Lymphatic invasion (LI) in primary cutaneous melanomas was recently found to be common. In this study, we evaluated LI as an independent prognostic factor.
This study included 251 patients with vertical growth phase (VGP) primary cutaneous melanomas who had paraffin-fixed lesional tissue and were in a prospective cohort seen between 1972 and 1991, had no clinical evidence of regional nodal disease at diagnosis and had at least ten years of follow-up. Dual immunohistochemistry (IHC) staining was used to detect lymphatic endothelium (podoplanin) and melanoma cells (S-100). Multivariate logistic regression for ten-year metastasis was used to define independent prognostic factors and a prognostic tree was developed to characterize and discriminate risk groups. Kaplan-Meier disese-free survival curves for those with and without LI within current AJCC stages were compared using the log-rank statistic.
LI was observed in 43% (108 of 251) of the study melanomas. The multivariate model for ten-year metastasis identified 4 independent prognostic factors: tumor thickness, mitotic rate (MR), LI, and anatomic site. The prognostic tree identified a group of patients with thin (≤1 mm thick) melanomas and poor prognosis: stage IB melanomas with LI. Survival curves for time to first metastasis demonstrated significantly poorer prognosis for patients with LI compared to those without it for both stages IB and IIA.
LI is common across the range of tumor thicknesses in primary VGP melanomas. It is an independent prognostic factor and significantly increases the risk of metastasis in patients in clinical stages IB and IIA.
We report the results of an association study of melanoma based on the genome-wide imputation of the genotypes of 1,353 cases and 3,566 controls of European origin conducted by the GenoMEL consortium. This revealed a novel association between several single nucleotide polymorphisms (SNPs) in intron 8 of the FTO gene, including rs16953002, which replicated using 12,313 cases and 55,667 controls of European ancestry from Europe, the USA and Australia (combined p=3.6×10−12, per-allele OR for A=1.16). As well as identifying a novel melanoma susceptibility locus, this is the first study to identify and replicate an association with SNPs in FTO not related to body mass index (BMI). These SNPs are not in intron 1 (the BMI-related region) and show no association with BMI. This suggests FTO’s function may be broader than the existing paradigm that FTO variants influence multiple traits only through their associations with BMI and obesity.
Sunbed/sunlamp use was recently classified as carcinogenic. This report considers characteristics of those who use sunbeds/sunlamps and the effect of sunbed/sunlamp use on their risk for melanoma within a large case-control study carried out in 1991–2. Females were more likely than males to have used sunbeds/sunlamps. Use by females increased strongly and significantly with younger ages and with the perceived ability to tan. For females the individual risk for melanoma increased with typical session time and frequency of sessions. Use before age 20, current use and years of use were not significant. The use patterns of occasional and frequent users were very different. We estimate that typical 5 minute sessions would increase the risk for melanoma by 19% for frequent users (10+ sessions) and by 3% for occasional users (1–9 sessions). Body sites that are not generally exposed to sunlight were more common sites of primary melanomas for frequent sunbed/sunlamp users. For males, measures of sunbed/sunlamp use were not significantly associated with melanoma risk.
sunbeds/sunlamps; risk factor; melanoma; UVR; dysplastic nevi
Regression in the radial growth phase (RGP) of primary cutaneous melanomas is common and has been shown to be an adverse prognostic factor. However, the underlying mechanism is unclear. We performed dual immunohistochemical staining of podoplanin and S100 on paraffin tissues from 321 patients with vertical growth phase (VGP) primary melanomas who had 10 years or more of follow-up. Lymphatic density (LD) and Lymphatic invasion (LI) was quantified and documented. The time to first metastasis and melanoma-specific death from the date of definite treatment was analyzed using univariate and multivariate Cox models. Among the 116 VGP melanomas that had regression in the adjacent RGP, 75 (23%) were classified as complete and 41 (13%) as partial. LD was significantly higher (p<0.001) in the 75 lesions with complete regression (mean ± SD, 23.7 ± 12.3/mm2) compared to the 41 with partial regression (15.5 ± 7.1/mm2) and was lower in 155 areas of adjacent normal dermis (7.3 ± 3.5/mm2) and 69 areas of distant normal dermis (5.5 ± 2.6/mm2). Patients whose lesions had areas of complete regression with LI and either high or low LD or had no LI with high LD, had shorter TFM (HRs = 2.5, 3.8, and 2.5, respectively) and increased risk of melanoma-specific death (HRs=3.1, 1.3 and 3.0, respectively) than those with no LI and low LD or those without areas of complete regression. These data indicate that complete RGP regression is associated with significantly increased LD, and that the adverse prognostic effect of RGP regression is at least partially mediated through lymphangiogenesis and LI in this area.
regression; lymphatic vessel density; lymphatic invasion; melanoma; prognosis
Melanoma is comprised of biologically distinct subtypes. The defining clinical, histomorphologic and molecular features are not fully established. This study sought to validate the association between genetic and histomorphologic features previously described, determine their reproducibility, and association with important clinical variables.
Detailed clinical and histomorphologic features of 365 primary cutaneous melanomas were assessed by 11 pathologists and correlated with mutation status of BRAF and NRAS. There was substantial agreement in the quantitative assessment of histomorphologic features showing similar or better interobserver reproducibility than the established WHO classification scheme. We confirmed that melanomas with BRAF mutations showed characteristic morphologic features (p<0.0001) and metastasized more frequently to regional lymph nodes (p=0.046). Importantly, melanomas without mutations were a heterogeneous group, with a subset having very similar features clinical and morphological features than those with BRAF mutation raising the possibility that they are biologically related.
Our study confirms an association between histomorphologic features, mutation status and pattern of metastasis, providing criteria for a refined melanoma classification aimed at defining biologically homogeneous disease subgroups.
melanoma; BRAF; mutation; classification; histomorphology
A melanoma case-control study was conducted to elucidate the complex relationship between sun exposure and risk.
960 population-ascertained cases, 513 population and 174 sibling controls recruited in England provided detailed sun exposure and phenotype data; a subset provided serum 25-hydroxyvitamin D3 levels.
Phenotypes associated with a tendency to sunburn and reported sunburn at ≥20 years of age were associated with increased melanoma risk (OR 1.56, 95% CI 1.23-1.99). Holiday sun exposure was not associated with an increased melanoma risk although this may be in part because reported sun exposure overall was much lower in those with a sun-sensitive phenotype, particularly among controls. Head and neck melanoma was associated with less sun exposure on holidays at low latitudes (OR 0.39, 95% CI (0.23-0.68) for > 13 hours /year compared to <3.1). Overall the clearest relationship between reported sun exposure and risk was for average weekend sun exposure in warmer months, which was protective (OR 0.67, 95% CI 0.50-0.89 for highest versus lowest tertile of exposure). Serum vitamin D levels were strongly associated with increased weekend and holiday sun exposure.
Sun-sensitive phenotypes and reported sunburn were associated with an increased risk of melanoma. Although no evidence was seen of a causal relationship between holiday sun exposure and increased risk, this is consistent with the view that intense sun exposure is causal for melanoma in those prone to sunburn. A protective effect of regular weekend sun exposure was seen, particularly for limb tumours, which could be mediated by photoadaption or higher vitamin D levels.
To describe associations of MC1R variants and melanoma in a US population and to investigate whether genetic risk is modified by pigmentation characteristics and sun exposure measures.
Melanoma patients (n=960) and controls (n=396) self-reported phenotypic characteristics and sun exposures via structured questionnaire and underwent a skin examination. Logistic regression was used to estimate associations of high [R] and low [r] risk MC1R variants and melanoma, overall and within phenotypic and sun exposure strata. A meta-analysis of results from published studies was undertaken.
Carriage of two [r] or any [R] variant was associated with increased risk of melanoma (odds ratio (OR) = 1.7; 95% CI, 1.0-2.8; OR=2.2; 95% CI 1.5-3.0, respectively). However, risk was stronger in or limited to individuals with protective phenotypes and limited sun exposure such as those who tanned well after repeated sun exposure (OR=2.4; 95% CI 1.6-3.6), had dark hair (OR=2.4; 95% CI 1.5-3.6), or had dark eyes (OR=3.2, 95% CI 1.8-5.9). We noted this same pattern of increased melanoma risk among persons who did not freckle, tanned after exposure to first strong summer sun, reported little or average recreational or occupational sun exposure, or reported no sun burning events. Meta-analysis of published literature supported these findings.
These data indicate that MC1R genotypes provide information about melanoma risk in those individuals who would not be identified as high risk based on their phenotypes or exposures alone.
melanoma; melanocortin-1 receptor; pigmentation phenotype; genetic variation
A cohort study was carried out to test the hypothesis that higher vitamin D levels reduce the risk of relapse from melanoma.
A pilot retrospective study of 271 patients with melanoma suggested that vitamin D may protect against recurrence of melanoma. We tested these findings in a survival analysis in a cohort of 872 patients recruited to the Leeds Melanoma Cohort (median follow-up, 4.7 years).
In the retrospective study, self-reports of taking vitamin D supplements were nonsignificantly correlated with a reduced risk of melanoma relapse (odds ratio = 0.6; 95% CI, 0.4 to 1.1; P = .09). Nonrelapsers had higher mean 25-hydroxyvitamin D3 levels than relapsers (49 v 46 nmol/L; P = .3; not statistically significant). In the cohort (prospective) study, higher 25-hydroxyvitamin D3 levels were associated with lower Breslow thickness at diagnosis (P = .002) and were independently protective of relapse and death: the hazard ratio for relapse-free survival (RFS) was 0.79 (95% CI, 0.64 to 0.96; P = .01) for a 20 nmol/L increase in serum level. There was evidence of interaction between the vitamin D receptor (VDR) BsmI genotype and serum 25-hydroxyvitamin D3 levels on RFS.
Results from the retrospective study were consistent with a role for vitamin D in melanoma outcome. The cohort study tests this hypothesis, providing evidence that higher 25-hydroxyvitamin D3 levels, at diagnosis, are associated with both thinner tumors and better survival from melanoma, independent of Breslow thickness. Patients with melanoma, and those at high risk of melanoma, should seek to ensure vitamin D sufficiency. Additional studies are needed to establish optimal serum levels for patients with melanoma.
Here we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E and D594G-mutated melanomas were found to exhibit constitutive levels of pERK, low levels of pMEK and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis, associated with mitochondrial depolarization and relocalization of AIF, whereas the BRAF-V600E mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal via CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2, and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.
melanoma; BRAF; CRAF; targeted therapy; sorafenib
Melanocortin-1 receptor (MC1R) variants have been associated with BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations in non-CSD (chronic solar-damaged) melanomas in an Italian and an American population. We studied an independent Italian population of 330 subjects (165 melanoma patients and 165 controls) to verify and estimate the magnitude of this association and to explore possible effect modifiers. We sequenced MC1R in all subjects and exon 15 of BRAF in 92/165 melanoma patients. Patients with MC1R variants had a high risk of carrying BRAF mutations in melanomas (odds ratio (OR) = 7.0, 95% confidence interval (CI) = 2.1–23.8) that increased with the number of MC1R variants and variants associated with red hair color. Combining these subjects with the originally reported Italian population (513 subjects overall), MC1R variant carriers had a 5- to 15-fold increased risk of BRAF-mutant melanomas based on carrying one or two variants (P<0.0001, test for trend), and regardless of signs of chronic solar damage. In contrast, no association with BRAF-negative melanomas was found (OR = 1.0, 95% CI = 0.6–1.6). No characteristics of subjects or melanomas, including age, nevus count, pigmentation, and melanoma thickness or location on chronically or intermittently sun-exposed body sites, substantially modified this association, although results could be affected by the small numbers in some categories. This study confirms that the known MC1R–melanoma risk association is confined to subjects whose melanomas harbor BRAF mutations.
The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer.
These four features were examined in 385 families with ⩾3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents.
Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, ⩾2 patients with MPM, median age at melanoma diagnosis ⩽40 years and ⩾6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only ⩾1 patient with MPM and age at diagnosis ⩽40 years simultaneously predicted the mutation risk.
The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer–CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.
; multiple primary melanomas; pancreatic cancer
Recent studies have shown that there is a considerable heterogeneity in the response of melanoma cell lines to MEK and BRAF inhibitors. In the current study, we address whether dysregulation of cyclin-dependent kinase 4 (CDK4) and/or cyclin D1 contribute to the BRAF inhibitor resistance of melanoma cells. Mutational screening identified a panel of melanoma cell lines that harbored both a BRAF V600E mutation and a CDK4 mutation: K22Q (1205Lu), R24C (WM39, WM46, and SK-Mel-28), and R24L (WM902B). Pharmacologic studies showed that the presence of a CDK4 mutation did not alter the sensitivity of these cell lines to the BRAF inhibitor. The only cell line with significant BRAF inhibitor resistance was found to harbor both a CDK4 mutation and a CCND1 amplification. Array comparative genomic hybridization analysis showed that CCND1 was amplified in 17% of BRAF V600E–mutated human metastatic melanoma samples, indicating the clinical relevance of this finding. As the levels of CCND1 amplification in cell lines are lower than those seen in clinical specimens, we overexpressed cyclin D1 alone and in the presence of CDK4 in a drug-sensitive melanoma line. Cyclin D1 overexpression alone increased resistance and this was enhanced when cyclin D1 and CDK4 were concurrently overexpressed. In conclusion, increased levels of cyclin D1, resulting from genomic amplification, may contribute to the BRAF inhibitor resistance of BRAF V600–mutated melanomas, particularly when found in the context of a CDK4 mutation/overexpression.
HMB45 is a mouse monoclonal antibody raised against Pmel17/gp100, a melanoma-specific marker, which is routinely used in the diagnosis of primary cutaneous malignant melanoma. The standard expression pattern for a positive HMB45 staining result on immunohistochemistry is based upon the results of chromogenic-based methods. We re-evaluated patterns of HMB45 staining across the 480-core “SPORE melanoma progression array” containing lesions representing the spectrum of melanocytic lesions ranging from thin nevus to visceral metastasis using the fluorescence-based staining technique and Automated Quantitative Analysis (AQUA) of the obtained digital images. The methods validated the expected cytoplasmic HMB45 staining pattern in 70/108 malignant lesions and in the epithelial components of nevus specimens. However, the fluorescence-based approach revealed a nuclear HMB45 localization present in the dermal component of all nevi that was not seen before. This nuclear localization could not be observed on routine chromogenic stains because the standard hematoxylin nuclear counterstain overwhelms the weak nuclear HMB45 stain. The thin (0.450±0.253) and thick (0.513±0.227) nevi had strongly positive mean ln(nuclear/non-nuclear AQUA score ratios), which are significantly higher than those from the group of malignant lesions (p<0.0001). This finding was reproduced on a smaller but independent progression array composed of nevi and melanomas from the Yale Pathology archives (p<0.01). The odds ratio associated with a sample being a nevus was 2.24 (95% CI: 1.87-2.69, p<0.0001) for each 0.1 unit increase of the ln(nuclear/non-nuclear AQUA score ratio) to yield an ROC curve with 0.93 units of area and a simultaneously maximized sensitivity of 0.92 and specificity of 0.80 for distinguishing benign nevi from malignant melanomas. Based on this preliminary study, we propose that the ratio of nuclear to non-nuclear HMB45 staining may be useful for diagnostic challenges in melanocytic lesions.
We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totalling 2019 cases and 2105 controls. Using pooling we identified a novel melanoma risk locus on chromosome 20 (rs910873, rs1885120), with replication in two further samples (combined P <1 × 10-15). The odds ratio is 1.75 (1.53, 2.01), with evidence for stronger association in early onset cases.
Lymphatic invasion by tumor cells has been noted infrequently in primary melanomas. Our primary hypotheses were that using immunohistochemical markers of lymphatic vessels and of tumor cells would improve detection of lymphatic invasion and that lymphatic invasion would correlate with regional nodal metastatic disease. This study included 106 patients who were diagnosed between 1972 and 1991 and who had ≥ 10 years of follow up. We performed dual immunohistochemical stains for podoplanin (for lymphatic vessels) and S-100 (for melanoma cells). Lymphatic invasion was identified by light microscopy and confirmed by multispectral imaging analysis. Lymphatic invasion was detected by morphology alone in 5 cases (4.7%) in contrast to immunohistochemical staining augmented by multispectral imaging analysis where 35 cases (33%) were identified (p <0.0001). Lymphatic invasion was significantly associated with time to regional nodal metastatic disease, as well as first metastasis and melanoma-specific death. “Local metastasis”, defined by immunohistochemistry-detected lymphatic invasion, satellites or neural invasion, identified 64% of those who had regional nodal metastatic disease within 5 years of diagnosis. Lymphatic invasion is an under-observed phenomenon in primary melanomas that can be better detected by immunohistochemical staining. The presence of lymphatic invasion may be a clinically useful predictor of regionally metastatic disease.