The cyclin-dependent kinase inhibitor 2 (CDKN2A; at chromosome 9p21) gene encodes two proteins, p16^CDKN2A and p14^CDKN2A (a product of an alternative splicing), that are known to function as tumor suppressors [21]. In particular, p16^CDKN2A is part of the G1–S cell cycle checkpoint mechanism that involves the retinoblastoma-susceptibility tumor suppressor protein (pRb). The p16^CDKN2A inhibits CDK4, which, in turn, phosphorylates pRb and allows progression through the G1–S checkpoint (Fig. 4.1). On the other hand, p14^CDKN2A interacts with the murine double minute 2 (MDM2) protein that targets p53 for degradation (Fig. 4.1) [22]. In particular, the p14^CDKN2A protein exerts a tumor suppressor effect by inhibiting the oncogenic actions of the downstream MDM2 protein, whose direct interaction with p53 blocks any p53-mediated activity and targets the p53 protein for rapid degradation. Impairment of the p14^CDKN2A-MDM2-p53 cascade, whose final effectors are the Bax/Bcl-2 proteins, has been implicated in defective apoptotic responses to genotoxic damage and, thus, to anticancer agents (in most cases, melanoma cells present concurrent high expression levels of Bax/Bcl-2 proteins, which may contribute to further increasing their aggressiveness and refractoriness to therapy) [23].

In normal conditions, expression levels of p53 within cells are low. In response to DNA damage, p53 accumulates and prevents cell division. Therefore, inactivation of the TP53 gene results in an intracellular accumulation of genetic damage which promotes tumor formation [24]. In melanoma, such an inactivation is mostly due to functional gene silencing since the frequency of TP53 mutations is low [25].

The CDKN2A mutations are more frequent in patients with a strong familial history of melanoma (three or more affected family members) relative to patients with no familial occurrence of melanoma [26]. A recent meta-analysis of studies conducted in independent populations indicated that multiple variants of the melanocortin-1 receptor (MC1R) gene increase the melanoma risk in CDKN2A mutation carriers [27].

The MC1R gene encodes a G-protein coupled receptor. In the skin, two types of melanin pigment, dark-protective eumelanin and red photoreactive pheomelanin, are present [28]. MC1R plays an important role in determining the ratio of eumelanin and pheomelanin production (Fig. 4.2). After stimulation by UV, keratinocytes produce alpha melanocyte-stimulating hormone (MSH) that binds to the MC1R on melanocytes and shifts the balance of these two pigments in the direction of eumelanin [28]. In particular, stimulation of MC1R by MSH mediates activation of adenylate cyclase, subsequent elevation of cAMP levels, and activation of the microphthalmia transcription factor (MITF; see below) (Fig. 4.2a). Activated MITF binds to a conserved region found in the promoters of the tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and DOPAchrome tautomerase (DCT) genes, stimulating the transcriptional upregulation of these proteins and inducing maturation of the melanosomes [29]. This ultimately results in increased eumelanin production and darkening of the skin or hair.

New findings have shed light on the mechanisms by which MC1R contributes to melanoma risk. In vitro studies showed that acute UV irradiation of melanocytes with impaired MC1R results in an increased production of free radicals [30]. Melanomas that arise on body sites only intermittently exposed to sun, and which therefore lack marked signs of chronic solar damage, were found to have a high frequency of BRAF mutations [18]. One could speculate that induction of BRAF mutations may occur only when solar exposure is not sufficiently prolonged to induce the striking tissue changes that generate the hallmark signs of solar damage. Several MC1R variants, which are impairing relevant protein function, have been associated with BRAF mutation in melanoma arising in Caucasian populations from the USA and Europe [31–34]. On the basis of such indications, it is possible that increased production of free radicals following UV exposure in combination with impairment of MC1R may induce mutations in the BRAF gene (Fig. 4.2b).

Additional mechanisms promoting susceptibility to pathogenetic mutations of the BRAF gene may however exist since there is no demonstrable association between germ line MC1R status and the prevalence of somatic BRAF mutations in melanomas from Australian population, even after classifying the melanomas by their location relative to intermittent and chronic sun exposure [35].

Phosphatase and tensin homolog deleted in chromosome ten (PTEN) has a key role in cellular signal transduction by decreasing intracellular phosphatidylinositol [3,4-bisphosphate (PIP2) and 3,4,5-trisphosphate (PIP3)] that are produced by the activation of phoshoinosite 3-kinase (PI3K) [36]. Active RAS exerts the strongest stimulus for PI3K activation (Fig. 4.1), resulting in an increase of PIP3 intracellular levels and a consequent conformational change of AKT [37]. Activated AKT in turn phosphorylates its substrate, the serine/threonine kinase mTOR, leading to increased synthesis of target proteins that regulate cell division and apoptosis [37]. The mechanisms associated with the ability of AKT to suppress apoptosis include the phosphorylation and inactivation of many proapoptotic proteins, such as BAD (Bcl-2 antagonist of cell death) and MDM2, as well as the activation of NF-kB [38] (Fig. 4.1).

In primary melanomas, inactivation of PTEN gene is mainly due to hypermethylation-based epigenetic mechanisms, with a low incidence (less than 10 %) of somatic mutations [39]. PTEN inactivation has been mostly observed as a late event in melanoma, although a dose-dependent downregulation of PTEN expression has been implicated in early stages of tumorigenesis [36]. In addition, alterations of the BRAF-MAPK pathway are frequently associated with PTEN-AKT impairment [2, 40]. In summary, the combined effects of the loss of PTEN and activation of the PI3K/AKT pathway may result in aberrant cell growth, apoptosis escape, and abnormal cell spreading and migration.

The microphthalmia-associated transcription factor (MITF) is a basic helix–loop–helix leucine zipper transcription factor that is considered to be the master regulator of melanocyte biology, since it is involved in melanoblast survival and melanocyte lineage commitment. MITF is activated by the MAPK pathway as well as by the cAMP pathway (Fig. 4.1) and leads to transcription of genes involved in pigmentation (TYR, TYRP1, and DCT; see above) as well as cell cycle progression and survival. A genome-wide analysis of copy number alterations in cancer identified MITF as an amplified locus in melanoma; MITF amplification correlated with increased resistance to chemotherapy and decreased overall survival [41].

The connection between MITF and melanoma development is complex because it plays a double role of inducer/repressor of cellular proliferation. High levels of MITF expression lead to G1 cell cycle arrest and differentiation, through induction of the cell cycle inhibitors p16^CDKN2A and p21 [42, 43] (Fig. 4.1). Very low or null MITF expression levels predispose to apoptosis, whereas intermediate MITF expression levels promote cell proliferation [41–43]. Therefore, it is thought that melanoma cells have developed strategies to maintain MITF levels in the range compatible with tumorigenesis. It has been shown that constitutive ERK activity, stimulated by ^V600EBRAF in melanoma cells, is associated with MITF ubiquitin-dependent degradation [44]. Nevertheless, continued expression of MITF is necessary for proliferation and survival of melanoma cells, because it also regulates CDK2 and Bcl-2 genes [45, 46]. It has been recently shown that oncogenic BRAF may control intracellular levels of the MITF protein through a fine balance of two opposite mechanisms: a direct reduction of MITF levels, by inducing protein degradation, and an indirect increase of MITF levels, by stimulating transcription factors which increase protein expression levels [47]. Oncogenic BRAF mutations are associated with MITF amplification in a low fraction (10–15 %) of melanomas [47] suggesting that other mechanisms are likely to be involved in ERK-dependent degradation of MITF.

Nuclear factor-kB (NF-kB) is important in inflammation and cell proliferation and survival [48]. A wide range of stimuli activates NF-kB which, upon activation, translocates to the nucleus to interact with kB sites located in regulatory regions of target genes [49].

Aberrant NF-kB regulation may cause alteration of the transcriptional activation of genes associated with cell proliferation, angiogenesis, tumor promotion, and suppression of apoptosis [50–52]. In melanoma, NF-kB is constitutively activated since expression of the inhibitor-of-kB (IkB) proteins, which form complexes sequestering NF-kB into the cytoplasm, seems to be significantly reduced in comparison to nevi [53]. In particular, activation of the AKT and/or MAPK pathways has been demonstrated to increase degradation of such complexes through activation of an IkB kinase (IKK) effector (Fig. 4.1), with subsequent release of NF-kB which may thus move to the nucleus and activate gene transcription [54–56].

Increased knowledge of molecular mechanisms in melanoma has prompted interest in how this may be applied to improve clinical decision making on the basis of more accurate diagnosis, refined prediction of probable future clinical course (prognostication), and the likelihood of a meaningful response to specific treatment modalities. These assessments currently are based on consideration of patient’s demographics, clinical presentation, and microscopic and immunohistochemical features of the individual melanoma. Clinical assessment relies on elicitation of a detailed clinical history and scrutiny of the skin tumor, possibly with the assistance of a dermoscope. If melanoma is suspected, the lesion is often examined in situ by ultrasound to assess depth of invasion [57]. Standard microscopic evaluation of tissue sections from a primary melanoma, stained by hematoxylin and eosin (HE), classifies the histology and cytology of any melanocytic proliferation present at the dermoepidermal interface, the presence and micrometer-measured extent of ulceration, and the measured thickness and depth of any dermal infiltration by tumor [58]. The tumor cells of junctional and dermal components are evaluated for pleomorphism, nuclear atypia, and the frequency and location of normal and abnormal mitoses. The associated stroma is assessed for the presence, density, and distribution of any peritumoral or intratumoral lymphocytic infiltrates. Evidence of vascular and/or lymphatic invasion sought and neurotropism and angiotropism recorded [59]. Prognosis is based on consideration of the presence and extent of ulceration, the micrometer-measured Breslow thickness, and the frequency of mitoses in the vertical growth phase [60].