25-Hydroxyvitamin D₂/D₃ levels and factors associated with systemic inflammation and melanoma survival in the Leeds Melanoma Cohort

In 2009, we reported evidence that lower serum 25-hydroxyvitamin D₂/D₃ levels at diagnosis to be independently associated with poorer outcome from melanoma in the Leeds Melanoma Cohort.1 As vitamin D levels are higher in fitter, healthier individuals,2 the relationship between vitamin D levels and melanoma survival has not yet been established to be causal: vitamin D levels might merely be a marker of healthier lifestyles. In vitro studies have shown evidence that vitamin D has an anti-proliferative effect on a proportion of melanoma cells in culture.3 Vitamin D has pleotropic effects in vivo, however, and importantly is a regulator of innate and adaptive immunity playing a role in the suppression of inflammation.4 We report here an evaluation of the potential role of systemic inflammation (as evidenced by associated factors such as aspirin usage and smoking) and its moderation by vitamin D in melanoma survival.

The role of tumor-related inflammation has been recognized as fostering a number of the “Hallmarks” of cancer as well as a process which can be targeted therapeutically.5 Whilst spontaneous immunological responses to melanoma are known to occur,6 and there is unequivocal evidence that immunologically mediated long term survival can be induced therapeutically,7 there is a growing body of evidence that excessive inflammation promotes cancer metastasis8 in part by suppressing adaptive immunity. Balkwill and colleagues9 have suggested that “smouldering” inflammation aids proliferation and survival of cancer cells, promotes angiogenesis and metastasis, subverts immune responses and alters responses to hormones and therapeutic agents. They also postulated that cancer and inflammation are connected by two pathways: intrinsic (activated by genetic events in the tumor itself) and extrinsic (inflammation in the tumor microenvironment).

There is clear evidence in melanoma that tumors drive inflammation (the intrinsic pathway). Driver mutations such as those in BRAF10 increase secretion of proinflammatory cytokines such as interleukin (IL) 6 and IL-8. There is also evidence in ulcerated tumors (ulceration is a potent poor prognostic factor for melanoma), that gene expression patterns associated with inflammation are increased.11 In our own data, gene expression patterns associated with a “wound healing” response were associated with tumor ulceration (Jewell et al. accepted 2014). That ulceration is associated with increased blood vessel density and increased macrophage infiltration12 suggests that ulceration may be a histopathological marker of considerable tumor-associated inflammation.

Inflammatory responses to melanoma in its microenvironment are therefore postulated to result from oncogenic changes in the tumors themselves and the responsive stromal changes, but in this study we have explored the possibility that systemic inflammation might also play a role in driving tumor progression. We have used data and samples from participants in the Leeds Melanoma Cohort.1

Systemic inflammation in the obesity-related chronic inflammatory syndrome (sometimes referred to as the “metabolic syndrome”) has been reported to increase the risk of cancers other than melanoma such as colorectal cancer13 and postmenopausal breast cancer.14 This syndrome is also associated with lower vitamin D levels.15-17 In fact serum levels of C-reactive protein (CRP) and 25-hydroxyvitamin D₂/D₃ are strongly negatively correlated.18 There is also some support for the view that vitamin D may at least in part moderate the deleterious effects of obesity in terms of the metabolic syndrome19, 20 and related immunological parameters.21, 22

In order to explore the hypothesis that potentially modifiable systemic inflammation reduces survival expectation for melanoma patients we looked for indirect evidence in the form of an association between factors reported to reduce inflammation such as use of nonsteroidal anti-inflammatories and aspirin, and vitamin D. We also looked for an association between co-morbidities associated with systemic inflammation such as diabetes, obesity and smoking in participants in the Leeds Melanoma Cohort. We then investigated the associations between these factors and tumor ulceration at presentation as tumor ulceration has been reported to be associated with inflammatory gene expression signatures in the tumor11 (Jewell et al. accepted 2014).

Material and Methods

Results

Discussion

Low levels of vitamin D are associated with increased death rates in many diseases, but there is limited evidence that supplementation reduces mortality. There is evidence from a meta-analysis of independent randomized clinical trials that supplementation reduces all-cause mortality,24 but individual trials in cancer prevention have to date been unconvincing. Low vitamin D levels are more common in the obese2, 17 and the physically inactive.2 It is therefore possible that the many associations reported between low levels of vitamin D and a variety of diseases may simply reflect low vitamin D levels as a marker of other lifestyle factors associated with disease. Serum vitamin D levels are strongly inversely correlated with serum levels of CRP, which is generally reported to be the most robust biomarker of systemic inflammation.18 This relationship might suggest that the associations between vitamin D and numerous diseases might represent vitamin D levels acting as a marker of inflammation rather than that vitamin D being causally related.25 There are data however, to suggest that vitamin D may also be an active moderator of inflammation and immune processes (reviewed by Guillot et al.4). Evidence for an immunological role for vitamin D includes the observation that seasonal variation in vitamin D levels is reported to be associated with variation in the human blood T-cell compartment26 and vitamin D is shown to regulate proinflammatory cytokine levels in healthy individuals.22 Low vitamin D levels are associated with higher circulating proinflammatory cytokines in HIV positive patients27 with increased CD4+ counts in those treated with vitamin D.28 In rheumatoid diseases there are data to support that vitamin D reduces proinflammatory T-cell subsets TH 1 and TH 17.29

In the Leeds Melanoma Cohort we collected data on exposures and concurrent illnesses associated with systemic inflammation. We have therefore tested the hypothesis that obesity and diabetes might be associated with increased mortality from melanoma and investigated whether vitamin D levels were statistically independently protective. We also tested whether exposure to drugs which have been reported to reduce inflammation, might be independent predictors of reduced mortality. A weakness of the study is that the detail available on usage of drugs such as NSAIDs and aspirin was limited and we hope to improve this over time.

The first question we asked however was whether factors known to be associated with systemic inflammation were associated with primary tumor ulceration, as a marker of inflammation within the tumor. In previous studies, evidence that ulceration of primary melanoma tumors is associated with gene expression and histopathological evidence of a proinflammatory milieu was reported (Jewell et al. accepted).11, 12 In this study we report that factors associated with systemic inflammation, higher BMI, diabetes and smoking were indeed predictive of ulceration of the primary tumor in univariable analysis. The relationship between BMI and diabetes did not persist as independent predictors in multivariable analysis although the ORs remained similar and statistical power issues may be relevant. Given the observation that ulcerated tumors have greater blood vessel density and increased numbers of macrophages12 and gene expression patterns associated with increased release of proinflammatory cytokines,11 this finding gives support to the hypothesis that systemic inflammation might play a role in determining ulceration (and implicit in that, inflammation within the tumor) and that this may in part be a biological explanation for the observation that ulceration is associated with poorer outcome for melanoma patients. Smoking increases evidence of systemic inflammation including CRP levels,30, 31 but a weakness of our study is that we report associations only and smoking could increase the likelihood of tumor ulceration via other mechanisms. Similarly, we can conclude good evidence that vitamin D levels are associated with reduced tumor ulceration but not that the association is causally related to reduction in inflammation. Berwick and Kesler32 previously reported that solar elastosis reported histologically in association with melanoma primaries was associated with a better outcome, which the authors speculated could reflect a protective effect of vitamin D or that chronic sun exposure might be associated with a biologically less aggressive tumor. We have reported previously that weekend sun exposure was associated with reduced melanoma risk,33 but found no evidence of an association between weekend sun exposure and outcome (data not shown).

We report evidence that supports the well-known association of ulceration with MSS in our data but that also that adjustment for vitamin D, smoking and to a lesser degree NSAID usage modulates this association albeit weakly. This suggests that in part at least these factors may influence melanoma survival through having an effect on ulceration. These factors have no effect once the model is adjusted for Breslow thickness, suggesting that the effect these factors may have on ulceration is captured by thickness of the primary.

We postulated that systemic inflammation might increase melanoma-related deaths but we did not show strong evidence that the presence of co-morbidities such as diabetes increased death from melanoma. We report only that diabetes was nonsignificantly associated with melanoma specific death (HR death 1.50, 95% CI 0. 94–2.40, p = 0.09) in a univariable analysis. We actually report evidence that overweight (but not obese) individuals had better survival than individuals in the normal weight range when survival models are adjusted for age and sex and this association persisted when the model is additionally adjusted for site and Breslow thickness, but there was no trend as the effect was not seen in the obese.

We do report however, evidence that increasing vitamin D levels were associated with lower risk of melanoma associated death in multivariable analysis, and that smoking was strongly associated with an increased risk of death. Reduced vitamin D levels and smoking are associated with increased CRP levels34, 35 and the data are therefore consistent with the view that systemic inflammation may play a part in determining melanoma survival. The study cannot be deemed to establish a causal relationship but does suggest that vitamin D and smoking may have a role in the determination of survival from melanoma patients. At the time of writing there are a lack of adequately powered studies to test an effect of smoking on cancer outcome generally, with some support for an effect of prostate cancer36, 37 and conflicting evidence for an effect on breast cancer.36, 38, 39 There was no significant association of NSAIDs with ulceration of the primary or with melanoma specific death. We had limited power to assess this as currently we have data only on ever/never use of NSAIDs and only 186 individuals had taken these drugs. It is clear however that there was no support at all for aspirin as a modifier of survival in this data, although use of aspirin did seem to be associated with reduced risk of ulceration of the primary.

The weaknesses of this study are that it was an association study, and that the effects of diseases such as diabetes and agents such as vitamin D and smoking have many complicated effects on health. Although this study is large at more than 2,000 cases, followed up for more than 7 years there is also insufficient statistical power to explore the small effects of multiple factors. Another weakness is that although more than half of the cases were recruited within 6 months of diagnosis for some cases there is potentially a delay in vitamin D sampling for anything up to 2 years postdiagnosis. Recently diagnosed patients may well alter their UVR exposure to such an extent in this time frame that the sampled serum level is not reflective of the original level at diagnosis.40 We therefore re-ran analyses of vitamin D in subsets of the data where the blood had been sampled no more than 1 year postdiagnosis and saw no material difference in the results (data not shown) suggesting that the delays in blood sampling did not materially affect our results.

Ulceration of the primary has an impact on outcome even for stage III melanoma patients (AJCC staging system41), which implies either that the ulcerated tumors are biologically different even when metastatic or that host factors affect both ulceration status and outcome. That obesity, diabetes, low vitamin D and smoking in this study were related to the presence of ulceration of primary melanomas is of note. It has been hypothesized that the presence of ulceration may be associated with suppression of adaptive immunity as a result of tumor-related inflammation,9 and the multivariable analyses performed here suggest that smoking and low vitamin D levels “explained” a small proportion of the deleterious effect of ulceration on outcome. It is possible therefore that cessation of smoking and correction of low vitamin D levels after diagnosis may be of benefit in terms of MSS, and the data provide some support for the hypothesis that the effects of smoking and vitamin D may be mediated through inflammation. This analysis however provided little evidence that medical conditions known to be associated with systemic inflammation modified MSS expectation, although low vitamin D and smoking remained as predictors of survival. The study therefore provided evidence more for a direct effect of vitamin D and smoking on melanoma survival than a mediation through inflammation as was postulated by Autier et al.25 but this needs now to be explored biochemically.