Human malignant melanoma (MM) is a life-threatening and highly aggressive tumour with a pronounced propensity to metastasize. The incidence and mortality rates of MM have been increasing worldwide. In Germany, there were an estimated 21,410 new MM cases and 3,042 deaths in 2016, whereas the reported MM incidence among women and men was reported to increase by >4% annually between 2003 and 2013(1). Although the 5-year survival rate for early-stage MM is >90% and drug therapy for metastatic MM has rapidly progressed in recent years, the 3-year overall survival decreases to <60% for advanced-stage MM with distant metastasis (2). Therefore, it is crucial to identify diagnostic markers to enable early identification of disease progression, which may be pivotal in improving survival outcomes for patients with advanced-stage MM. However, only a few clinically useful serological markers are currently available, and the prognostic outcome is mainly determined by tumour thickness, lymph node involvement, ulceration status of the excised primary tumour and identification of distant metastasis. To date, the only biomarker used in the American Joint Committee on Cancer (AJCC) staging system is lactate dehydrogenase (LDH), the most validated and strongest independent prognostic factor for MM with distant metastasis (3). In addition to LDH, S100 calcium-binding protein B (S100B) is another prognostic and diagnostic serum marker in MM patients with distant metastasis for monitoring tumour response to treatment and identifying recurrent disease (4). Several other diagnostic serum factors have been investigated in MM with distant disease, such as melanoma inhibitory activity protein, galectin-3, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9, but none have reached clinical relevance (5). Therefore, there are currently no validated serum markers for identifying MM progression at its earlier stages.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-estradiol, which is generated by catechol-O-methyltransferase (6). Recent studies have revealed that the serum levels of 2-ME are low in non-pregnant women and physiologically increase during pregnancy, reaching a maximum during the last trimester, whereas 2-ME level is suppressed in women with preeclampsia (7). A number of studies suggested that 2-ME is critical for placental homeostasis by regulation of cytotrophoblast differentiation and oxygen tension during normal pregnancy, whereas impaired 2-ME levels have been implicated in the pathophysiology of preeclampsia (8). Interestingly, beside these physiological effects in pregnancy, it has been found that 2-ME exerts anticancer effects by inducing growth arrest or caspase-dependent and -independent apoptosis in a number of tumour cell lines (e.g., melanoma, bladder cancer, leukaemia and ovarian cancer) in vitro and in mouse experiments in vivo (9-12). Additionally, 2-ME has been shown to inhibit angiogenesis by modulating angiogenesis-related genes, such as hypoxia inducible factor-1α (HIF-1α) and VEGF (13,14). Therefore, clinical trials with 2-ME in cancer treatment are underway (15). Furthermore, it has been found that 2-ME levels were reduced in patients with endometrial cancer compared with those in healthy controls (16). In melanoma cell 2-ME was able to inhibit cell growth and to induce apoptosis (17).

In view of this data and with the knowledge, that angiogenesis plays a crucial role in melanoma progression it was hypothesized that 2-ME serum levels could be reduced in patients with malignant melanoma. Therefore, the aim of the present pilot study was to analyse 2-ME serum levels in melanoma patients compared to healthy volunteers (negative control) and pregnant woman (positive control). We analysed whether 2-ME serum levels of patients with MM are correlated with the tumour stage and if they can be used as prognostic/diagnostic serum markers during disease progression. The evaluation of 2-ME as a treatment option in melanoma patients was not addressed in this study.

MM is a potentially life-threatening disease with a poor prognosis in the advanced stages (2). Improvement of survival outcomes of patients with MM largely depends on early detection. To date, there are only two clinically approved serum markers for the monitoring of patients with MM: S100B and LDH. Unfortunately, the prognostic value of both markers is limited and therefore, there is a great need to identify new prognostic biomarkers.

From preclinical data, 2-ME appeared to be a promising molecule to investigate as a prognostic marker in melanoma. It has been demonstrated that 2-ME exerts anti-angiogenic effects through downregulation of VEGF and HIF-1α and induces apoptosis in endothelial cells (13,14,18). It has been reported that 2-ME inhibits cell proliferation by inducing apoptosis and promoting G2/M phase arrest of MM cells (11,17). Additionally, exogenic applied 2-ME has been shown to be effective against MM growth, independent of the BRAF mutational status, and significantly decreased cell proliferation in a 3D skin reconstruction model (11). Based on these data and findings from a recent study on patients with endometrial carcinoma who exhibited reduced endogenous levels of 2-ME compared to healthy subjects, we decided to assess 2-ME serum levels in patients with MM compared to healthy volunteers (negative control) and pregnant woman (positive control) (16). Our intention was to evaluate the potential of endogenously generated 2-ME, as a prognostic serum marker and therefore patients were not exposed to exogenous 2-ME. As expected, the 2-ME serum levels were elevated in pregnant healthy women compared to healthy volunteers and MM patients. The 2-ME serum levels of the MM patients were slightly, but not significantly, lower compared with those of healthy controls. In the present study, 2-ME levels were not found to be correlated with any of the analysed prognostic relevant clinicopathological parameters, such as tumour thickness, AJCC stage, S100B and LDH serum levels.

Although a relatively large number of 78 patients with malignant melanoma in different tumour stages were examined in this study, the tumour subgroups were in part relatively small, so that a further subdivision was not possible. Since no trend could be derived from the analyses so far, it is likely, that further subgroup analyses would not have yielded any additional findings. A further limitation is that the serum levels were determined only at one time point of the disease, regardless of the state of treatment. Future studies should address several time points of the disease and additionally evaluate treatments and tumour burden in order to rule out possible confounders.

In conclusion, these results are the first to indicate that endogenous 2-ME serum levels are not reduced in patients with MM. Therefore, in contrast to the potential treatment effects of exogenously administered 2-ME, endogenous 2-ME serum levels cannot be used as diagnostic or prognostic marker in MM. In our opinion, further investigations should focus on the therapeutic applications of systemically administered 2-ME in patients with MM, rather than the prognostic potential of 2-ME serum levels.