LDH catalyzes the conversion of pyruvate to lactate in hypoxic or anoxic conditions. An elevated level of LDH is believed to be due to spillage into the bloodstream when melanoma cells outgrow their blood supply [4]. High levels of LDH are associated with worse prognoses, independently of site or number of metastases [28]. In the American Joint Committee on Cancer (AJCC) melanoma staging system, patients with distant metastasis and elevated LDH levels are considered stage IV M1c [4]. Patients with stage IV disease and normal serum LDH at initial staging have 1-year OS of 65% and 2-year OS of 40%. With elevated LDH levels, 1-year and 2-year OS are 32 and 18%, respectively [4]. Apart from its prognostic value, in patients treated with a combination of dabrafenib and trametinib, LDH was shown to be associated with poorer outcomes [29]. Moreover, when LDH decreases by more than 27.3% from the baseline, this has been associated with radiological response to immunotherapy [30].

S100 proteins are implicated in a multitude of cell functions. As early as the 1980s, S100 β was found to be expressed in human melanoma cell lines and was proposed as a marker that could aid in diagnosis of melanoma [31].

However, S100 β can be found in abnormal levels in many pathological conditions, including liver, brain and renal injury, inflammatory and infectious processes [32].

In 2008, Mocellin et al. published a meta-analysis of 22 series, with a total of 3393 patients with malignant melanoma at all stages. This revealed that positive serum 100B was associated with reduced survival (hazard ratio [HR]: 2.23; connfidence interval [CI]: 1.92–2.58; p < 0.0001) [33]. Abraha et al. displayed a correlation between serum S100B levels and Breslow thickness. Serum S100 β > 0.2 μg/L and primary melanoma tumour thickness > 4 mm combined had sensitivity of 91% and specificity of 95% as predictors for disseminated disease, and consequently may inform prognosis at the point of diagnosis [34]. However, this was not confirmed in other studies of multivariate analyses, where levels of serum S100 β did not show clinical prognostic value [35]. Overall, the evidence for routine use of serum S100 β as a prognostic marker in melanoma is limited. This is due to small sample sizes, lack of proven significance in multivariate studies and mismatch of disease stages across studies [33]. However, measurement of serum S100 β in patients with Breslow > 1 mm lesions is recommended every 3–6 months in German and Swiss guidelines [36,37].

Melanoma inhibitory activity (MIA) is secreted by melanoma cells and is a regulatory growth factor [38]. MIA was proposed as a melanoma biomarker, because it is not expressed in benign human melanocytes or benign melanocytic nevi, but is strongly expressed in malignant melanoma cells [39]. Higher levels of MIA were linked with more advanced stages of melanoma and worse prognosis [5]. This was first shown in a German study that included over 830 blood samples of 326 patients with malignant melanoma of all stages. The cutoff was set at 9.8 ng/mL. In stage I and II patients, elevated MIA concentrations were found in 5.6%. This increased to 60% in patients with stage III and 89.5% in stage IV melanoma. Patients at stage III or IV that underwent resection or treatment with irradiation or chemotherapy prior to the study had MIA levels below the cutoff. Notably, all patients with reduced MIA levels in all stages did not develop further metastases during the follow-up period. In patients displaying a significant increase in MIA levels, metastases were detected either at the point of analysis, or in the following 2–6 months [40]. A comparative study in 373 melanoma patients among MIA, S100B, LDH and albumin showed that MIA was not superior to the use of S100 β or LDH; specifically, S100 β had the higher sensitivity (0.86) in newly diagnosed metastatic melanoma while MIA had the second highest (0.80); LDH sensitivity was lower at 0.48 and albumin lowest at 0.15. However, MIA had the lowest specificity (0.62), whilst albumin had the highest (0.99) [6].

VEGF is elevated in patients with advanced-stage melanoma. This was associated with negative immune effects, such as impaired dendritic cell function [41,42]. It was also linked with both elevated and decreased T helper 2 (Th2) cytokines. These were found to result in suppression of effective antitumour immunity. VEGF inhibitors can lead to improved dendritic cell function and reverse Th2 dominance, leading to Th1 polarity. These changes should in theory enhance tumour rejection [43]. Ugurel et al., in a study including 125 patients with stages I-IV melanoma, concluded that VEGF was found to be an independent prognostic marker for OS [44]. However, VEGF has not been found to be effective as a marker of disease progression. This was replicated in a study in 2005, although healthy individuals were found to have higher VEGF levels [45]. When used to monitor patients, VEGF potentially has high negative predictive value (90%) with low sensitivity, specificity and positive predictive value of 57.1%, 78% and 34.5% respectively.

Apart from those already discussed, there is a multitude of other promising serum biomarkers. Tumor associated antigen 90 immune complex (TA90IC) and its utility in melanoma has been indicated in several studies. In a comparative study between TA90IC, MIA and S100 β in stage III melanoma patients undergoing adjuvant immunotherapy, TA90IC was the earliest elevated marker and an independent predictor for survival and recurrence of melanoma [15]. Further studies support this, indicating that antiTA90 IgM can be an independent prognostic factor for melanoma [46]. The expression of TA90IC can be elevated in inflammatory processes, such as hepatitis with liver cirrhosis [15]. Tyrosinase is a marker specific for melanocytes and Schwann cells, which are normally not found in peripheral blood [47]. Several studies have been performed, with conflicting results. Some have shown that the presence of microRNAs (miRNA) in tyrosinase correlates with melanoma relapse progression [48,49]. However, variable levels of miRNA in stage III and IV melanoma indicate that blood tyrosinase level is not a dependable marker in metastatic disease [50,51]. Osteopontin’s role in cell death, tumour cell growth and recruitment of tumor promoting stromal cell has also been described [52,53,54]. In Maier et al., it was shown that a combination of S100 β and osteopontin may correlate with disease relapse and help identify patients at high risk of metastasis [55]. However, osteopontin can also be elevated in several autoimmune conditions. Interleukin-8 is a chemokine associated with inflammatory processes; it has been shown to promote angiogenesis and correlate with disease stage, survival, tumour burden and response to treatment [56]. Melanoma Antigen Gene A3 protein (MAGE-A3) is part of a family of proteins whose genes are normally silent, except in male germline cells. This is not the case in melanoma and other tumours though [57]; indeed, elevated levels are found in early melanoma stages. Nevertheless, its use as a prognostic factor has yet to be proved [58]. YKL-40 is a glycoprotein secreted by activated neutrophils and macrophages. Elevated levels can be seen in non-malignant diseases, but were also found to be an independent prognostic biomarker for poor survival in breast, lung, colon, ovary and kidney cancers [59]. Their use alone or in conjunction with LDH as an independent prognostic marker was shown by Schmidt et al. [60]. In the same study, serum level of YKL-40 at diagnosis was found to be an independent factor for survival [61]. However, YKL-40 has yet to receive approval by the Food and Drug Administration (FDA) to be used as a biomarker in the United States [62]. In addition, medications such as IL-2 and IFN-a2b increase YKL-40 expression and can cause false negative results [63]. Cytoplasmic melanoma-associated antigen (CYT-MAA) is produced in normal and tumour cells alike. However, its levels are elevated in melanoma. Although it is not sensitive or specific, it has been linked with disease recurrence and progression, as well as potentially with response to immunotherapy [64]. Melanotransferrin (MTF) is expressed in normal adult, fetal and tumour cells [65]. It is commonly found in exocrine tissues, such as salivary glands and the pancreas, as well as the epididymis [66]. Its exact role in melanoma is not yet known. However, it is thought to contribute to angiogenesis, tumour proliferation and tumour genesis [66,67]. Microphthalmia-associated transcription factor (MITF) contributes to the regulation of melanocytes’ development, differentiation and function [68]. MITF is sensitive and specific in identifying melanoma cells [69]. Its levels are inversely proportional to melanoma cell invasiveness [70]. The identification of MITF after treatment indicates metastatic disease and worse outcomes in melanoma patients [71]. Glycoprotein 100 (gp100) is normally expressed in adult melanocytes. However, levels are increased in neonatal cells and melanoma, albeit those levels vary [72]. Despite this, gp100 is not specific and was not proven to correlate with response to treatment [73,74]. Lastly, elevated C reactive protein (CRP) and interleukin 6 (IL-6) levels were found to be linked to reduced survival and treatment resistance [74,75]. Despite this, CRP was found to be an independent predictor for survival [76].

Interestingly enough, protein levels can vary significantly between serum and plasma, due to the storage conditions, the method of blood fractionation and the properties of the specific analysed proteins. As such, there are significant discrepancies in the literature regarding protein levels in plasma and serum; nevertheless, it has been shown that plasma has better reproducibility in protein measurement. Table 1 summarises the serum biomarkers that were described here. Among them, at least S100 β, MIA, VEGF, osteopontin, and interleukin 8 (IL-8) are relevant in plasma [77].