Metastatic melanoma represents one of the most aggressive and deadly forms of skin cancer, characterized by its ability to metastasize rapidly to other organs and tissues. Despite significant advances in the understanding and treatment of melanoma, metastatic disease remains a major clinical challenge with a rather limited prognosis. This article aims to explore advances in the identification and use of serological, histological, microRNA, and circulating tumor cell (CTC) biomarkers in the treatment of metastatic melanoma, providing a comprehensive view of the current situation and future prospects.

In terms of the epidemiology of stage IV melanoma, it stands out as being responsible for the majority of skin cancer-related deaths, despite constituting only a small percentage of all skin cancers. It should be noted that its incidence has increased significantly in recent decades, especially in populations of Caucasian origin. It is estimated that approximately 100,000 new cases of invasive melanoma will be diagnosed in the United States in 2024, with a mortality rate of approximately 8,000 cases per year. The incidence varies geographically, being higher in regions with greater exposure to ultraviolet radiation, such as Australia and New Zealand (1,2).

It should be noted that different risk factors include exposure to ultraviolet (UV) radiation, a family history of melanoma, the presence of dysplastic nevi, and the phenotype of fair skin. Intermittent and intense exposure to sunlight, which causes sunburn, is strongly associated with an increased risk of melanoma. In addition, the use of tanning beds has also been identified as a significant risk factor (2). On the other hand, genetic factors also play a crucial role in melanoma susceptibility. Mutations in genes such as CDKN2A and BRAF are associated with an increased risk of melanoma. In this sense, the CDKN2A gene, which encodes the proteins p16INK4A and p14ARF, is particularly relevant in familial melanoma, while BRAF mutations are common in sporadic melanomas (3,4).

The process of melanoma metastasis involves multiple steps, including local invasion, lymphovascular invasion, and proliferation in the new microenvironment. The ability of melanoma cells to perform these steps is mediated by a variety of molecular and cellular factors, including genetic and epigenetic alterations, interactions with the tumor microenvironment, and the ability to evade the immune system. All of this leads to histological aggressiveness that is quite prominent among all tumors. Despite this, survival from metastatic melanoma has improved in recent years thanks to advances in targeted therapies and immunotherapies. However, long-term survival remains limited. According to recent data, the five-year survival rate for metastatic melanoma is approximately 22.5%, although this figure varies depending on the location of the metastases and the tumor burden. Brain metastases, for example, are associated with a particularly poor prognosis (5,6).

In this sense, the identification of subgroups of patients who may benefit from specific treatments is an active area of research. Factors such as mutational load, PD-L1 expression, and the presence of mutations in genes such as BRAF and NRAS are being studied to better understand the heterogeneity of metastatic melanoma and develop more personalized therapies. Mutations in the BRAF gene, present in approximately 50% of melanomas, have allowed the development of targeted inhibitors such as vemurafenib and dabrafenib, which have shown improvements in survival. In addition, immune checkpoint inhibitors such as ipilimumab, nivolumab, and pembrolizumab have revolutionized the treatment of advanced melanoma by stimulating the body's own immune response against tumor cells (7). Despite these advances, many patients with metastatic melanoma do not respond to current therapies or develop resistance, underscoring the need to identify new biomarkers that can predict response to treatment, monitor disease progression, and develop personalized therapies.

This is why biomarkers play a crucial role in precision medicine, as they allow for better stratification of patients and more effective monitoring of the disease. In the context of metastatic melanoma, serological, histological, microRNA, and circulating tumor cell biomarkers offer a promising area of research and clinical application with the goal of improving diagnostic accuracy, treatment follow-up, and prognosis of patients with metastatic melanoma.

To date, LDH has been the only serological biomarker used to monitor tumor response to treatment, relating increasing values to disease progression. However, it has an important bias since an increase in these levels can also occur due to other causes (8–10).

According to the study carried out by Mancuso et al (8), it was observed that several serological markers have been associated with a worse prognosis in melanoma, and it can be determined that, in terms of tumor activity, high levels of Th2 cytokines [interleukin-4 (IL-4), IL-5 and IL-13)] and decreased levels of Th1 cytokines [IL-2 and interferon γ (IFN-γ)-interferon-γ] will condition the suppression of antitumor immunity.

In other studies such as that of Paganelli et al (11), the behavior of IFN-γ can be studied in depth, demonstrating that patients with an early stage in diagnosis (I–II) had elevated levels of IFN-γ in the blood, which progressively decreased with the metastatic development of the disease. Thus demonstrating the importance of IFN-γ in the antitumor immune response.

It has also been observed (8,11) that the presence of both the proinflammatory cytokine IL-17A and high levels of IL-10 (mainly immunosuppressive activity) and transforming growth factor beta (TGF-β) are associated with a poor prognosis [findings that can also be observed in studies (12–14)].

In this section, it is important to highlight the role of S100 proteins, particularly S100B, which are of special interest for use as diagnostic markers in melanoma due to their higher expression in tumor tissues compared to healthy tissues. Recently, elevated levels of S100B in the plasma of melanoma patients have been associated with a poorer prognosis, underscoring their relevance in assessing disease progression (15–18).

On the other hand, it has been observed that increasing levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) can promote tumor cell migration in vitro and in vivo in multiple cancer types. Conversely, some studies have suggested that GM-CSF has inhibitory effects on tumor progression (19). Despite the lack of consensus on the exact role this factor plays, it has a component in tumor progression, which supports its inclusion in biomarker studies. The role of serum dermcidin (an antimicrobial peptide that can stimulate keratinocytes for the production of cytokines through the G protein and the subsequent activation of protein kinase) as a marker of metastatic progression should be highlighted (8).

Other studies reviewed, such as that of Paganelli et al (11), have corroborated the information described above, identifying the direct relationship between the levels of certain cytokines in the blood with the clinical course of the disease, and there may be a direct association with tumor progression.

Therefore, with the information provided by the different studies reviewed, we can conclude that the joint evaluation of elevated serum levels of IL-4, as well as the decrease in levels of GM-CSF, IFN-γ and DCD would be associated with a worse prognosis of the disease. Despite this, to date there are still no validated serological markers for determining the prognosis of the disease, since the marker that shows the greatest sensitivity in tumor staging is still the Breslow index.

Histologic markers are crucial for diagnosing and classifying melanoma, as well as for assessing its prognosis and determining appropriate treatment. It is important to recognize that immunohistochemistry (IHC) should be interpreted in the context of clinical and histological findings and not in isolation. The following are some key histologic markers used in the study of melanoma, which are summarized in Table I.

Melanocytic lesions encompass a variety of skin tumors, from various benign nevi to malignant melanoma. IHC plays an important role in the treatment of cutaneous melanocytic tumors because the presence of melanin pigment indicates melanocytic differentiation, and in most cases, IHC is unnecessary. However, in difficult cases, IHC helps confirm the diagnosis. In some cases, pigment production may be minimal or absent, which can lead to a misdiagnosis such as carcinoma or poorly differentiated lymphoma. Immunohistochemical markers for melanocytic differentiation, such as Melan A, MART-1 (melanoma antigen recognized by T lymphocyte 1), HMB-45 (black human melanoma 45) (all cytoplasmic), and S-100 (nuclear), are frequently positive in melanocytic tumors but negative in epithelial or mesenchymal tumors (20,21). Among these markers, S-100 is the most sensitive, showing almost universal positivity in melanocytic tumors (22). Melan A and HMB-45 are more specific for melanocytic differentiation but less sensitive. HMB-45 can distinguish between benign and malignant melanocytic lesions; deeper parts of a nevus show reduced expression of HMB-45, while melanoma exhibits uniform positivity (23). It is important to note that some malignant melanoma variants, such as desmoplastic melanomas, are almost always negative for Melan A and HMB-45, but show S-100 expression. Therefore, the use of a combination of melanocytic markers is advisable in complicated diagnostic cases. SOX10, a member of the Sry HMG box (Sox) family of transcription factors, is a crucial transcription factor in the differentiation of neural crest cells. It is a newly described marker for melanocytic tumors, showing strong and diffuse nuclear positivity in these tumors. In reference to MITF, SOX10 directly regulates the expression of MITF, playing a crucial role in melanocyte development and melanoma progression. It is also useful for diagnosing desmoplastic melanoma and differentiating it from other spindle cell and scar cell tumors (24,25). Like other melanocytic markers, SOX10 should be used in conjunction with other markers and not in isolation.

IHC is usually not required to differentiate between benign melanocytic nevi and melanoma. The presence of pigmented nevi cells at various stages of maturation is usually sufficient for diagnosis. However, in certain cases, such as nevoid melanomas, differentiation of nevi such as Spitz's nevus is necessary, which exhibits transepidermal migration and pagetoid dissemination. Proliferative markers may be particularly important in these situations, as melanomas exhibit a much higher proliferation rate compared to nevi. Although there is no universally accepted cut-off point, the mean Ki-67 proliferation rate in Spitz's nevus is usually less than 2%, while in melanomas, the proliferation rate is 10% or more (26).

There are prognostic and predictive markers in melanoma that are crucial in selecting the most appropriate treatment for patients. Melanoma is often characterized by activation of the RAS-RAF-MAP kinase signaling pathway. The BRAF mutation is considered a key initial event in 50–60% of melanoma cases. Detection of the BRAF mutation is of great clinical importance because BRAF inhibitors are used to treat advanced melanoma. The most commonly seen BRAF mutation in melanoma is BRAF V600E. Genetic sequencing is the gold standard for detecting BRAF mutations. However, a commercially available antibody (VE1 clone) can now detect this specific mutation with high sensitivity and specificity (27). Tumor-infiltrating lymphocytes (TILs) are important for tumor immunosurveillance, as they can inhibit tumor growth. The TIL response in melanoma can be classified into three types: absent, non-intense, and intense. Programmed death ligand 1 (PD-L1) expressed by tumor cells can interact with PD receptors on T cells, leading to their inactivation. PD-1 inhibitors, such as pembrolizumab, have been introduced as a treatment for metastatic melanoma as part of immunotherapy. The use of this drug requires prior detection of PD-L1 expression by tumor cells and TILs, as it predicts response to treatment (28,29). IHC is essential for detecting PD-L1 expression, although staining patterns and diagnostic criteria vary across clones (30).

Therefore, proper application and interpretation of histologic and immunohistochemical markers, combined with clinical and histologic findings, are vital for accurate diagnosis, prognosis, and treatment selection in the treatment of melanoma. The continued evolution and integration of these markers into clinical practice promises significant improvements in the outcomes of melanoma patients (30).

Table I presents a summary of the main histologic biomarkers used in the diagnosis, surveillance, and prognosis of metastatic melanoma. It includes details on the type, usefulness and references of each biomarker, providing a comprehensive overview of clinical and research applications (30).

It should be noted that there is a relationship between genetic, epigenetic, and environmental factors that justify the increase in the incidence of cutaneous melanoma since the 1960s (31,32). Currently, phenotypic characteristics, such as fair skin, freckles and red hair (phototypes I and II) are identified as risk factors for the development of this neoplasm. Likewise, ultraviolet radiation (UVR) represents a direct risk factor for direct DNA damage as it induces several mutations in the different molecular pathways, thus conditioning the development of one of the types of melanoma. These processes are factors that facilitate the appearance of malignant melanoma and, therefore, currently represent a public health problem (31,33).

Exposure of the skin to ultraviolet radiation has positive and negative effects. Among the effects of exposure to ultraviolet radiation are the regulation between the local neuroimmunoendocrine system and central homeostasis. Among the mechanisms proposed to reinforce this conception is what is stated in Slominski et al (34), where it is proposed that ultraviolet radiation, both UVA and UVB, produce physicochemical changes (at the level of pH, ions, reactive oxygen species and reactive nitrogen species) and that these changes could induce the activation or alter the functioning of sensory nerve endings, resulting in the transmission of signals to the brain or stimulation of the sympathetic nervous system (SNS) or autonomic nervous system (ANS), resulting in reflex-based responses.

If ultraviolet exposure is not controlled, as Cole et al (35) commented, these physicochemical changes will accumulate in skin cells and trigger cascades of signaling pathways that cause alterations in collagen homeostasis, being the link with skin aging, thus leading to immunosuppression (35–38).

A result of ultraviolet radiation is that, when absorbed by DNA, it will carry out the production of pyrimidine cis-syn cyclobutane (CPD) dimers (39) as well as the photoproduct 6–4PPs (40), leading to oxidative stress (37). The accumulation of DNA damage can lead to mutations in key genes to maintain homeostasis, such as the p53 gene, which acts as a tumor suppressor (41).

Dysregulated melanogenesis, together with the depletion of essential cellular antioxidants and the production of reactive oxygen species (ROS), as well as the direct influence of quinone and semiquinone intermediates on RNA, DNA, and regulatory proteins, will result in an environment conducive to mutations that favors melanomagenesis (42,43).

To understand melanogenesis, it is essential to consider melanin as a pigment whose production is regulated by exposure to ultraviolet radiation, hormones, and cytokines (42). As a result, it should be noted that in addition to its protective role against UVR, melanin may play a role in the malignant transformation of melanocytes (44,45). There are two main types of melanin: eumelanin and pheomelanin (46,47). While eumelanin offers radiation protection and photoprotection by acting as an antioxidant and an efficient sunscreen, pheomelanin, due to its lower stability in the face of light, can create an environment prone to mutations after exposure to shortwave ultraviolet radiation (44,48,49).

According to the 2018 WHO classification, we can find three types of melanoma: those associated with cumulative sun damage (CSD), those not associated with cumulative sun damage, and nodular melanoma (50). Melanoma pathways associated with CSD include superficially disseminated, lentigo maligna, and desmoplastic melanomas, with a subdivision between high sun damage and low sun damage based on histopathological findings (38PO). Melanomas not associated with CSD are subclassified into spitzzoid, acral, mucus, and uveal melanomas and melanomas arising in congenital and blue nevi. This new classification describes in more detail the mutagenic changes found in the formation of melanoma.

Each subtype of melanoma is the result of the evolution of a precursor pathway (e.g., mutations in BRAF and NRAS are not accepted as features of the ultraviolet light-induced mutation (32,50). Each precursor has a variable risk of progression. It should be noted that nodular melanoma is classified in isolation in the new WHO classification because it can originate from any precursor pathway (51).

The role of fibroblasts in the evolution of melanoma has been reviewed, noting that the removal of reactive oxygen species interferes with the accumulation of HIF-1 in fibroblasts associated with hypoxia and reduces the expression of IL-6, VEGF-A and SDF-1. This phenomenon suggests that hypoxia-driven oxidative stress plays a crucial role in regulating the angiogenic and inflammatory response during melanoma progression (52).

Therefore, molecular and genetic markers have been postulated as a great tool for the diagnosis and targeted treatment of melanoma, facilitating the development of new lines of treatment (53). All these biomarkers are summarized in Table II.

Circulating tumor cells (CTCs) are cancer cells that break off from a primary tumor and travel through the bloodstream or lymphatic stream. They are markers of cancer spread and can be detected in peripheral blood. Studies of CTCs in melanoma in recent years have made it possible to assess tumor progression, disease recurrence, and response to treatment without the need for tumor biopsies (83).

Cell surface-specific melanoma-associated antigens (MAAs) are very limited, with limited availability of antibodies specific to these antigens. Analysis of MAA biomarkers by RT-PCR suggests that many CTCs are present in the peripheral blood of melanoma patients. The different levels of expression between MAA biomarkers during follow-up demonstrate the heterogeneity of CTCs. The utility of MAA multi-marker RT-PCR provides clinical information that correlates with disease outcomes in treated and untreated patients. From a clinical point of view, the enrichment of CTCs is crucial to increase the concentration of these cells in peripheral blood samples, facilitating their detection and analysis. Advances in enrichment methods have allowed for standardized and reproducible evaluations of multiple genetic alteration profiles of DNA, mRNA, and MAA. For example, the CELLSEARCH^® system has been approved by the FDA to analyze CTCs in metastatic cancers such as breast, colon, and prostate, using ferrofluids with CD146 antibodies to enrich CTCs and a specific antibody against MAA for detection in melanoma (84).

One study (85) evaluated the detection of CTCs in patients with metastatic melanoma using techniques such as immunocytochemistry, RT-PCR, and digital droplet PCR to analyze both the presence of CTCs and circulating tumor DNA (ctDNA). High heterogeneity was identified among melanoma CTCs, with multiple distinct subpopulations. Detection of CTC in this study was associated with shorter, progression-free overall survival. In addition, correlations were observed between CTC scores and plasma ctDNA concentrations, suggesting that these biomarkers may have prognostic utility.

Assessment of tumor response in patients with advanced melanoma treated with checkpoint inhibitors (CIIs) or combination therapies is a challenge without real-time biomarkers, considering the possibility of objectifying pseudoprogressions in radiological imaging evaluations. A prospective study evaluated whether the molecular profiling of CTCs in blood can facilitate the follow-up of patients with metastatic melanoma during their treatment with CII (86). Panels of mRNA/DNA biomarkers were used in patients with melanoma during treatment, including LDH, CTC-mRNA, and tumor BRAF. These panels allow patients to be stratified into two groups: low-risk and high-risk. High-risk patients had lower disease-free survival and shorter overall survival. Patients who overexpressed beta catenin 1 on RNA sequencing had an increased risk of disease progression in contrast to patients who responded completely to treatment. On the other hand, CTC sequencing allowed the identification of subclinical disease in patients who developed progressive disease during treatment and follow-up.

In a study conducted in China (87), it was investigated whether there was a correlation between the number of CTCs before and after treatment with immunotherapy and BRAF-targeted therapy in patients with melanoma, including 49% in stages III–IV. It was found that an elevated baseline CTC count was associated with deep local invasion, nodal metastases, and distant metastases, as well as poorer overall survival, progression-free survival, and disease-free survival. These findings suggest that baseline CTC count may be an important prognostic and predictive factor in patients with advanced melanoma.

Hoon et al (88) demonstrated the usefulness of CTCs in patients with stage I–IV cutaneous melanoma, positively correlating MAA markers detected by RT-PCR with stage, disease progression, and survival. They highlight the transcription factor associated with microphthalmia (MITF), crucial in the development of melanocytes and the growth of melanoma. The detection of MITF increases with tumor stage and its presence after treatment is associated with lower disease-free and overall survival (89).

Studies such as that of Koyanagi et al (90) have explored the usefulness of CTC screening as a predictor of response to treatment in metastatic melanoma. They evaluated the expression of MAAs mRNA biomarkers (B4GALNT1, MAGEA3, MITF, MLANA, and PAX3) by RT-PCR in a prospective multicenter phase II clinical trial. They observed a decrease in the number of MAA-positive CTCs during treatment, which was associated with improved response and increased survival. Surgical treatment has also been shown to significantly reduce the number of CTCs at all stages of melanoma (I–IV) compared to healthy patients (91).

The analysis of CTCs is constantly expanding thanks to the development of new technologies for their enrichment and detection. Recent advances in molecular profiling have the potential to enrich the information derived from CTCs, thereby increasing their clinical utility for patients with advanced melanoma.

MicroRNAs (miRNAs) were first discovered in 1993 as a group of non-coding RNAs with a length of 21 to 23 nucleotides (92). Since their discovery, miRNAs have become an exciting area of research. In recent years, the value of miRNAs in melanoma according to their tumor inhibitory properties has been studied, classifying them as oncogenes or tumor suppressor genes (93). Importantly, miRNA expression levels have been clinically related to the response rate, efficacy, and side effects of treatments (94). Some key aspects of the involvement of miRNAs in melanoma are described below.

Fibroblasts play a suppressive role in the early development of melanoma. However, with tumor growth, fibroblasts are reprogrammed into cancer-associated fibroblasts (CAFs) to promote tumor progression. Before the invasion of melanoma cells, melanoma cells release some melanocytes into the dermis. These melanoma-derived exosomes contain a large amount of non-coding RNAs that are biologically active. The absorption of these miRNAs by fibroblasts can lead to the formation of CAFs, which are especially important in the formation of the metastatic niche (95).

Therefore, miRNAs can act as oncogenes or tumor suppressors. In this sense, miR-21 is frequently overexpressed in melanoma, which favors cell proliferation and resistance to apoptosis (96), while miR-34a acts as a tumor suppressor (97). On the other hand, some miRNAs, such as miR-10b and miR-182, are associated with the promotion of invasion and metastasis in melanoma cells, facilitating tumor progression to more aggressive stages (98).

It should also be noted that miRNAs are also implicated in resistance to conventional and targeted therapies. For example, miR-200c (99) has been linked to resistance to BRAF inhibitors, limiting the first-line therapeutic arsenal in these patients.

In addition, miRNA expressions in blood or biopsies in melanoma patients have been shown to be closely related to response to immunotherapy treatments, suggesting that miRNAs could serve as practical biomarkers to predict the response rate or survival time of patients treated with immunotherapy (100,101).

miRNAs can modulate the activity of different immune cells. For example, miR-155 is crucial for the function of T cells and macrophages, as it influences the immune response against melanoma. In this context, it has been observed that a high level of miR-155 expression after receiving anti-PD-1 treatment correlates with prolonged overall survival in patients with melanoma (102,103). In addition to miR-155, an elevated level of other miRNAs has been shown to indicate a positive response to PD-1 inhibitors. The expression of miR-100-5p and miR-125-5p has been shown to be positively correlated with the overall survival of melanoma patients treated with PD-1 inhibitors (104). Therefore, the interaction of miRNAs with immune checkpoint inhibitors, such as PD-1 and CTLA-4 inhibitors, is an area of great interest. miRNAs can affect the efficacy of these therapies and also be involved in the appearance of adverse effects related to the immune system.

In summary, miRNAs are crucial components in melanoma biology and have the potential to revolutionize both the diagnosis and treatment of this aggressive disease.

In other sense recent research, including studies by Scheau et al (105), has significantly expanded our understanding of the neuroendocrine factors involved in melanoma pathogenesis, highlighting their roles in directly influencing tumor cell proliferation and metastasis or indirectly through modulating immune and inflammatory processes that impact disease progression. This emerging evidence suggests that neuroendocrine factors, including neurotransmitters like catecholamines and neuropeptides such as alpha-MSH, may offer novel serological markers for melanoma (105,106).

The intricate interactions of melanoma cells with the neuroendocrine system, as described in Scheau's comprehensive review, underscore a complex network where melanoma cells not only respond to neuroendocrine signals but also produce these factors to modulate their microenvironment. For instance, the production of CRH and ACTH by melanoma cells can lead to systemic effects, potentially altering the HPA axis and influencing systemic immune responses. This phenomenon could explain the elevated levels of certain hormones and neurotransmitters in patients with advanced melanoma, which might serve as potential biomarkers for disease status and progression (105,106).

Moreover, the ability of melanoma to drive systemic changes through neuroendocrine manipulation suggests that these markers could be pivotal for understanding the tumor's influence on systemic disease mechanisms.

However, in line with previous references to serological biomarkers, the study of serological and neuroendocrine biomarkers opens up a wide horizon. The reviewed studies observed that cross-evaluating the levels of serological markers alongside the Breslow index offers greater prognostic value in staging metastatic melanoma. Integrating these insights not only aids in the detection and monitoring of melanoma but also enhances our understanding of its systemic impact. This holistic approach is crucial for developing more precise and effective management strategies for patients with this aggressive cancer.

Treatment of metastatic melanoma remains a significant challenge due to its aggressive nature and ability to spread rapidly to other organs. Although advances in targeted therapies and immunotherapies have improved the outlook, the long-term survival rate remains low. However, the identification and use of various biomarkers offer a promising avenue to improve the diagnosis, follow-up and personalized treatment of this disease.

Serological biomarkers, such as LDH and certain cytokines, have been shown to be useful in providing information about tumor progression and response to treatment. However, more research is needed to validate its clinical application routinely. On the other hand, histological biomarkers, such as Melan-A, HMB-45, and S-100, are critical in the diagnosis and classification of melanoma, as they help distinguish between benign and malignant lesions and identify specific subtypes.

Similarly, microRNAs and circulating tumor cells (CTCs) have shown great potential to improve the treatment of metastatic melanoma. MicroRNAs can act as oncogenes or tumor suppressors, influencing disease progression and response to treatment. While CTCs allow tumor progression and response to treatment to be evaluated without the need for invasive biopsies, representing a less invasive tool for continuous patient monitoring.

In summary, advances in the identification and utilization of serological, histological, microRNA, and CTC biomarkers represent a very promising area of research and clinical application. These biomarkers are essential for improving the diagnosis, follow-up, and prognosis of metastatic melanoma, and have the potential to offer more precise and personalized treatments, significantly improving the approach to patients with metastatic disease.