolonged treatment with topical imiquimod is associated with higher efficacy. In this study we prospectively treated 27 patients suffering from LM of the face with imiquimod 5% cream using an intensive treatment regimen consisting of daily applications for 12 weeks inducing at least 10 weeks skin inflammation. Twenty-four patients completed the treatment as recommended, 23 were available for follow-up (mean 39 months). Clinical and histopathological clearance was observed in 20 patients after on average 14 weeks of treatment. Notably, histopathological examination of a skin biopsy showed clearance of the LM in all 24 patients, including those who still showed some hyperpigmentation at 4 weeks off treatment. A clinical recurrence occurred in only 1 of the 24 treated patients. These findings suggest that the efficacy of imiquimod can be improved by implementing a more intensive treatment regimen. Randomized controlled trials are needed to confirm our results and establish the role of topical imiquimod in the treatment of LM.
[Show abstract][Hide abstract] ABSTRACT: We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS). Results were combined using random effects meta-analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04–1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01–1.39, p = 0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS.
Although staging systems predict outcome fairly well for melanoma, survival still varies among individual patients. In this meta-analysis, the authors found that inheritance of a rare genetic variant of PARP1 was associated with improved survival of melanoma patients. Increased expression of PARP1 has been associated with poorer outcome, and depletion of PARP1 may reduce both melanoma growth and angiogenesis. The identification of this and other germline variants that affect survival may help to identify key biological pathways active in host/tumor interactions, which may lead to the discovery of new therapeutic targets for treating advanced melanoma.
International Journal of Cancer 02/2014; 135(7). DOI:10.1002/ijc.28796 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An association between low serum vitamin D levels and poorer melanoma survival has been reported. We have studied inheritance of a polymorphism of the GC gene, rs2282679, coding for the vitamin D-binding protein, which is associated with lower serum levels of vitamin D, in a meta-analysis of 3137 melanoma patients. The aim was to investigate evidence for a causal relationship between vitamin D and outcome (Mendelian randomization). The variant was not associated with reduced OS in the UK cohort, per-allele hazard ratio (HR) for death 1.23 (95% CI 0.93,1.64. In the smaller cohorts, HR in OS analysis was 1.07 (95% CI 0.88,1.3) and for all cohorts combined, HR for OS was 1.09 (95% CI 0.93,1.29). There was evidence of increased melanoma specific deaths in the 7 cohorts for which these data were available. The lack of unequivocal findings despite the large sample size illustrates the difficulties of implementing Mendelian randomization. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.
[Show abstract][Hide abstract] ABSTRACT: The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome-wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome-wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.
[Show abstract][Hide abstract] ABSTRACT: Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFβ) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1's ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFβ activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFβ therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.Oncogene advance online publication, 25 February 2013; doi:10.1038/onc.2013.30.
[Show abstract][Hide abstract] ABSTRACT: This chapter describes a study in which the pattern of numerical chromosomal alterations in cutaneous anaplastic large cell lymphoma (C-ALCL) tumor samples was defined using array-based comparative genomic hybridization (CGH). First, the array-based CGH technique applied is outlined in detail. Next, its application in the analysis of C-ALCL tumor specimens is described. This approach resulted in the identification of highly recurrent chromosomal alterations in C-ALCL that include gain of 7q31 and loss on 6q16-6q21 and 13q34, each affecting 45% of the patients. The pattern characteristic of C-ALCL differs markedly from chromosomal alterations observed in other CTCL such as mycosis fungoides and Sézary syndrome and yielded several candidate genes with potential relevance in the pathogenesis of C-ALCL.
[Show abstract][Hide abstract] ABSTRACT: Cutaneous melanoma, a type of skin tumor originating from melanocytes, often develops from premalignant naevoid lesions via a gradual transformation process driven by an accumulation of (epi)genetic lesions. These dysplastic naevi display altered morphology and often proliferation of melanocytes. Additionally, melanocytes in dysplastic naevi show structural mitochondrial and melanosomal alterations and have elevated reactive oxygen species (ROS) levels. For this study we performed genome-wide expression and proteomic analysis of melanocytes from dysplastic naevus (DNMC) and adjacent normal skin (MC) from 18 patients. Whole genome expression profiles of the DNMC and MC of each individual patient subjected to GO-based comparative statistical analysis yielded significantly differentially expressed GO classes including "organellar ribosome," "mitochondrial ribosome," "hydrogen ion transporter activity," and "prefoldin complex." Validation of 5 genes from these top GO classes revealed a heterogeneous differential expression pattern. Proteomic analysis demonstrated differentially expressed proteins in DNMC that are involved in cellular metabolism, detoxification, and cytoskeletal organization processes, such as GTP-binding Rho-like protein CDC42, glutathione-S-transferase omega-1 and prolyl 4-hydroxylase. Collectively these results point to deregulation of cellular processes, such as metabolism and protein synthesis, consistent with the observed elevated oxidative stress levels in DNMC potentially resulting in oxidative DNA damage in these cells.