Abstract
Melanoma progression is well defined in its clinical, histopathological and biological aspects, but the molecular mechanism involved in and the genetic markers associated to metastatic dissemination are only beginning to be defined. The recent development of high throughput technologies aimed at global molecular profiling of cancer is switching on the spotlight at previously unknown candidate genes involved in melanoma, such as WNT5A and BRAF. In fact, several tumor suppressors and oncogenes have been shown to be involved in melanoma pathogenesis, including CDKN2A, PTEN, TP53, RAS and MYC, though they have not been related to melanoma subtypes or validated as prognostic markers. Here we have reviewed the published data relative to the major genes involved in melanoma pathogenesis, which may represent important markers for the identification of genetic profiles of melanoma subtypes.

Keywords: Melanoma, progression, BRAF,RAS, CDKN2A, PTEN, TP53.

1. Introduction
Metastatic melanoma progression generally evolves from the local growth of a cutaneous lesion to regional metastasis at the draining lymph node/s, sometimes with 'in transit' metastases, and then to a disseminated disease involving cutaneous widespread and/or visceral metastases at any organ, commonly involving lungs, liver and brain [1].

From a histopathologic point of view melanoma progression first occurs in a crucial switch from a radial to a vertical phase of local growth. Melanoma cells can then spread either through lymphatics or directly by the hematogenous route to distant sites giving rise to life threatening metastatic disease. In fact, the major prognostic factors are the thickness and the ulceration of the primary lesion and which directly opens up to vascular access.

The biological changes occurring in melanoma progression are monitored and shaped by the immune system and the vast majority of mutations generated during tumorigenesis are likely to be selected as a consequence of the complex interactions between malignant cells and host factors. Cell characteristics acquired in metastatic progression consist of uncontrolled autocrine growth, resistance to apoptosis, and the achievement of invasive properties including adhesive, motility, proteolytic and angiogenic capacities [2].

From a genetic perspective, in which neoplastic transformation is viewed as a disease of genes, melanoma progression is associated to identifiable genetic mutations, which drive cancer cells to abnormal growth and dissemination [3]. It is conceivable that genetic background may alter both the susceptibility to melanoma development and the disease course. The identification of susceptibility genes is actively pursued to improve disease prevention, while it is clear that the ideal targets for the development of effective therapeutics are the genes determining disease progression.

Malignant melanomas are defined by different clinico-pathological cellular subtypes, which have not been related to independent prognostic or therapeutic significance [4]. Numerous genetic evidences point to the existence of different melanoma subtypes that differ in their clinical behavior, but their genetic traits are only beginning to be determined. In an effort aimed at the definition of genetic markers associated to melanoma progression and prognosis we have reviewed here the literature relative to the major tumor suppressors and oncogenes involved in melanoma pathogenesis, which possibly represent the starting point for the identification of genetic profiles of melanoma subtypes.

2. Global genetic analysis of melanoma progression by Comparative Genomic Hybridization (CGH)
Different patterns of chromosomal aberrations have been shown by the analysis of DNA copy number changes in several hundreds of primary melanomas using chromosome and array based CGH. Recurring, nonrandom chromosome abnormalities recognized in melanoma mostly include losses in chromosomes 1, 6, 9 and 10 and gains in chromosomes 6, 7 and 8, occurring in over 95% of the cases [5, 6]. Different types of melanomas have been shown to display distinct patterns of chromosomal aberrations. Marked differences in the genetic make-up have been related primarily on anatomical location and sunlight exposure pattern. Acral lentiginous melanomas (ALM), that arise on glabrous skin of the palms and soles or under the nails, and are considered not related to UV radiations, show very early and multiple gene amplifications compared to superficial spreading melanomas (SSM) [7]. In contrast, SSM and Lentigo Maligna (LM), occurring more frequently on chronically sun damaged skin, show frequent aberration of 13q and 17p regions spanning the locations of excision repair gene ERCC5 and p53, respectively [8]. Age dependency of copy number changes at chromosomes 5, 17 and 18 are also associated to LM, a melanoma type that generally presents in older patients and shows a protracted clinical course and a longer tumor progression [9]. Mucosal melanomas also have been shown to harbor peculiar chromosomal imbalances including 1q, 6p and 8q gains [10].

The amplified chromosomal regions frequently contained known oncogenes such as HRAS, CDK4, cyclin D1, HTERT, and BRAF but also involve regions with unknown candidate genes. For example, Cyclin D1 shows only occasional amplification in LM and SSM melanomas and revealed different mechanisms regulating protein over-expression beside increased copy number, since it was detected also in some cases with normal copy number [11].

Although the data of extensive analysis of primary lesions are available, few metastatic lesions have been studied and thus the maintenance of subgroup differences at the metastatic level is unclear. The analysis of karyotypic pathways in melanoma by the application of several statistical methods to 362 published melanoma karyotypes lead to the identification of two major cytogenetic pathways initiated with chromosome 3 losses and 6p gains and both including as later events alterations at chromosomes 1, 8, 9, 10, 11 and 15 [12]. In the metastases increasing number of genetic alterations were detected illustrating a higher level of genetic instability. The appearance of sequential chromosome alterations including structural mutations and large deletions has long been recognized in melanoma [13]. A few paired primary and metastatic lesions analyzed by CGH revealed shared and acquired abnormalities confirming that the accumulation of multiple genetic alteration accompanies the disease progression [14]. In this line, isolated blood circulating tumor cells have been shown to carry several changes in DNA copy number [15].

The rapidly evolving genomic profiling techniques on DNA microarrays will soon enable genotyping of melanoma cells with BAC precision which will lead to a sharp definition of primary tumor genetic subtypes and, through the analysis of metastatic lesions, will bring new information relative to the frequency of multi-lineage progression in melanoma.

3. BRAF gene
In 2002 the efforts of the Human Genome Project in the screening of cancer genes and the high-throughput genomic technologies lead to the identification of mutations detected at high frequency in melanoma in the BRAF oncogene [16], acting downstream the RAS pathway, which had been described 20 years before as the retroviral homologous [17].

An explosion of published data relative to BRAF alterations in melanoma has followed the first report by Davies. From all the studies published up to now (June 7th 2004), including more than 1300 specimens and more than 250 cell lines (listed in Table 1), the most common BRAF gene mutation V599E - which should be called V600E on the basis of the correct sequence derived from NCBI gene bank Acc. No. NT_007914, after Kumar et al. [39] - represents a molecular marker for about 50% melanomas, which is the tumor type with the higher frequency of mutations at this gene. A possible explanation for the high frequency of BRAF mutations in melanoma relative to cancers lies in the role of BRAF in mediating the MSH activation of the MAPK pathway, by signaling via cAMP/PKA and IP3/DAG/PKC G protein-coupled receptors [48].

Table 1. BRAF mutations in nevi and melanoma lesions

Lesion typea	BRAF V599E	other BRAFb	NRAS mutationsc	Methodd	References
Benign melanocytic nevi (total)	157/228 (68%)	1/158 (0.6%)*	6/6 (39%)	A, P, H, S, R	[18-22]
Intradermal nevi	74/92 (80% )	0/69 (0%)	3/39	A, P, H, S	[18-20]
Compound nevi	34/50 (68%)	1/50 (2% )*	1/11	A, P, H	[18,20]
Junctional nevi	2/6 (33%)	0/6 (0%)	2/6	H	[20]
Papillomatous nevi	20/27 (74%)	nt	nt	S	[19]
Blue nevi	0/20 (0%)	nt	nt	S	[19]
Spitz nevi	0/120 (0%)	nt	nt	P, S	[19,23,24]
Congenital nevi	12/20 (60%)	nd	3/7	A, P, S	[18,19]
Dysplastic or atypical nevi	21/56 (37%)	nd	nd	A, P, S, R,	[18,19,22]
Primary Melanomas (total)	213/698 (30%)	33/491 (6%)	82/285 (28%)e	A, P, S, R, D	[16,18,19,21,25-34]
	Tot BRAF mutations: 238/698 (36%)
SSM	31/68 (45%)	2/29 (6%)	0/2	P, S	[31,35,36]
NM	7/17 (41%)	1/13 (7%)	0/8	P, S	[31,35,36]
LM	1/12 (8%)	0/3 (0%)	nd	P	[31,35]
ALM	5/27 (18%)	1/4 (25%)	1/4	P, S	[31,36]
Mucosal	3/38 (7%)	nd	nd	A, P	[34,37]
RGP	2/20 (10%)	nd	18/18	P	[21]
VGP	12/29 (41%)	1/8 (12%)	3/3	A, P	[21,34]
Metastases (total)	252/617 (40%)	104/464 (22%)	73/377 (19%)	A, P, S, R, D	[18,19,21,25,27,29,30,33,36,38-43]
	Tot BRAF mutations: 395/685 (57%)f
Nodal	37/102 (36%)	0/33 (0%)	3/54	P, S	[18,36,42,43]
Cutaneous	35/69 (50%)	0/15 (0%)	10/40	A, P, S, R	[18,27,36]
Visceral	10/27 (37%)	3/27 (11%)	2/16	P	[43]
Cerebral	2/5 (40%)	1/5 (20%)	1/5	P, S	[36]
Short Term Cell lines (total)	42/74 (56%)	10/74 (13%)	6/41 (14%)	P, D	[16,29,44]
Primary	4/13 (30%)	3/13 (23%)	3/8	P, D	[29,44]
Metastases	27/46 (58%)	6/46 (13%)	3/33	P, D	[29,44]
Cell lines (total)	128/215 (59%)	10/213 (4%)	27/194 (13%)	P, S, D	[16,29,36,40,43,45-47]
Primary	23/40 (57%)	3/40 (7%)	1/35e	P, S, D	[36,46,47]
Metastases	6/10 (60%)	0/10 (0%)	1/5	P, S, D	[36,47]

^a SS: superficial spreading melanoma; NM: nodular melanoma; LM: lentigo maligna; ALM: acral lentiginous melanoma; RGP: radial growth phase; VGP: vertical growth phase; Short Term Cell lines: less than passage 15 in colture.
^b Analysis of exon 11 and 15; ^* D593V mutation identified by sequence analysis of exon 15; for nevi, exon 11 was analized for mutation only by Kumar et al. [20], but nomutation was detected. nd: not detected by sequence analysis of exon 15; nt: not tested. In melanomas other mutations include V599K/R/D/G/-604 del, K600E/del, L596Q/S/V, D593V/E, K474E, G468S/R/E, G465E/A/R, G463R, R444R, K438Q, and a 6 bp insertion between codons 598 and 599, coding for two Thr residues, and a silent ACA to ACT mutation at codon in the same sample.
^c Detected by sequence analysis of exon 1 and 2.
^d A: AS-PCR; P: PCR sequence analysis; H: PCR-SSCP/heteroduplex analysis; S: PCR-SSCP; R: PCR-RFLP; D: PCR+D-HPLC.
^e NRAS mutation with BRAF V599E mutation reported in one cell line and one primary melanoma [28,46].
^f Total BRAF mutations include also melanoma samples in Shinozaki et al.[32]

Moreover, some data indicate the over-representation of certain BRAF polymorphic variants in both sporadic and familial melanoma patients [49]. Germline mutations have been found only in 4 patients [29] out of more than 300 patients from melanoma families tested [42, 47, 50]. BRAF mutations in melanoma appear to be an early event, as they are detectable in early lesions such as RGP, and to be associated to melanoma progression, since invasive melanoma and metastases show a higher frequency of mutations compared to early lesions and to primary melanomas (Table 1). In addition, the maintenance of the mutated BRAF gene in the studied primary and metastatic lesions from the same individuals and the detection of mutant BRAF in metastases in patients with a primary tumor showing wild type BRAF gene [32, 33], clearly point to the association between mutated BRAF and the metastatic capacity of melanoma cells.

In vitro transfer of mutant BRAF gene in melanocytes has been shown to induce the acquisition of a transformed phenotype with regard to in vitro aggressive growth and tumor formation upon transplantation in nude mice [51].

Paradoxically, BRAF mutations may even precede the neoplastic transformation, since all types nevi besides Spitz and Blue nevi show BRAF alterations at a high frequency, with dysplastic nevi showing a notable lower frequency. Whether the examined nevi samples were obtained mostly from melanoma patients or from individuals at high risk for melanoma because of familiar susceptibility has not been specified in these studies. Indeed BRAF is the first gene resulting mutated in such a high number of melanoma lesions and of nevi.

BRAF mutations have been shown to be more common in younger patients [32, 39] and in melanomas arising in intermittently exposed body sites compared to chronically sun damaged skin, while are rare in ALM and LM melanomas and in mucosal melanoma [31, 33] and completely absent in uveal melanoma as well as in its metastases [25, 26, 34, 38, 41]. Whether these associations would imply that BRAF mutations are mostly linked to UV carcinogenesis in certain genetic backgrounds (for example MC1R variants) remains to be studied [52].

In short term cell lines (STC) the difference between frequency in primary and metastases is maintained whether long term cultured cell lines show similar frequencies.

Mutation V599E is far the most represented, though other mutations involving the same base, and other positions in exon 15 and in exon 11 have been detected, most of them only occasionally. In this regard, it is notable that substitutions V599K and V599R have been found frequently in melanoma (27 and 14 cases respectively in all the studies reported in Table 1) and that the V599K substitution has not been detected in other tumor types nor in nevi, although most of the studies on nevi assessed only the V599E mutation by the use of AS-PCR or RFLP-PCR methods.

The BRAF gene resides on 7q, which is gained frequently in melanoma and its region 7q34 has been shown to be amplified by CGH in 24/68 tested primary tumors, either showing mutated or wild type alleles, but it is yet not clear whether 7q amplification are associated to a particular melanoma subgroup [31]. Also, the functional effect of BRAF gene amplification has not yet been evaluated: the detection of different levels of gene expression [36] and of protein expression [44] awaits further experimental evaluations.

Finally, the BRAF mutation represents an important target for the development of therapeutic opportunities, so urgently needed for the treatment of the metastatic disease, and also for the development of prophylactic interventions in individuals at high risk for melanoma development. Suppression of BRAF V599E expression in melanoma cells with siRNA abrogates melanoma cell growth in vitro [53] and a variety of agents interfering with the RAF pathway have been discovered, some which have entered phase III trials [54]. Moreover, the V599E mutated peptide may represent a potential antigen for immunotherapeutic strategies [55]. The identification by microarray expression profiling of the genes which are exclusively expressed in BRAF melanoma will also open new possibilities of identifying novel therapeutic targets in the BRAF expression signature [45].

4. Signaling through the MAPK pathway
The BRAF gene encodes for a serine/threonine kinase which is regulated by the binding with RAS protein [56]. The activating V599E substitution lies within the kinase domain and has been shown to mimic the phosphorylation of the BRAF protein thus inducing the activation of the downstream MEK resulting in an over-activation of the RAS-ERK pathway. The V599 mutants have an augmented kinase activity, but also other mutants endowed with weak kinase activity have been shown to stimulate ERK activation through the interaction with CRAF, another member of the RAF protein trio [33, 57]. BRAF and NRAS mutations appear to be equivalent in the constitutive activation of the MAPK pathway, and are generally found with mutual exclusion in melanoma. Coupled BRAF and NRAS mutations have been detected in six cases of all the samples reported in Table 1 [28, 33, 43, 46], either involving exon 15 and 11 BRAF mutants.

RAS genes mutations have long been recognized in melanoma samples. From the recent re-evaluation of the frequency of NRAS mutations carried out along with the extensive BRAF gene analysis, it is clear that about 20% melanoma lesions carry mutated NRAS. NRAS mutations occur in nevi as well. Other RAS genes, KRAS and HRAS, have been shown involved only occasionally in melanoma [58]. From the parallel analysis of BRAF and NRAS gene performed in most of these studies a small melanoma subgroup has been identified lacking mutation at either genes and for which activation of the MAPK pathway should be caused through other genes which remains undefined at the moment. A role of PTEN gene inactivation in this context was proposed since PTEN exerts a regulatory effect on the MAPK activation; however, more recent data indicate that PTEN mutations are associated to BRAF mutations thus pointing to the parallel activation of the AKT pathway along with the MAPK in melanoma progression [40, 44]. BRAF and NRAS mutations have been shown inversely associated with allelic loss on chromosome 9p in primary melanomas: however, if only microsatellite markers located in the CDKN2A locus are considered, most of the studied cases show BRAF/RAS mutation along with LOH at CDKN2A [28]. Mutational profiles associated with BRAF mutations in STC were shown to involve mutations at CDKN2A, PTEN and TP53 tumor suppressors, as shown in Fig. 1: although the p16/ARFdel+BRAF^V599E profile was the most represented, BRAF/RAS mutations were also associated to TP53 mutations or to mutations at multiple tumor suppressor genes. The p16/ARFdel+BRAF^V599E profile was associated with a longer survival while complex mutational profiles including BRAF mutations and alterations at multiple tumor suppressor genes were detected in aggressive disease with poor survival [44].

Figure 1.
Distribution of mutational profiles detected in lesions from melanoma patients with survival < 2 years or > 2 years from lymph node surgery. All the different combinations of affected genes are shown in the legend [44].

BRAF V599E mutation has been shown to be associated with a longer disease-free survival but also to a shorter duration of response to treatment [39]; others have found an association with a poor prognosis when metastases are considered [33] and no difference in the survival between primary showing or not the BRAF/NRAS mutations [32]. Clearly more data are needed in this regard in order to link mutated BRAF and the other major genetic aberrations frequently occurring in melanoma to disease outcome.

One characteristic of melanoma cells is the production of autocrine growth factors, such as bFGF and MGSA/GRO, that activate the MAPK signaling.

The activation of the MAPK pathway is a frequent and early event in melanoma [59, 60]. It has been hypothesized that ERK activation is necessary for tumor metastases in melanoma. A regulatory role of MEK/ERK pathway on the expression of proteases involved in the matrix degradation and on cell adhesion molecules associated to metastatic spread has been detected in different studies, along with a regulatory role on Mitf transcription factor that is involved in melanoma cells survival [61, 62].

NME1/NM23 gene encoding a nucleoside diphosphate kinase is a prototypical metastases suppression genes [63], identified in a murine melanoma cell line by subtractive hybridization and then analyzed in melanoma in numerous studies, generally reporting lower expression in melanomas with higher tumor thickness and a correlation between expression and survival [64, 65]. Nucleoside diphosphate kinase recently has been shown to phosphorilate KSR protein, a kinase suppressor of RAS acting as a scaffold protein for MAPK pathway thus potentially affecting ERK activation [66].

5. Melanoma Susceptibility Genes in melanoma progression
Melanoma susceptibility genes include p16/INK4A, CDK4, ARF and a gene, as yet undefined and located at the 1p22 region [67]. Besides CDK4, which has been shown to be rarely mutated both at the germinal and at somatic level, and the unknown gene mapping at 1p22, CDKN2A genes have been largely studied in melanoma and recently reviewed [68].

Although the incidence of CDKN2A gene mutations in 25-40% melanoma prone families is well established, their role in sporadic melanoma has long been a question of debate, because the mutation analysis only rarely evidenced mutated sequences in primary and metastatic lesions, while melanoma cell lines show CDKN2A mutations in most, if not all, tested samples (Table 2). This gap has been partially explained by the evidence that the majority of the lesions display allelic loss at microsatellite markers mapping in the CDKN2A locus, thus indicating that deletions are the principal mutation events. p16 loss has been shown to occur in melanoma also by transcriptional silencing by gene methylation and by post-transcriptional events. In addition, a discontinuous pattern of genetic alterations at CDKN2A during tumor progression was revealed by the analysis of primary tumors and the metastastic lesions deriving from it, implicating the accumulation of further deletions at CDKN2A as secondary genetic changes [87, 88]. Several studies detected a similar pattern of LOH in matched primary and metastatic lesions suggesting that allelic loss occurs before tumor dissemination and plays a role in the development of melanoma. Moreover, LOH studies indicated the existence of additional tumor suppressors loci mapping to chromosome 9p21-24 involved in melanoma [76, 89].

Table 2. CDKN2A mutations, allelic loss and p16 expression in nevi and melanoma

Lesion type	Mutations	LOH	Positive by IHCd	References
Benign nevi	0/22 (0%)	0/56 (0%)c	43/43 (100%)	[69-72]
Dysplastic nevi	3/31 (10%)	14/95 (14%)c		[69, 73-75]
Melanoma samplesa		68/112 (60%)		[76]
Primary melanomas (total)	24/195 (12%)	57/104 (54%)	175/443 (39%)	[71, 72, 76-81]
Metastases	17/293 (5%)	80/168 (47%)	18/79 (22%)	[69, 76, 78, 82]
Short Term Cell Lines	30/41 (73%)b		5/42 (11%)	[44]
Cell lines	110/127(86%)b		11/60 (18%)	[83-86]

^a Include both primary and metastatic lesions.
^b Including homozygous deletions.
^c Microsatellite markers mapping at 9p21 but not in the CDKN2A locus.
^d Nevi and melanoma lesions from patients with family history of melanoma are included [71].

Two recent papers reported the experimental conditions for the analysis of p16 protein expression by immunohistochemistry (IHC) [71, 80] and from the analysis of a large number of cases (including some old studies carried out with the same method) p16 clearly results lost in melanoma but not in nevi, at a higher frequency in metastatic lesions either in sporadic and in familial melanoma patients (Table 2). These results define the relation between p16 inactivation and melanoma progression, which is observed in the vast majority of metastases albeit not in all the cases. The higher occurrence of allelic loss and of protein loss in thick as compared to thin primary melanomas further underline the importance of p16 inactivation for melanoma progression [71, 77].

Few data are available on p14/ARF, the second product of the CDKN2A locus. It has been established that exclusive p14/ARF mutations are very rare and that the gene may be silenced by hypermethylation or by post-transcriptional modifications, but the role p14/ARF in melanoma progression remains substantially untested. The only published IHC study reports positive staining for 11/14 benign nevi, 3/12 melanomas and 0/6 metastases, strongly suggesting that p14/ARF and p16 loss may go together in melanoma progression [90].

Other families at increased risk for melanoma include Xeroderma Pigmentosum, Retinoblastoma, Werner syndrome and Li-Fraumeni families, carrying XP, Rb, RECQL2 and TP53 germline mutations respectively. In addition, individuals carrying mutations at BRCA2 show increased melanoma risk along with other cancer types [91]. Mutations at these genes have not been studied in melanoma with the exception of TP53 gene which is considered below.

6. PTEN gene and AKT signaling pathway
10q region is the second most frequent site of allelic loss in melanoma after 9p, frequently altered by monosomy. The PTEN tumor suppressor gene is located in 10q23 and encodes for a lipid/protein phosphatase with dual specificity: by the lipid phosphatase activity PTEN signals down the PI3K/AKT pathway regulating G1 progression and apoptosis, and by the protein phosphatase activity PTEN inhibits MAPK signaling [92]. Cowden syndrome patients bearing germ line mutations at the PTEN gene are at high risk for developing breast and thyroid carcinomas but may also develop melanomas [93].

Mutations at PTEN gene are quite common in melanoma cell lines but not in STC lines and in specimens; on the contrary, over 30% samples reveal LOH, and IHC data indicate that a similar fraction of lesions lack normal protein expression (Table 3). Two issues should be considered that may explain the discrepancies in the results: first, the existence of a highly conserved processed PTEN pseudogene that may mask as a mutant allele and complicate the interpretation of the results of the genetic analysis [105, 106]; second, the existence of epigenetic mechanisms of gene silencing and of post-transcriptional events abrogating protein expression evidenced by studies of gene and protein expression carried out in parallel [44, 94]. Although a limited information is available in this regard, LOH frequency appears similar in primary and in metastatic lesions thus suggesting that PTEN occur in melanoma initiation, as also suggested by cytogenetic studies. Few data are available about PTEN in nevi: the loss of 10q has been detected in dysplastic nevi [107], protein expression by IHC was detectable in 38/39 samples [101] and the analysis of acquired melanocytic nevi and melanoma tissue specimens failed to reveal difference in protein expression as evaluated by semi-quantitative RT-PCR [99].

Table 3. PTEN gene mutations, allelic loss and protein expression in melanoma

Lesion type	Mutations	LOHb	Loss of proteinc	References
Melanoma samplesa	1/83 (1%)	20/83 (24%)	5/34 (14%)	[94, 95]
Primary tumors	7/68 (10%)	11/26 (42%)	41/122 (33%)	[96-101]
Metastases	16/150 (10%)	37/95 (38%)		[96-99, 102, 103]
Short Term Cell Lines	3/41 (7%)	nd	3/41 (7%)	[44]
Cell lines	29/177 (16%)	52/137 (38%)	nd	[95, 102-104]

^a Including both primary and metastatic lesions.
^ab For 28/175 samples both LOH and gene mutations are reported; for 19/97 samples homozygous deletion of the PTEN gene is reported.
^ac Protein loss as detected by IHC in tumor specimens and by Western Blot in cell lines.

The inter-relation of PTEN mutations with alteration in other genes important in melanoma pathogenesis is largely unexplored. Besides the NRAS and PTEN mutations studied in melanoma cell lines discussed above, our study performed on STC showed lack of PTEN protein in the presence of gene expression revealed by RT-PCR in cases showing wild type BRAF and RAS genes, possibly indicating that loss of PTEN may affect MAPK activation. Unexpectedly, the samples showing PTEN mutations, but not the samples showing only PTEN protein loss, carried also associated PT53 mutations [44]. The concomitant mutations at the two tumor suppressor genes in melanoma appear noteworthy, because they are exclusive in other malignancies.

One consequence of the loss of PTEN is represented by the activation of AKT. Phosphorilated AKT has been detected in most metastatic melanoma lesions but not in nevi [108]: AKT activation has been shown to be PI3K dependent and to play an important role also through the activation of NF-kB transcription factor. Although a high frequency of mutations of the PIK3CA gene has been detected in several human cancers, melanoma showed absence of mutations in PI3KCA [109, Bardelli personal communication].

Although a role of PTEN gene in melanoma pathogenesis is evident in these studies a better understanding of the PTEN signaling events in disease progression need to be obtained.

7. TP53 gene
Due to its short half life, p53 protein is undetectable in normal tissues including the skin but mutated variants show increased stability and the augmentation of protein levels determines IHC detection. In most studies, nevi show absence and melanoma show p53 overexpression by IHC; in general, p53 staining increase with tumor thickness and is more frequent in metastases (Table 4). In melanoma associated to nevi, p53 is expressed only in the malignant cells [134].

Table 4. TP53 gene mutations and protein expression in nevi and melanoma

Lesion type	Positive by IHC	TP53 Mutations	References
Benign nevia	17/150 (11%)	2/39 (5%)	[69, 70, 110-115]
Dysplastic nevi	2/69 (3%)	4/46 (9%)	[63, 69, 113, 114, 116]
Primary melanomas	230/847 (27%)	20/418 (5%)	[69, 78, 110, 111, 115-127]
SSM	53/168 (32%)	6/102 (6%)	[69, 111, 117-119, 125, 126]
NM	140/663 (21%)	9/86 (11%)	[69, 70, 110, 111, 115, 116, 119, 120-126]
VGP	37/190 (20%)		[78]
Metastases	111/315 (35%)	22/199 (11%)	[69, 78, 110, 113, 116, 120, 121, 123, 125, 128, 129]
Short Term Cell Lines	11/42 (26%)	11/49 (22%)	[44, 69]
Cell lines	26/42 (62%)	21/124 (16%)	[69, 120, 125, 130-133]

^a Congenital and Spitz nevi are included.

However, a correlation between expression of detectable protein levels and presence of TP53 gene mutations shown in other tumor types was not observed in melanoma, and generally melanoma expressing detectable p53 protein revealed absence of gene mutations by the analysis of exons 5-8, the usually mutated exons, indicating that other mechanism of protein stabilization exist. The mechanisms at the basis of p53 stabilization remains unclear, though it may be simply explained by the presence of mutations at other gene regions not analyzed in most of the studies but reported in melanoma [114, 126]. The frequency of TP 53 mutations in few studies is close to 25% [44, 125] while the overall frequency is about 10% (Table 4).

Mutations of TP53 show preponderance for C to T transitions or tandem CC to TT changes, strongly implicating UV light as a mutagen, although one study detected C to T transitions also in mucosal melanomas [126]. In a population based study, p53 positive melanoma was strongly associated with inability to tan, history of other skin cancer and melanoma arising at sun exposed skin (such as head and neck), while p53 negative melanoma was associated to nevus density and freckling tendency [122]. From these data the authors hypothesized the existence of two pathways for melanoma development, one associated to chronical sun exposure and the other to melanocyte proliferation. Similarly, Maldonado and collaborators reported that BRAF mutations are more common in melanoma arising in intermittently sun exposed skin [31]. Further studies are needed to evaluate whether other genetic markers such as MC1R variants can be associated to melanoma arising at body sites with different level of sunlight exposure [52].

Few studies report a significant association between TP53 mutations or protein expression and major clinical data, risk of metastases and survival [78, 115, 129].

Concomitant mutations at TP53 and p16 genes were shown in melanoma cell lines [128, 135] and high levels of p53 immunostaining in metastases showing CDKN2A deletion [129], indicating that the inactivation of both genes is not unusual in melanoma. In STC we found TP53 mutations either associated or alternative to mutations at CDKN2A genes, always associated to BRAF mutations, and preferentially associated to poor survival (as shown in Fig. 1).

In conclusion, the role of TP53 in melanoma is only partially understood. Due to the central role of TP53 in tumor pathogenesis through the control of genomic stability, cell cycle arrest, induction of apoptosis and transcriptional activity on genes regulated in tumor progression, its involvement in melanoma should be clearified.

8. Beta catenin, c-MYC, APC and WNT pathway
Mutations in CTNNB1 gene coding for ß-catenin were identified in melanoma cell lines [136]. Although down regulation of ß-catenin expression was detected by IHC in a consistent fraction of primary and metastatic lesions compared to nevi [137-139], several studies have shown that CTNNB1 gene mutations occur rarely in melanoma specimens [140-142].

The expression of APC gene, the major regulator of ß-catenin, has been shown to be reduced by hypermethylation of its promoter in about 15% melanoma samples and cell lines [143]. ß-catenin is a key effector in the WNT signaling transduction pathway inducing tumors to become invasive by disrupting cellular communication [144].

WNT5A, a gene recently identified by cDNA microarrays as highly expressed in a subclass of melanoma metastases characterized by a high motile and invasive in vitro phenotype [145], is associated with aggressive melanoma behavior mediated through the activation of PKC pathways which are associated to cytoskeletal organization and invasion [146].

c-MYC constitutive transcription can be induced by mutated or over-expressed b catenin [147]. c-MYC has been shown frequently to be overexpressed in melanoma and c-MYC extra copies have been reported in nodular melanoma, in metastases and shown absent in nevi [148, 149]. A high frequency of patients with primary tumors showing c-MYC amplification revealed visceral metastases, but also conflicting data have been reported about the prognostic significance of c-MYC over-expression [150-152]. MXI1 is a negative regulator of c-MYC located at 10q close to PTEN locus in a chromosomal region showing LOH with higher frequency in metastases as compared to primary tumors [153].

c-MYC is at the center of a transcription factor network that regulates cell proliferation, differentiation and apoptosis and show links with transcriptional regulatory machinery, such as STAT proteins, chromatin remodeling and DNA repair, such as BRCA1 [154]. It has recently been shown that c-MYC activation can induce DNA damage thus accelerating tumor progression via genetic instability [155]. In the light of these data, the role of c-MYC in melanoma progression requires further evaluation.

Acknowledgements
We thank Giorgio Parmiani and Marco A. Pierotti for critical reading of the manuscript.

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