Cutaneous melanoma (CM) is a particularly aggressive form of cancer that originates from melanocytes, pigment-producing cells derived from the neural crest [1]. Despite representing a mere 4% of all skin cancers, CM accounts for up to 75% of skin cancer-related deaths [2]. However, with early detection and proper intervention, over 90% of the cases could be cured [3].

The pathogenesis of CM is multifactorial, involving both genetic and environmental factors [4]. Ultraviolet radiation (UVR), either from natural light or artificial sources, is the most important environmental risk factor for CM. Additionally, individuals with lighter complexion have the highest risk of developing CM due to lower levels of melanin which make these individuals more likely to develop sunburns. A higher number of nevi are also associated with an increased risk. A familial history of CM further increases this risk, possibly due to shared sun exposure behaviors or hereditary genetic mutations [5].

Patient survival is strongly correlated with an early detection of the disease. Among various prognostic factors, the depth of invasion remains the most critical determinant of survival in numerous studies. Intraepidermal (in situ) melanomas can be cured by excision alone, and thin melanomas have minimal metastatic potential [6]. On the contrary, thick CMs still have very high mortality rates [7].

2. Melanoma Pathogenesis

Ultraviolet radiation exposure is a major risk factor for CM due to the UVR capacity to damage DNA, causing somatic mutations [8]. The exposure can be classified as intermittent or chronic, the latter being mostly occupational. Both these exposure patterns are associated with an increased risk of CM, but it appears that the risk is higher for intermittent exposure ^[8][9][8,9]. This may be explained by the fact that intermittently exposed individuals have lower melanin levels and are more likely to develop sunburns [10]. Nevertheless, there are cutaneous melanomas, such as acral melanomas, which arise in skin that is not exposed to UVR. In this context, according to the 2023 WHO Classification of Tumors, cutaneous melanomas are classified as melanomas arising in sun-exposed skin and melanomas arising in sun-shielded sites (Table 1) [11]. Apart from UVR exposure, hereditary predisposition is another risk factor for cutaneous melanomas. However, familial cases encompass around 10% of all melanomas [12]. In this respect, several high-penetrance genes such as CDKN2A, CDK4, or BAP1 are the most mutated in hereditary melanomas [13]. Individuals with germline mutations in the CDKN2A, a tumor suppressor gene, have a very high lifetime risk of developing CM, this mutation being encountered in up to 40% of melanoma-prone families [12]. Nevertheless, these mutations are relatively rare and are responsible for just around 2% of all CM cases [8]. In addition to these high-penetrance genes, some medium-penetrance genes such as MITF and MC1R are also involved in hereditary CM [12]. Furthermore, MC1R can be considered a “melanoma modifier gene” as it also increases the penetrance of CDKN2A [14]. As hereditary melanomas are relatively rare, most cutaneous melanomas are characterized by a remarkably high burden of somatic genetic mutations ^[15][16][15,16]. Identifying these genetic mutations can serve both diagnostic and prognostic purposes. Genetic testing is particularly useful for the diagnosis of dedifferentiated CMs which lack typical morphological and immunohistochemical features. In such cases, the diagnosis can be established by identifying melanoma-specific mutations [17]. The most frequent mutations in CM affect genes involved in the aberrant activation of the RAS/RAF/MEK/ERK signaling pathway, also known as the mitogen-activated protein kinase (MAPK) pathway, and the phosphoinositol-3-kinase (PI3K)/AKT pathway [18]. These mutated genes include BRAF, NRAS, NF1, PTEN, KIT, TP53, CDKN2A, and TERT ^[19][20][19,20]. The MAPK pathway is involved in the transduction of extracellular signals to the nucleus, thus activating genes that regulate cell proliferation and differentiation ^[21][22][21,22]. This aberrant activation is responsible for several cellular dysfunctions, such as the deregulation of the cell cycle and inhibition of apoptosis ^[21][23][24][21,23,24]. MAPK is the most frequently dysregulated pathway in cutaneous melanoma [25]. Up to 90% of all melanoma cases exhibit an abnormal activation of the MAPK pathway. The second most frequently activated pathway in CM is the PI3K pathway which plays a crucial role in maintaining cellular homeostasis ^[26][27][26,27]. As the MAPK pathway is the most affected in CM, numerous mechanisms contribute to its abnormal signaling, including BRAF mutations ^[18][28][18,28]. Between 37% and 60% of cutaneous melanomas harbor a somatic mutation in this gene, with the highest frequency observed in CM associated with low CSD [29]. The majority of BRAF mutations in cutaneous melanoma are missense, resulting in amino acid substitutions at the valine 600 position. Approximately 80% are V600E mutations (glutamic acid substitution), while 5–12% are V600K mutations (lysine substitution). Less common mutations include V600D (valine to aspartic acid) or V600R (arginine substitution). Additionally, BRAF non-V600 mutations can occur in around 5% of cases [30]. The BRAF gene encodes a protein kinase with three distinct domains: two regulatory and one catalytic. The latter is involved in the phosphorylation of MEK and in maintaining the protein inactive through a hydrophobic interaction. [31]. In the BRAF V600E mutation, the hydrophobic valine residue is substituted by a polar, hydrophilic glutamic acid which induces a conformational change in the catalytic domain, resulting in a constitutively active kinase ^[32][33][32,33]. BRAF non-V600E mutations generally operate through a similar mechanism, enhancing BRAF kinase activity [33]. Acknowledging these mutations is clinically significant for treatment and prognosis. BRAF V600-mutated melanomas can be treated with BRAF/MEK inhibitors, with response rates higher in V600E-mutated cases compared to V600K-mutated cases. Furthermore, even though the evidence is still limited, BRAF non-V600-mutated melanomas may still benefit from BRAF/MEK inhibitors [30]. The second most prevalent cause of aberrant MAPK pathway signaling In cutaneous melanoma is attributed to activating mutations in the NRAS gene. These mutations occur in 15–30% of melanomas and are predominantly missense, most often affecting codon 61 ^[34][35][34,35]. These mutations perpetuate aberrant signaling through both the MAPK and PI3K pathways ^[18][36][37][18,36,37]. It is noteworthy that NRAS and BRAF mutations are generally considered to be mutually exclusive, although co-mutations have been observed in rare instances [37]. NRAS- and NRAS-BRAF-co-mutated melanomas have a less favorable prognosis than BRAF-mutated ones as there are no target therapies for NRAS mutations [17]. Neurofibromin 1 (NF1) is a tumor suppressor gene, mutated in 10–15% of CM, making it the third most common mutation in this pathology ^[38][39][38,39]. NF1 alterations are more frequent in melanomas associated with high CSD. These cases tend to possess a high mutational burden, including a co-occurrence of BRAF or NRAS mutations ^[19][40][19,40]. The NF1 protein serves as a regulator of the RAS family, attenuating downstream RAS signaling [41]. Consequently, loss-of-function mutations in NF1 result in the hyperactivation of NRAS, leading to increased signaling through both the MAPK and PI3K pathways ^{[19][38][39][41]}[19,38,39,41]. Analyzing NF1 mutation status has some prognostic value even though there are no target therapies for NF1-mutated melanomas, but such cases respond favorably to immunotherapy [42]. Moreover, NF1 analysis can offer important diagnostic information as this mutation is particularly common in dedifferentiated lesions which can be difficult to diagnose otherwise [17]. The receptor tyrosine kinase KIT plays a crucial physiological role in the proliferation and survival of melanoma cells, through the PI3K and the MAPK signaling cascades. KIT mutations are found in 2–8% of melanoma cases and are more common in acral melanomas and melanomas associated with low CSD ^[43][44][43,44]. Recognizing these mutations is important as such cases can benefit from tyrosine kinase inhibitors [45]. Mutations in the TERT promoter confer a proliferative advantage to melanoma cells and are common in advanced disease, being associated with a less favorable prognosis. Nevertheless, this mutation could become a potential therapeutic target ^[46][47][46,47]. TP53-mutated melanomas are also associated with advanced disease [46]. Assessing the status of TP53 is important as these mutations have been associated with MAPK inhibitor resistance but they can also become potential therapeutic targets ^[48][49][48,49]. The PTEN gene, a tumor suppressor gene, is commonly dysregulated in the vertical growth phase of melanoma and in metastatic lesions, occurring in 10–30% of cutaneous melanomas ^[18][50][18,50]. PTEN alterations tend to be mutually exclusive with NRAS mutations but often co-occur with mutations in BRAF ^[51][52][51,52]. This co-occurrence has been hypothesized to increase PI3K pathway activation ^[51][52][51,52], mimicking the effects of an NRAS-only activation ^[51][53][51,53]. Additionally, PTEN loss-of-function is involved in acquired resistance to BRAF inhibitors in BRAF-mutated melanomas [54]. As mentioned before, BRAF-mutated melanomas may respond to BRAF inhibitors. However, therapeutic success is often temporary, as patients usually experience disease progression at some point or may even exhibit primary resistance to this target therapy. In this respect, acquired genetic mutations affecting the MAPK and PI3K signaling pathways play a central role in resistance to both chemotherapy and targeted therapies ^[55][56][57][55,56,57]. In this context, targeted PTEN therapy could improve the outcomes of the patients [49]. Having taken everything into consideration, due to this extraordinary genetic heterogeneity of melanomas, a multi-faced diagnostic and therapeutic approach including the identification of molecular biomarkers and genetic aberrations is imperative for optimizing patient outcomes.

3. Emerging Biomarkers in CM

Due to the extraordinarily heterogenous histopathological, immunohistochemical, and molecular landscape of CM, this disease continues to pose important challenges in terms of diagnosis and treatment. Therefore, several new biomarkers have become increasingly studied in recent years in the hope of improving the understanding of CM pathogenesis and management. Unlike immunohistochemical and genetic testing, these emerging biomarkers are expected to improve the early detection and subsequent monitoring of CM in rapid, cost-effective, and non-invasive ways as they can easily be analyzed from blood samples. Furthermore, these new biomarkers may also serve as potential therapeutic targets.

3.1. MicroRNA

MicroRNA (miRNAs) represent non-coding RNAs involved in degrading mRNAs ^[58][96]. They have been increasingly recognized as critical modulators of oncogenic processes, including various stages of cancer progression such as melanoma ^[59][60][111,112]. In melanomas, miRNA dysregulation is involved in promoting cell proliferation, resistance to apoptosis and invasion, angiogenesis, and metastasis ^[61][113]. Additionally, miRNAs have also been associated with resistance to BRAF and MAPK inhibitors ^[62][63][114,115]. In this context, due to their detectability in both intra- and extracellular compartments and their stable levels even in unfavorable conditions, miRNAs have attracted considerable attention as emerging biomarkers in oncology ^[64][116]. At present, miRNA levels can be assessed from various sources, such as resected primary or metastatic tumors, as well as arterial or venous plasma and serum. Importantly, the data derived from these different sources have shown no significant divergence. Depending on the type of cancer under investigation, abnormal miRNA expression profiles have been found to correlate with various disease stages, overall prognosis, tumor recurrence, and potential responsiveness to therapeutic interventions ^[59][65][111,117]. In patients with melanoma, different miRNAs can be either up- or down-regulated, and have been correlated with progression-free survival and overall survival ^[66][67][68][118,119,120]. Interestingly, serum levels of various microRNAs can discriminate between melanoma stages with increased accuracy compared to S100B or LDH ^[69][121]. For instance, miR-137, miR-148, and miR-182 downregulate MITF expression and promote tumor invasion ^[70][122]. miR-221 plasma levels are increased in melanoma patients, and are correlated with stage, recurrence, and disease progression ^[71][123]. Rigg E. et al. demonstrated that miR-146a-5p is overexpressed in melanoma brain metastases and its knockdown results in a reduction of metastatic lesions ^[72][124]. On the contrary, other types of microRNA such as mirR-211, miR-542 3p, or miR-152-3p are downregulated in invasive melanomas ^[73][125]. In vitro studies demonstrated that increasing miR-152-3p expression inhibits the proliferation and invasiveness of melanoma cells ^[74][126]. miR-542 3p is involved in epithelial-to-mesenchymal transition (ETM), and its experimental upregulation inhibited ETM and metastatic spread ^[75][127]. miR-143 also bears anti-tumoral effects as it has been linked to promoting apoptosis and inhibiting the proliferation of melanoma cells ^[76][128]. Similar anti-tumoral effects have been reported for miR-224-5p which can additionally overturn acquired resistance to BRAF inhibitors ^[77][129]. Several other microRNA seem to play a role in resistance to target or conventional chemotherapy. A downregulation of miR-7, miR-579 3p, and miR-126 3p was found in melanomas resistant to BRAF/MAPK inhibitors ^[78][79][80][130,131,132], while miR-31 downregulation is associated with chemoresistance ^[81][133]. Importantly, the experimental upregulation of miR-7 and miR-126 3p restored responses to BRAF inhibitors in melanoma cell lines ^[78][79][130,131].

3.2. Exosomes

Exosomes are extracellular vesicles secreted by cells, encompassing a unique molecular signature that reflects the cell type from which they originate. Given their traceable cellular origins and facile isolation, exosomes can be regarded as potential biomarkers for diagnosis and prognosis in various cancers, including melanoma ^[82][134]. They are readily available through minimally invasive methods, as they can be isolated from a variety of biological fluids such as blood, plasma, urine, and cerebrospinal fluid ^[83][135]. Exosomes extracted from melanoma cell lines have been shown to contain distinct mRNA, miRNA, and protein profiles ^[82][83][134,135]. Exosome analysis can offer important diagnostic and prognostic information, as various exosomal components are significantly altered in cutaneous melanomas ^[82][84][85][134,136,137]. In this respect, Surman M. et al. found increased exosome concentrations in melanoma cases but those levels were not correlated with disease stage ^[82][134]. On the contrary, Boussadia Z. et al. reported a higher exosome concentration in metastatic melanoma compared to non-metastatic cases ^[86][138]. The complex relationship between exosomal components and melanoma progression is not entirely understood, but various mechanisms have been proposed. For instance, exosomes can carry and modulate the activity of matrix metalloproteinases (MMPs), as well as alter cell adhesion and activate fibroblasts to become cancer-associated fibroblasts, thus stimulating melanoma invasiveness ^{[82][87][88][89]}[134,139,140,141]. Exosomal components have also been shown to enhance metastatic potential in CM by promoting epithelial-to-mesenchymal transition (ETM) ^[90][142], angiogenesis ^[82][91][134,143], and lymphangiogenesis ^[92][144]. As melanoma is particularly prone to brain metastases, exosomes may also at least partially explain this characteristic by damaging endothelial cells and the blood–brain barrier, and activating glial cells ^[93][145]. Some exosomal components have also been linked to resistance to therapy in CM, and targeting these molecules may improve therapeutic response ^[94][146]. In this context, exosomes can influence the melanoma microenvironment by altering the function of lymphocytes and stimulating tumor-associated macrophages (TAMs) to become M2-polarized and secrete pro-tumorigenic cytokines ^[95][96][97][147,148,149]. These effects can affect the response to immunotherapy, and targeting TAMs could improve the outcome of melanoma patients ^[98][150]. Furthermore, exosomes can be used as a means for administering therapy ^[99][151]. In this respect, exosomes containing BRAF siRNA were shown to have increased anti-tumoral activity compared to siBRAF in melanoma cell lines ^[100][152]. Similarly, cord-blood-derived exosomes produced significant genotoxicity and a decrease in survival time for melanoma cells and lymphocytes from melanoma patients, apparently by delivering anti-oncogenic miR-7. These results are particularly important as the exosome caused no significant damage to normal lymphocytes ^[101][153]. While these findings underscore the promising role of exosomes as diagnostic, prognostic, and treatment tools in melanoma, additional research is required to comprehensively delineate their utility. Future studies may aim to validate these biomarkers in larger patient cohorts, completely elucidate the roles of exosomal components in melanoma progression, and assess the feasibility of incorporating exosomal markers into existing diagnostic, prognostic, and therapeutic frameworks. The complex effects of exosomal components in CM pathogenesis are presented in Figure 1.