Conventional cytotoxic drugs are largely ineffective in melanoma. Dacarbazine (DTIC), until 2011 the only standard therapy, has a low objective response rate (<20%) in metastatic disease (7). Numerous other chemotherapeutic agents including temozolomide, taxanes, fotemustine, and platin derivatives have been unsuccessfully trialed as single agents, combinations, or in conjunction with immune-modulators such as IFNα-2b or IL-2 at the cost of significant toxicity (14, 15).

An activating mutation in the BRAF^V600 gene on the long arm of chromosome 7 is found in approximately 50% of melanomas (16, 17). This change results in the constitutive activation of BRAF kinase, likely to promote RAS–RAF–MEK–ERK pathway-induced melanoma cell survival and proliferation. Vemurafinib is a pathway inhibitor drug that inhibits the mutated form of the BRAF kinase. Although originally designed to block the V600E (glutamate for valine) mutated form of BRAF, it can also inhibit other mutant forms such as the V600K (lysine for valine) (7, 18–20). In stage IV disease, vemurafenib has been shown to prolong progression-free survival (12.5 vs. 9.5 months) and OS (13.6 vs. 9.7 months) in comparison to dacarbazine (DTIC) (21). Vemurafenib, the first pathway inhibitor therapy approved for melanoma in 2011, represented the first significant change in outlook for stage IV disease in decades. However, following treatment, the majority of treated patients develop secondary resistance to the drug, thought to be associated with mechanisms overcoming the blockade of BRAF kinase inhibitors and with alternative activation of the MAPK kinase pathway (22). Treatment has also been linked to an enhanced risk of keratoacanthomas, squamous cell carcinomas (SCC), and the development of new melanomas (23–25) implying manipulation of other signaling cascades. Clinical trials are ongoing for BRAF inhibitors in the adjuvant setting and for combination therapies such as with BRAF and MEK inhibitor drugs. Recently, two trials reported that the combination of a BRAF inhibitor and a MEK inhibitor significantly improve progression-free survival (26, 27). Standard clinical evaluation now includes screening for the presence of the BRAF mutation in excised high risk and metastatic melanoma deposits. This test, now used for selecting patients to receive pathway inhibitor treatment, may be considered as a co-diagnostic tool for this treatment.

The identification of new mutations such as alternative BRAF, NRAS, c-kit, GNA11, and GNAQ mutations has led to the development of further kinase inhibitors and a number of these are being assessed pre-clinically and in clinical trials.

The efficacy of immune therapies such as pegylated IFNα-2b and high dose IFNα-2b has been known for a number of years. These adjuvant treatments have shown improved disease-free survival (DFS), relapse-free survival (RFS) but only marginal improvements for OS (14, 28, 29). Their cost and toxicity, however, mean that their routine use is extremely limited. Likewise, high dose IL-2 was, until recently, the only effective therapy in stage IV disease, capable in rare cases of inducing prolonged remission (15). High toxicity confined treatment to a small subset of selected patients and in specialist clinical centers.

Ipilimumab is an IgG1 monoclonal antibody that recognizes the immune checkpoint cell surface molecule CTLA-4 on T cells. The antibody blocks the binding of the immunomodulatory molecule CTLA-4 to CD80/CD86 on the surface of antigen-presenting cells, preventing negative CTLA-4-mediated signals. This can result in universal activation of T cells, including some capable of specifically recognizing melanoma cells. Ipilimumab has shown efficacy in several clinical trials in untreated and treated metastatic patients in combination with a range of therapies such as peptide vaccines and as a mono-therapy (8, 30, 31). Some long-term responses are observed, albeit in a minority of patients and use of this agent is associated with autoimmune-like toxicities, most likely due to universal activation of CTLA-4-expressing T cells. The agent was approved by regulatory authorities in metastatic melanoma in 2011.

Based on the promise demonstrated by anti-CTLA-4 treatment, antibodies against other immune regulatory proteins such as PD-1, PD-L1, CD137, OX40, and CD40 are being investigated (32, 33). Administration of the anti-PD-1 antibody nivolumab demonstrated impressive objective responses in approximately 30% of individuals treated in a phase I trial (34). Combination therapy with nivolumab and ipilimumab in a dosing study demonstrated clinical safety and showed enhanced efficacy that appeared to be superior compared to data published on either antibody alone (35). Hamid et al. reported that the monoclonal antibody lambrolizumab (anti-PD-1) can induce sustained tumor regression in patients that had refractory disease despite ipilimumab treatment (36). Based on early clinical trial data, the anti-PD-1 antibody pembrolizumab was approved for the treatment of advanced melanoma in 2014 (37, 38).

The emergence of these new targeted and immune therapies, their success in only subsets of patient groups, as well as the need to assess patient benefit with combination treatments, all highlight the need to develop a range of tools, not only for prognosis but also to assist prediction and monitoring of treatment responses (39).

The enzyme LDH is a clinical serological biomarker in melanoma. It remains to date the strongest prognostic indicator found to be elevated with tumor burden (67). LDH is mostly released upon cell damage or death, with both phenomena indicating higher tumor burden and disease progression. An increase in serum LDH is, however, not specific to malignancy, but also occurs in many other settings such as hemolysis, infection, infarction, and inflammation. Therefore, the positive predictive value in melanoma is limited by this false-positive rate (68). Recent studies have shown that LDH is less sensitive in early stage disease, but has negative predictive value for metastatic relapse (69–71). It is nonetheless at present a useful and clinically available tool for indication of tumor progression in patients at later disease stages and it is therefore incorporated in the TNM classification (Table 1).

Tyrosinase is part of the biosynthesis process of melanin and is constitutively expressed in melanocytes and melanoma cells. Tyrosinase mRNA levels are detectable in the blood of melanoma patients with advanced metastatic disease detected by nested RT-PCR (42, 43). Initial evaluations revealed that tyrosinase is an independent prognostic marker for tumor progression (42, 43, 72). Samija et al. demonstrated that tyrosine mRNA is associated with a decrease in OS (44). However, high variability in the levels of serum tyrosinase have been reported, most likely due to difficulties in sample processing and the transient presence of metastasizing tumor cells in the blood (43). Therefore, it is not surprising that several studies could not confirm a significant prognostic utility for tyrosinase (73). This includes a recent study that showed no differences in tyrosinase serum levels when comparing patients to healthy volunteers (74).

Growth factors in combination with interleukins are major regulators of inflammatory conditions in the tumor microenvironment. Vascular endothelial growth factor (VEGF) is known to support tumor-associated angiogenesis, and to contribute to inflammatory conditions, which promote immunosuppression and redirection of effective anti-tumoral immunity. Ugurel et al. reported that VEGF was an independent prognostic marker for overall and progression-free survival in their cohort (125 stage I–IV patients) (50). Unfortunately, several subsequent studies could not confirm this finding. Although associations between VEGF and the disease were found, no correlation with disease progression was attained (51, 75).

Osteopontin, a secreted integrin-binding glycol-phosphoprotein, has been described to reduce apoptosis, enhance tumor growth, and be a major component for the recruitment of tumor-promoting stromal cells from the bone marrow (76, 77). In a recent study, Maier et al. demonstrated that osteopontin in combination with S100B can help to differentiate patients who are likely to subsequently relapse and develop metastatic disease from those who do not (55). Yet, the presence of osteopontin is also associated with other medical conditions such as autoimmune diseases and this may translate to false-positive readouts in patients with melanoma.

YKL-40 is a glycoprotein secreted by many cells including cancer cells and by immune cells such as macrophages and activated neutrophils (78–80). The physiological functions of YKL-40 are yet not fully understood and reports of its functionality as an independent prognostic marker vary (41, 57–59). More importantly, immunomodulatory drugs such as IL-2 and IFN-α2b stimulate YKL-40 expression and therefore its use as a biomarker can yield false-negative readouts during treatment (81).

The small protein melanoma-inhibitory activity (MIA) is secreted by melanoma cells and is involved in cell–cell contact by interacting with the extracellular matrix. It is also thought that MIA promotes tumor cell invasion and metastasis (82). Equally, it was reported that MIA has a high sensitivity and specificity compared to other clinically relevant biomarkers such as LDH (59, 69). Elevated levels of MIA correlated with more advanced disease stages, poorer prognosis, and decreased DFS (59, 69, 77, 83). However, different studies using multivariate analysis demonstrated higher rates of false-positive readouts in women, suggesting an alternative source of MIA may exist and that this protein may participate in and be influenced by other biological processes unrelated to malignancy (60, 69, 84).

The family of S100 proteins has been of special interest as diagnostic markers in melanoma over the last decade, since the expression and secretion of these proteins is much higher in malignant compared to healthy tissues (85–87). S100 proteins and in particular S100B were recently found to be elevated in the serum of melanoma patients with these elevated levels being associated with poorer prognosis (63, 64), DFS, and OS (88). In a more recent prognostic study of patients treated with IFN-α2b versus observation in stage II and III patients, investigators showed that S100B is associated with worse OS and distant metastasis-free survival (DMFS). Interestingly, S100B in patients’ sera correlated over time with disease progression, increasing further in parallel to increases in disease burden (62). False-positive results can occur after brain, liver, or renal injury as well as during infectious diseases (89–91). However, data acquired to date point to the merits of further evaluation of S100 as a potential clinical tool.

Interleukin-8 (IL-8), also known as CXCL8, is a chemokine produced by malignant cells and associated with inflammatory responses; it can induce neutrophil chemotaxis and is also thought to be a potent promoter of angiogenesis. A recent study has shown that IL-8 correlated with tumor burden, stage, survival, and response to therapy such as BRAF inhibitors (66). These data show that IL-8, perhaps in combination with other chemokines and cytokines that participate in tumor inflammation, has potential as a prognostic marker and warrants further investigation.

Cyclooxygenases (COX1–3) are a group of proteins that are important modulators in the human body, affecting essential pathways such as the catabolic metabolism. COX1–3 also converts arachidonic acid into prostaglandin. Of this group, COX-2 can be induced in tumor cells (92, 93). Becker et al. showed a correlation between COX-2 staining intensity and Breslow thickness in melanoma (46). Furthermore, Kuzbicki et al. reported a higher COX-2 staining intensity in melanoma lesions compared to benign nevi (45).

The Galectin-3 molecule is mainly secreted by inflammatory cells and is associated with both tumor progression and metastasis in melanoma (94). However, using a multifactorial Cox regression analysis, Brown et al. showed an inverse association between galectin-3 with tumor size (thin tumors had more galectin-3) and improved OS (56).

Matrix metalloproteinases play an important role in remodeling the tumor tissue microenvironment as they are responsible for proteolytically breaking components of the local tissue architecture, promoting tissue remodeling, and facilitating tumor cell migration (95, 96). For these reasons, the proteins are over-represented in tumor tissues. Nikkola and colleagues showed that MMP-1- and MMP-3-positive melanoma metastases correlate with decreased DFS (47). Furthermore, Rotte et al. confirmed a higher expression of MMP-2 in melanoma when compared to normal and dysplastic nevi. In this study, the levels of MMP-2 expression positively correlated with tumor progression and worse survival (49). It is, however, noteworthy that this study was conducted using tumor tissue microarrays through the application of peroxidase developed with 3,3’-diaminobenzidine (DAB – a brown substrate), making the assessment of MMP-2 difficult to distinguish from melanoma cells in pigmented lesions.

Cancer-associated cell adhesion proteins are proteins that adhere to different cells, show altered expression levels in cancer, and may participate in tumor cell migration, immune cell evasion, and angiogenesis (97). Molecules such as the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) are known to foster interactions between somatic cells and immune cells. It was demonstrated that expression of CEACAM on tumor cells inhibits immune responses and leads to tumor progression (61). Investigation of CEACAM-1 expression suggested that it may be an independent factor to predict the risk of metastasis (98). Studies have also suggested associations between serum levels of cell adhesion molecules with the development of metastasis (99–101).

The chondroitin sulfate proteoglycan 4 (CSPG4), also known as the high molecular weight melanoma-associated antigen (HMW-MAA) or the melanoma-associated chondroitin sulfate proteoglycan (MCSP) is a glycoprotein–proteoglycan complex expressed on the surface of melanoma, glioma, neuroblastoma, certain breast carcinomas, and acute leukemias (102). CSPG4 is thought to play crucial roles in cell adhesion, melanoma migration, and metastasis (103). Although CSPG4 is over-expressed in over 80% of all melanomas, it is found at all disease stages and data so far have not yielded any correlations with disease progression (104). However, a recent study reported that a cytoplasmic form of the protein appears to correspond with response to treatment with a melanoma vaccine, indicating that CSPG4 may potentially be evaluated as a marker of response or progression (105).