Vitamin D-VDR Signaling Inhibits Wnt/ß-Catenin-Mediated Melanoma Progression and Promotes Antitumor Immunity.

1α,25-Dihydroxyvitamin D3 signals via the vitamin D receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As patients with melanoma commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D-VDR signaling and replicated the findings in The Cancer Genome Atlas metastases. VDR expression was independently protective for melanoma-related death in both primary and metastatic disease. High tumor VDR expression was associated with upregulation of pathways mediating antitumor immunity and corresponding with higher imputed immune cell scores and histologically detected tumor-infiltrating lymphocytes. High VDR-expressing tumors had downregulation of proliferative pathways, notably Wnt/ß-catenin signaling. Deleterious low VDR levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (<25 nmol/L ∼ 10 ng/mL) shortened survival in primary melanoma in a VDR-dependent manner. In vitro functional validation studies showed that elevated vitamin D-VDR signaling inhibited Wnt/ß-catenin signaling genes. Murine melanoma cells overexpressing VDR produced fewer pulmonary metastases than controls in tail-vein metastasis assays. In summary, vitamin D-VDR signaling contributes to controlling pro-proliferative/immunosuppressive Wnt/ß-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of a causal relationship between vitamin D-VDR signaling and melanoma survival, which should be explored as a therapeutic target in primary resistance to checkpoint blockade. SIGNIFICANCE: VDR expression could potentially be used as a biomarker to stratify patients with melanoma that may respond better to immunotherapy.

Transcriptomic Analysis Reveals Prognostic Molecular Signatures of Stage I Melanoma.

PURPOSE: Previously identified transcriptomic signatures have been based on primary and metastatic melanomas with relatively few American Joint Committee on Cancer (AJCC) stage I tumors, given difficulties in sampling small tumors. The advent of adjuvant therapies has highlighted the need for better prognostic and predictive biomarkers, especially for AJCC stage I and stage II disease. EXPERIMENTAL DESIGN: A total of 687 primary melanoma transcriptomes were generated from the Leeds Melanoma Cohort (LMC). The prognostic value of existing signatures across all the AJCC stages was tested. Unsupervised clustering was performed, and the prognostic value of the resultant signature was compared with that of sentinel node biopsy (SNB) and tested as a biomarker in three published immunotherapy datasets. RESULTS: Previous Lund and The Cancer Genome Atlas signatures predicted outcome in the LMC dataset (P = 10-8 to 10-4) but showed a significant interaction with AJCC stage (P = 0.04) and did not predict outcome in stage I tumors (P = 0.3-0.7). Consensus-based classification of the LMC dataset identified six classes that predicted outcome, notably in stage I disease. LMC class was a similar indicator of prognosis when compared with SNB, and it added prognostic value to the genes reported by Gerami and colleagues. One particular LMC class consistently predicted poor outcome in patients receiving immunotherapy in two of three tested datasets. Biological characterization of this class revealed high JUN and AXL expression and evidence of epithelial-to-mesenchymal transition. CONCLUSIONS: A transcriptomic signature of primary melanoma was identified with prognostic value, including in stage I melanoma and in patients undergoing immunotherapy.

Genetic and Environmental Determinants of Immune Response to Cutaneous Melanoma.

The immune response to melanoma improves the survival in untreated patients and predicts the response to immune checkpoint blockade. Here, we report genetic and environmental predictors of the immune response in a large primary cutaneous melanoma cohort. Bioinformatic analysis of 703 tumor transcriptomes was used to infer immune cell infiltration and to categorize tumors into immune subgroups, which were then investigated for association with biological pathways, clinicopathologic factors, and copy number alterations. Three subgroups, with "low", "intermediate", and "high" immune signals, were identified in primary tumors and replicated in metastatic tumors. Genes in the low subgroup were enriched for cell-cycle and metabolic pathways, whereas genes in the high subgroup were enriched for IFN and NF-κB signaling. We identified high MYC expression partially driven by amplification, HLA-B downregulation, and deletion of IFNγ and NF-κB pathway genes as the regulators of immune suppression. Furthermore, we showed that cigarette smoking, a globally detrimental environmental factor, modulates immunity, reducing the survival primarily in patients with a strong immune response. Together, these analyses identify a set of factors that can be easily assessed that may serve as predictors of response to immunotherapy in patients with melanoma. SIGNIFICANCE: These findings identify novel genetic and environmental modulators of the immune response against primary cutaneous melanoma and predict their impact on patient survival.See related commentary by Anichini, p. 2457.

Prevalence and predictors of germline CDKN2A mutations for melanoma cases from Australia, Spain and the United Kingdom.

BACKGROUND: Mutations in the CDKN2A and CDK4 genes predispose to melanoma. From three case-control studies of cutaneous melanoma, we estimated the prevalence and predictors of these mutations for people from regions with widely differing latitudes and melanoma incidence. METHODS: Population-based cases and controls from the United Kingdom (1586 cases, 499 controls) and Australia (596 early-onset cases, 476 controls), and a hospital-based series from Spain (747 cases, 109 controls), were screened for variants in all exons of CDKN2A and the p16INK4A binding domain of CDK4. RESULTS: The prevalence of mutations for people with melanoma was similar across regions: 2.3%, 2.5% and 2.0% for Australia, Spain and the United Kingdom respectively. The strongest predictors of carrying a mutation were having multiple primaries (odds ratio (OR) = 5.4, 95% confidence interval (CI: 2.5, 11.6) for 2 primaries and OR = 32.4 (95% CI: 14.7, 71.2) for 3 or more compared with 1 primary only); and family history (OR = 3.8; 95% CI:1.89, 7.5) for 1 affected first- or second-degree relative and OR = 23.2 (95% CI: 11.3, 47.6) for 2 or more compared with no affected relatives). Only 1.1% of melanoma cases with neither a family history nor multiple primaries had mutations. CONCLUSIONS: There is a low probability (<2%) of detecting a germline CDKN2A mutation in people with melanoma except for those with a strong family history of melanoma (≥2 affected relatives, 25%), three or more primary melanomas (29%), or more than one primary melanoma who also have other affected relatives (27%).

Melanoma risk for CDKN2A mutation carriers who are relatives of population-based case carriers in Australia and the UK.

BACKGROUND: CDKN2A mutations confer a substantial risk of cutaneous melanoma; however, the magnitude of risk is uncertain. METHODS: The study estimated the hazard ratio (HR) and the average age specific cumulative risk (ie, penetrance) of reported melanoma for CDKN2A mutation carriers in case families using a modified segregation analysis of the first and higher degree relatives of 35 population-based cases. The study sample included 223 relatives of 13 melanoma cases diagnosed when aged 18-39 years from Melbourne, Sydney and Brisbane, Australia, and 322 relatives of 22 melanoma cases diagnosed at any age from Yorkshire, UK. RESULTS: The estimated HR for melanoma for mutation carriers relative to the general population decreased with regions of increasing ambient ultraviolet (UV) irradiance, being higher for the UK than Australia (87, 95% CI 50 to 153 vs 31, 95% CI 20 to 50, p=0.008), and across Australia, 49 (95% CI 24 to 98) for Melbourne, 44 (95% CI 22 to 88) for Sydney, and 9 (95% CI 2 to 33) for Brisbane (p=0.02). Penetrance did not differ by geographic region. It is estimated that 16% (95% CI 10% to 27%) of UK and 20% (95% CI 13% to 30%) of Australian CDKN2A mutation carriers would be diagnosed with melanoma by age 50 years, and 45% (95% CI 29% to 65%) and 52% (95% CI 37% to 69%), respectively, by age 80 years. CONCLUSIONS: Contrary to the strong association between UV radiation exposure and melanoma risk for the general population, CDKN2A mutation carriers appear to have the same cumulative risk of melanoma irrespective of the ambient UV irradiance of the region in which they live.

Vitamin D receptor gene polymorphisms, serum 25-hydroxyvitamin D levels, and melanoma: UK case-control comparisons and a meta-analysis of published VDR data.

We have carried out melanoma case-control comparisons for six vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) and serum 25-hydroxyvitamin D(3) levels in order to investigate the role of vitamin D in melanoma susceptibility. There was no significant evidence of an association between any VDR SNP and risk in 1028 population-ascertained cases and 402 controls from Leeds, UK. In a second Leeds case-control study (299 cases and 560 controls) the FokI T allele was associated with increased melanoma risk (odds ratio (OR) 1.42, 95% confidence interval (CI) 1.06-1.91, p=0.02). In a meta-analysis in conjunction with published data from other smaller data sets (total 3769 cases and 3636 controls), the FokI T allele was associated with increased melanoma risk (OR 1.19, 95% CI 1.05-1.35), and the BsmI A allele was associated with a reduced risk (OR 0.81, 95% CI 0.72-0.92), in each instance under a parsimonious dominant model. In the first Leeds case-control comparison cases were more likely to have a higher body mass index (BMI) than controls (p=0.007 for linear trend). There was no evidence of a case-control difference in serum 25-hydroxyvitamin D(3) levels. In 1043 incident cases from the first Leeds case-control study, a single estimation of serum 25-hydroxyvitamin D(3) level taken at recruitment was inversely correlated with Breslow thickness (p=0.03 for linear trend). These data provide evidence to support the view that vitamin D and VDR may have a small but potentially important role in melanoma susceptibility, and putatively a greater role in disease progression.

Frequent p16-independent inactivation of p14ARF in human melanoma.

BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.

CDKN2A and CDK4 variants in Latvian melanoma patients: analysis of a clinic-based population.

Germline mutations of the CDKN2A and CDK4 genes explain a significant proportion of familial melanoma. To date, there have been few published estimations of the prevalence of such mutations in sporadic melanoma patients. In this study, we investigated CDKN2A and CDK4 exon 2 for germline mutations in 125 consecutive cutaneous malignant melanoma patients recruited through the Latvian Oncological Center, using amplicon melting analysis and sequencing. No disease-related CDKN2A germline mutations were identified in any of the melanoma patients analysed but the previously described CDK4 mutation, Arg24His, was found in one patient with a family history of melanoma. CDKN2A polymorphisms were studied as putative low penetrance susceptibility genes. The proportion of cases with polymorphisms in this Latvian melanoma population was Ala148Thr (c.442G>A) (6%), 500 C/G (c.*29C>G) (18%), and 540 C/T (c.*69C>T) (20%); however, only the frequency of the Ala148Thr polymorphism was higher in melanoma patients than in 203 controls (6 versus 1%, P=0.03). Ala148Thr has also been reported in association with melanoma in a Polish series but not in an English series. We therefore examined the Ala148Thr carrier's haplotype in 10 Latvian and 39 Polish samples. No significant difference was seen between these populations and the predominant haplotype observed in English samples, giving no indication that the discrepancy could be explained by population differences in linkage disequilibrium. In summary, our results show that germline mutations at the CDKN2A locus are rare in sporadic melanoma in Latvia. The study does, however, provide some additional evidence for a role for the CDKN2A polymorphism Ala148Thr as a low penetrance susceptibility gene. The detected CDK4 exon 2 mutation was found in only the seventh family identified worldwide with a germline CDK4 mutation.

Prevalence of 9p21 deletions in UK melanoma families.

Although the CDKN2A gene has been shown to be the major genetic determinant governing high-penetrance susceptibility to melanoma, there remains a significant proportion of melanoma pedigrees in which germline mutations of CDKN2A have not been identified. We have therefore studied the prevalence of germline 9p deletions encompassing the CDKN2 locus in melanoma pedigrees, using multiplex ligation-dependent probe amplification. Germline deletions were found in 3 of 93 UK pedigrees, with no previously identified CDKN2A mutations. A hemizygous deletion of CDKN2A exon 1beta previously reported by this group was confirmed in one family and identified in a second. Microsatellite analysis determined that these two families were ancestrally related. In the third family, a novel p16 hemizygous deletion involving CDKN2A exons 1alpha, 2, and 3 was detected. An additional 9p21 deletion reported previously in a USA melanoma-neural system tumor family was shown to involve CDKN2A exon 1beta, and not p16. The CDKN2A exon 1beta deletions provide further evidence that this tumor suppressor gene is important in melanoma-neural system tumor susceptibility, but do not exclude the possibility of a novel gene or regulatory element also being deleted in this region. Deletions at 9p21 are rare and explain only a small proportion of melanoma susceptibility. This study is the first to comprehensively exclude deletions in melanoma-prone families with no previously identified CDKN2A mutations.

A mutation hotspot at the p14ARF splice site.

Germline mutations of CDKN2A that affect the p16INK4a transcript have been identified in numerous melanoma pedigrees worldwide. In the UK, over 50% of pedigrees with three or more cases of melanoma have been found to carry mutations of CDKN2A. Mutations that affect p14ARF exon 1beta exclusively are very rare. This has led to the suggestion that it is p16INK4a and not p14ARF that plays the critical role in melanoma predisposition. We report the identification of a cluster of five different germline mutations at the p14ARF exon 1beta splice donor site in melanoma pedigrees. All the five splice site variants showed evidence of being causal mutations. Three of the variants were demonstrated to result in aberrant splicing of the p14ARF mRNA, confirming their role in melanoma predisposition. No other point mutations were identified in the coding region of p14ARF. The p14ARF transcript of CDKN2A is clearly important in disease predisposition in a subset of melanoma pedigrees. Curiously, the only mutations so far reported to affect p14ARF exon 1beta exclusively have been knockout mutations. Further investigation into the spectrum of mutations observed in this gene may help clarify the exact role of p14ARF in melanoma predisposition.

Intronic sequence variants of the CDKN2A gene in melanoma pedigrees.

Germ-line mutations of the tumor-suppressor gene CDKN2A predispose individuals to melanoma in families worldwide. However, coding mutations of CDKN2A have not been detected in a significant proportion of those affected. The identification of a disease-associated intronic mutation of CDKN2A in UK families, which has proved to be the most common CDKN2A mutation as yet identified in this population, has highlighted the possibility that additional causal mutations may lie within the intronic sequence of the gene. In this article, we describe the comprehensive screening of 109 English and 26 Australian melanoma pedigrees for intronic mutations of CDKN2A. In total, 24 sequence variants were identified across the two introns of the gene. We show evidence that two of the CDKN2A intronic variants (IVS1 + 1104 C > A and IVS1 - 1104 C > G) predispose to melanoma. IVS1 + 1104 was shown to result in the aberrant splicing of both p16(INK4a) and p14(ARF) mRNA. Overall, however, the proportion of English melanoma families with these variants is small.

The effect of sun exposure in determining nevus density in UK adolescent twins.

We report a study of 221 teenage twin pairs to examine the genetic and environmental determinants of nevi representing the most potent phenotypic risk factor for melanoma. Our published heritability analysis estimated that nevi are mainly genetically determined. In this paper we examine the role of sun exposure. We report a correlation between nevus density and sun exposure, particularly that acquired in hotter countries than in the UK (mean nevus density 41 per m2 in those in the highest quartile of exposure vs 24 per m2 in those with no exposure, p<0.0001). We were not able to demonstrate a protective effect for either sun protection cream or shirt wearing. By including phenotypic variables and reported sun exposure into the heritability analysis, we conclude that 66% of the total variance of nevus count is attributable to genetic effects: 7% associated to eye color, 6% to hair color, and 1% to reported skin type, which leaves 52% as to yet unidentified genetic factors. Of the 25% of variation attributable to environmental influences, one-third is estimated to be because of sun exposure on hot holidays.