The MDCS is a population-based cohort study, and, therefore, a potential selection bias compared to the general population must be considered [24]. Since participants were>= 45years at baseline, mean age at diagnosis was higher than in the average population. Hence, in light of the fact that high age is often associated with more advanced melanoma [10], the comparatively low proportion of cases with advanced melanoma is somewhat unexpected, and might indicate an increased awareness and tendency to seek medical attention earlier among study participants. In our study, women were significantly younger at diagnosis than men. Although the life-time risk of melanoma is higher in males than in females, a reversed tendency is seen in adolescents and young adults, where the rate of increase of the incidence of melanoma is higher in females than in males [25,26]. This might be explained by the fact that intentional tanning, not least the use of tanning beds, is more frequent in younger women [27-29]. Unfortunately, information on tanning habits is not available for participants in the MDCS.

The observation that Ki67 expression was a significant predictor of an adverse clinical outcome in men but not in women, also when adjusted for other factors, is intriguing. Notably, gender had no modifying effect on the prognostic value of mitotic index, another tumour characteristic reflecting tumour proliferation, and Breslow thickness had a similar impact on survival in both sexes. A prognostic role for Ki67 expression is supported by several studies [30-33], and, moreover, the rate of proliferation has been demonstrated to decrease when melanoma cells enter the dermis, corresponding to the transition from in situ to invasive radial growth, and to increase again with the onset of the vertical growth phase [33]. Information on growth phase was not available for the patients in our study, and Ki67 expression was assessed according to the estimated proportion of all melanoma cells, without further fine-tuning according to lesional compartment, since this would have required analysis of full-face sections. Sex-related differences have been demonstrated in the time course and pattern of melanoma metastasis [14], and our findings also indicate a potential influence of sex hormones on the balance between invasion and proliferation in the earlier phases of melanoma progression. Notably, there was no significant difference between sexes in the distribution or associations of mitotic index, tumour thickness and Ki67 expression, that might explain the differential prognostic impact of the latter in women and men. There were however sex-related differences in the associations of Ki67 expression with ulceration and Clark level, both being strongly associated with Ki67 expression in women but not in men. Moreover, in men, but not in women, ulceration was an independent prognostic factor for both DFS and OS. Of note, ulcerated melanoma has been proposed to constitute a biologically distinct subtype of melanoma [34], and, hence, our findings are of potential interest, as they suggest that the clinical course of this phenotype might be influenced by endocrine factors.

Use of the TMA technique for biomarker studies in melanoma has several limitations, not least related to technical difficulties to obtain qualitative tissue cores from small lesions, thereby creating a selection bias towards larger tumours available for analysis. The majority of melanomas in our study were however thinner than 1mm, also in the TMA cohort, and Ki67 remained an independent predictor of an impaired DFS and OS in men even after adjustment for tumour thickness, thus supporting its prognostic value also in thinner melanomas. Another potential limitation to the TMA technique is that heterogenously expressed markers might not be reliably determined. The only investigative biomarker in this study, Ki67, has been demonstrated to show variable expression in melanoma, depending on the growth phase [33]. We did however check for staining heterogeneity of Ki67 expression by comparing full-face sections and TMA cores from a subset of the tumours and found no obvious difference.

Another limitation to our study is that the comparatively low number of recurrences did not allow for a meaningful analysis of sex-related differences in the predilection of local, regional or distant recurrence. However, while the recurrence rate did not differ significantly between the sexes, the finding of a significantly prolonged survival in women compared to men after the first recurrence is consistent with previous studies [14].