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[119]
membrane receptors to downstream pathways controlling the cell cycle, growth, apoptosis, and senescence .
The HRAS, KRAS, and NRAS oncogenes were the first human oncogenes to be discovered [120] . In the case
[29]
of CM, NRAS mutations are found in 17% of the cases, according to COSMIC database . NRAS hot-spot
mutations are mutually exclusive of BRAF hot-spot mutations. HRAS and KRAS mutations are much less
frequent in CM. Regarding NRAS, the most common mutations cause a change of the amino acid at position
61, lying at the GTP-binding domain. These substitutions disrupt the GTPase activity of the protein, locking it
[121]
in its active conformation .
NF1
NF1 is a tumour suppressor gene encoding for a direct negative regulator of RAS signaling [122] . In particular,
NF1 is a GTPase-activating protein known to downregulate RAS activity by stimulating the hydrolysis of
GTP and returning the protein to its inactive form. A significant enrichment of NF1 mutations was found in
BRAF and NRAS wild-type melanomas [17,18] . In the TCGA study NF1 was found as the third most frequently
observed significantly mutated gene of the MAPK pathway [123] . Mutations in the NF1 gene are loss of
function mutations, mainly nonsense point mutations [124] , which can be considered as an alternative way to
activate the MAPK signaling pathway.

TERT
The TERT gene encodes for the telomerase reverse transcriptase, the catalytic subunit of the telomerase
ribonucleoprotein, essential for the maintenance of telomeres and chromosomal stability. Recurrent
somatic mutations in the TERT promoter have been characterised in CM, with high frequency in sporadic
melanoma. Specifically, the two hot-spot mutations, located at -124 and -146bp relative to the transcriptional
start site, are C>T transitions, consistent with a UV signature mutational profile [125,126] . In a recent study
exploiting whole genome sequencing, 86% of CM cases were found mutated at one or more out of four
[22]
positions upstream of the transcriptional start site . All these mutations are mutually exclusive and create
new binding sites for the E26 transformation-specific family transcription factor GA-binding protein. Recent
evidence suggests that TERT promoter mutations result in TERT over expression [127,128] . They are established
after MAPK-pathway activating mutations, but still during the early stage of melanoma progression [129] .

CDKN2A
CDKN2A is a well characterised tumour-suppressor gene, found to harbour somatic alterations in a wide
variety of different tumour types [128] . Regarding CM, in addition to its association with familial melanoma,
somatic alterations resulting in CDKN2A inactivation are also frequently observed in sporadic melanoma.
The most frequent alteration is the deletion of the CDKN2A gene, reported in 41% of CM [129] . CDKN2A
expression is additionally regulated at the epigenetic level, mainly by methylation of its promoter and
subsequent gene silencing. The two proteins encoded by CDKN2A, p16 and p14, have distinct roles in the
regulation of the cell cycle. p16 modulates G1 to S phase transition by inhibiting the kinase activity of cyclin
dependent kinases 4 and 6 (CDK4 and CDK6), while p14 acts through TP53 stabilisation. Biallelic loss of
CDKN2A and subsequent disruption of the G1/S checkpoint, is believed to be a crucial step in melanoma
progression towards transition to the invasive phenotype [127] .

TP53
TP53 is a well-known tumour suppressor gene, involved in the transcriptional regulation of several target
genes. TP53 is mutated in 27 different types of cancer [130] . Regarding melanoma, 15% of cases harbour
[29]
mutations in TP53 . Based on mutational studies, comparing primary melanomas and metastases, TP53
was found to be more frequently mutated in melanoma metastases, indicating that TP53 mutations may arise
later during melanoma progression [118] .

PTEN
PTEN is a tumour-suppressor gene, coding for the phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase.