Inherited Variation at MC1R and Histological Characteristics of Primary Melanoma

Variation in the melanocortin-1receptor (MC1R) gene is associated with pigmentary phenotypes and risk of malignant melanoma. Few studies have reported on MC1R variation with respect to tumor characteristics, especially clinically important prognostic features. We examined associations between MC1R variants and histopathological melanoma characteristics. Study participants were enrolled from nine geographic regions in Australia, Canada, Italy and the United States and were genotyped for MC1R variants classified as high-risk [R] (D84E, R142H, R151C, R160W, and D294H, all nonsense and insertion/deletion) or low-risk [r] (all other nonsynonymous) variants. Tissue was available for 2,160 white participants of the Genes, Environment and Melanoma (GEM) Study with a first incident primary melanoma diagnosis, and underwent centralized pathologic review. No statistically significant associations were observed between MC1R variants and AJCC established prognostic tumor characteristics: Breslow thickness, presence of mitoses or presence of ulceration. However, MC1R was significantly associated with anatomic site of melanoma (p = 0.002) and a positive association was observed between carriage of more than one [R] variant and melanomas arising on the arms (OR = 2.39; 95% CI: 1.40, 4.09). We also observed statistically significant differences between sun-sensitive and sun-resistant individuals with respect to associations between MC1R genotype and AJCC prognostic tumor characteristics. Our results suggest inherited variation in MC1R may play an influential role in anatomic site presentation of melanomas and may differ with respect to skin pigmentation phenotype.


Introduction
Inherited variation in the melanocortin-1 receptor (MC1R) gene is a robust genetic marker for moderately increased risk of melanoma [1]. We hypothesize that variation in MC1R influences the occurrence of melanomas that can be distinguished by histology or other tumor characteristics. However, evidence supporting a consistent association between MC1R variation and melanoma tumor characteristics is limited. Direct cross-study comparisons are hindered due in part to a lack of standardized measures and characterization of MC1R risk variants [2], coupled with differences in categorization of melanoma characteristics (e.g. collapsing of anatomic site presentation).
To more thoroughly address whether MC1R variants are associated with tumor characteristics, we present results from individuals diagnosed with a first incident primary tumor in a large population-based case-control study of melanoma: the Genes, Environment and Melanoma (GEM) Study. We examined associations between variation in MC1R and American Joint Committee on Cancer (AJCC) established tumor characteristics that are associated with prognosis: Breslow thickness and presence of mitoses and ulceration [3][4][5][6][7][8], as well as with presence of tumor infiltrating lymphocytes (TILs), a purported prognostic factor [9]. We also evaluated other histopathological tumor features for associations with MC1R variation in an effort to further characterize potential etiologic heterogeneity.

GEM Study
The GEM Study is a population-based case-control study that enrolled a large series of individuals diagnosed with a first incident invasive primary cutaneous melanoma. Study participants were identified from eight population-based cancer registries and one hospital center in Australia, Canada, Italy and the United States. Detailed study recruitment methods have been previously described [10,11]. The human research oversight committees at each of the GEM study sites, including those at the British Columbia Cancer Agency, Vancouver, BC, CA; Cancer Care Ontario, Toronto, ON, CA; Centro per la Prevenzione Oncologia, Torino, IT; Memorial Sloan Kettering Cancer Center, New York, NY, US; Menzies Cancer Center, Hobart, TAS, AU; University of California, Irvine, CA, US; University of Michigan, Ann Arbor, MI, US; University of North Carolina, Chapel Hill, NC, US; and University of Sydney, Sydney, NSW, AU, approved the study protocol. Written and signed informed consent was obtained from all participants.
Diagnostic pathology reports were obtained for each participant with a first incident primary melanoma (n = 2,424) from the appropriate ascertainment center, and data corresponding to histological subtype, lesion thickness, and anatomic location of lesion were abstracted. Tumor tissue slides for 2,105 (86.8%) participants with a diagnosis of first incident melanoma were available for centralized pathological review, performed in large part by one of three study pathologists (KB, LF, PG). Standardized pathologic review of slides included evaluation of: histologic subtype, Breslow thickness, Clark level, mitoses, solar elastosis, TILs, presence of satellite lesions, presence of coexisting nevi, presence of pigmentation, evidence of lesion regression, ulceration, and vertical growth phase. Melanomas were classified according to established histopathological criteria [12,13]. Since Breslow thickness was both abstracted from the pathology report and recorded during the centralized pathologic review, the measure corresponding to the deepest reading was chosen to represent the value of most biological relevance. Using a glossy colored guide to aid in differentiating between nevi and other skin lesions, participants were asked to have the nevi on their backs counted by a family member or friend; logistic models were adjusted for this continuous variable. A phenotypic index was derived using data collected from a study participant self-administered questionnaire [14], and was based on: hair color (black or dark brown = 1; light brown or blond = 2; red = 3), eye color (black or brown = 0; all other colors = 1), and relative inability to tan in response to sun exposure (no = 0; yes = 1) [15]. Phenotypic index scores of 1 and 2 indicate relatively darker cutaneous phenotypes and lower phenotypic melanoma risk; an index score of 3 indicates medium phenotypic risk. Hereinafter, we refer to individuals with any of these three scores as having a "sun-resistant" phenotype. Phenotypic index scores of 4 and 5 indicate relatively fairer cutaneous phenotypes and higher phenotypic risks for melanoma, hereinafter referred to as "sun sensitive".

Statistical Analysis
For this report, we include only those GEM participants with first incident primary melanomas who were successfully genotyped for MC1R and who self-reported their race as white (n = 2,160). We used SAS (SAS Institute, Cary, NC) to perform multinomial logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between MC1R variant status and tumor characteristics while adjusting for sex, age at most recent melanoma diagnosis, study ascertainment center, phenotypic index and number of nevi on the back. For tumor characteristics that were modeled dichotomously, results are equivalent to those obtained from a binomial logistic regression model. We also conducted analyses stratified by sunresistant and sun-sensitive phenotypes; we then evaluated the Wald p-value of the interaction term for sun sensitivity by MC1R to assess heterogeneity of effect between sun-sensitive and sun-resistant phenotypes. All statistical tests were two-sided with an alpha level of 0.05.

Results
Overall, tumor characteristics were not associated with genotyping success (data not shown).
In univariate analyses, we compared the distributions of MC1R genotype risk categories across strata of prognostic tumor characteristics including: Breslow thickness and presence of mitoses, ulceration, or TILs. No statistically significant associations were noted among these tumor characteristics. We did observe a statistically significant association between anatomical site and MC1R variant carriage based on low-[r] and high-[R] risk variant carriage (p = 0.002) ( Table 1). Our findings with respect to MC1R variant carriage number were consistent with no association (data not tabulated).
Multivariate analyses are also presented in Table 1. No statistically significant associations were noted among prognostic tumor characteristics. However, our adjusted analyses revealed a strong association between carriage of more than one MC1R [R] variant and melanoma development on the arms (OR = 2.39; 95% CI: 1.40, 4.09) when compared to individuals who developed melanomas on the trunk or pelvis. Associations between MC1R variants and strata of other melanoma tumor characteristics were consistent with no association after adjustment.
Because previous reports have indicated that melanoma risk associated with carriage of high-risk [R] MC1R variants is particularly informative among individuals with darker phenotypic characteristics [21], we explored associations between MC1R variants and the four prognostic tumor characteristics by skin pigmentation phenotype. We noted statistical heterogeneity between individuals with sun-sensitive and sun-resistant phenotypes for the associations between MC1R variants and Breslow thickness (p = 0.03), presence of mitoses (p = 0.03), presence of ulceration (p = 0.04), as well as presence of TILs (p = 0.01) ( Table 2). We observed relatively stronger associations between Breslow thickness and MC1R among sun-sensitive individuals. Similarly, we noted pronounced positive associations between carriage of only [r] variants (vs. carriage of only consensus) and presence of mitoses and ulceration among sun-sensitive individuals, whereas carriage of only [r] variants among sun-resistant participants showed little or no association with presence of mitoses and an inverse association with presence of ulceration. We found carriage of [R] variants was more prevalent among sunsensitive individuals with non-brisk TILs observed in their melanomas compared to sunresistant individuals with non-brisk TILs. In contrast, both [r] and [R] variants were more prevalent among sun-resistant cases with brisk TILs observed in their lesions compared to sunsensitive cases with brisk TILs.

Discussion
The GEM Study provides well-annotated histopathological data for melanomas and complete sequencing of participant DNA at the MC1R locus, which allows for a comprehensive examination of the associations between variants and histopathological tumor characteristics. In this study we report no pronounced or statistically significant main effect associations of MC1R with AJCC accepted prognostic factors of Breslow thickness, presence of ulceration, or presence of mitoses overall. Similarly, we did not observe an association between variation in MC1R and TILs, which were shown to be an important independent prognostic feature of melanoma in GEM [9].
However, we did find a persistent positive association between carriage of more than one [R] variant and melanoma presentation on the arms after adjustment. After stratification by skin pigmentation phenotype, this observed association was limited to individuals with sunresistant phenotypes. There are several previous reports of MC1R variation in association with anatomical site of melanoma, but they generally grouped sites together on the basis of sunexposure before analyses and/or categorized MC1R variants differently, [22][23][24][25][26] and are not directly comparable to this study. We did attempt to draw a comparison between our results and results from a case-control study of sporadic and familial melanoma in a Swedish population [25], which reported an increased association between carriage of 1 MC1R variant and melanoma presentation on the trunk (OR = 1.54; 95% CI: 1.01, 2.37). After recapitulating their coding for anatomic site, MC1R, and other covariates to the best of our ability, we were unable to replicate that finding (data not shown    ORs are adjusted for center, sex, age at melanoma diagnosis, phenotypic index, and total body mole density doi:10.1371/journal.pone.0119920.t001    [27]. Interestingly, prior studies have noted more favorable prognoses among melanomas presenting on the extremities [28,29]. Future studies of variation in MC1R related to anatomical melanoma presentation are necessary to validate our findings. We explored effect modification by phenotypic index only among the prognostic measures of Breslow thickness, ulceration, mitoses, and TILs to limit the potential for false discovery. We observed significant differences between phenotypically sun-resistant and sun-sensitive individuals with respect to all four prognostic tumor factors. Interestingly, sun-sensitive cases demonstrated stronger associations across Breslow thickness, mitoses and ulceration compared to those observed among sun-resistant individuals. These results are thought-provoking considering that it is among individuals with more sun-resistant phenotypes that MC1R has been associated with increased risk for melanoma [21,30]. However, we did note generally stronger associations between brisk TILs and MC1R among individuals with a sun-resistant phenotype compared to sun-sensitive cases. Although associations between MC1R variant carriage and all four prognostic variables were significantly different between phenotypic classifications, we were likely underpowered to detect associations within strata of phenotypic index despite the large sample size available in the GEM Study.
This investigation of tumor characteristics among 2,160 first incident cases of melanoma is the largest such study to examine associations with germline variation in MC1R. A strength of this study is the population-based nature of the parent GEM Study, from which a large number of incident cases were drawn from nine international ascertainment centers, improving generalizability of results to persons of European ancestry living in a variety of climates. Other advantages of this investigation were the centralized histopathological review conducted by expert pathologists and the ability to adjust for the potential impact of skin pigment and number of nevi. However, we do acknowledge the possibility that false positive findings may have arisen due to multiple hypothesis testing and the exploratory nature of associations examined between MC1R variation and tumor factors stratified by phenotypic index; thus, these findings should be validated in larger study populations before more meaningful interpretations can be made.