UV radiations are very toxic agents for skin. The wave lengths of absorption depends upon molecular structure, being shorter around 200 nm for conjugated bonds in organic molecules and longer around 300 nm, in the range of solar spectrum, for linear repeats or ring structures. Therefore, DNA containing many ring structures, i.e., bases, and conjugated bonds represents the most relevant absorber of UV in the cell with consequent gene mutations [30]. The effects of UV on human skin can be considered acute or chronic. Between the acute effects, DNA damage is essentially due to the induction of primarily cyclobutane-type pyrimidine dimers and pyrimidine pyrimidone photoproducts [31–33].

Therefore, base modifications occur where a tandem of pyrimidine exists. Radiation induces primarily C  →  T and CC  →  TT transitions that are the hallmark of UV-induced mutagenesis [34]. Subsequently, when DNA polymerase cannot interpret the DNA template, the enzyme inserts A residues by default [35]. In addition, UV induces also cytosine photohydrates, purine photoproducts, and single-strand breaks in the DNA and the production of specific reactive oxygen species (ROS), responsible for the induction of single-strand breaks, DNA–protein cross-links, and altered bases in DNA [36, 37]. Acute UV-induced skin damage is also responsible for the induction of p53 expression through different mechanisms. p53 is a critical protein that causes cell cycle arrest, either favoring the cellular repair pathways to remove DNA damage before DNA synthesis and mitosis [38–40] or promoting apoptosis in cells with excessive DNA damage [41]. p53 protein increases in epidermal cells after exposure to UV radiation, as demonstrated in SKH-hr1 mice [42]. In details, it seems to be induced when DNA damage has characterized by pyrimidine dimers and DNA stands break [42]. In addition, p53 phosphorylation is induced by UV radiation through involvement of the ATM, ATR, and p38, and of the ERK1/2 and JNK-1 mitogen-activated protein (MAP) kinases-dependent pathways [43–46]. Thus, serine residue phosphorylation promotes stabilization of p53 by (1) disrupting the interaction of p53 with Mdm2, (2) enhancing p53 transcriptional activity, and (3) favoring p53 nuclear localization. The final effects of p53 activation are essentially cell cycle arrest followed by DNA repair occurring through two major pathways: non-excision repair (NER) and base excision repair (BER). In details, in NER pathway p53 transactivates the two xeroderma pigmentosum-associated gene products (p48XPE and XPC) and GADD45; [47–49] in BER pathway, p53 plays a role in regulating DNA polymerase, which is directly involved in DNA repair/synthesis [50]. When DNA damage is too deep, activated p53 in damaged skin induces apoptosis, through activation of a wide variety of transcriptional factors related to activation of the apoptosomal complex, such as the Bcl-2 family members Bax, p53-upregulated modulator of apoptosis (PUMA), Noxa, p53-upregulated apoptosis-inducing protein 1 (p53AIP1), and PIGa (galectin-7) [51–53]. Moreover, it has been demonstrated that UVB-induced apoptosis is related also to activation of Fas–Fas ligand, potent activators of pro-caspase 8 cleavage and, at the same time, of the cytochrome c release from mitochondria, followed by activation of the Apaf-1–pro-caspase 9 complex that both induce apoptosis [54]. Finally, UV-induced acute skin damage can also stimulate cell survival mechanisms and induce cell proliferation in the form of cell hyperplasia. In fact, UV triggers proliferation through activation of growth factor receptors such as erbB receptors and cytokine receptors, such as receptors for tumor necrosis factor (TNF) and interleukin-1 (IL1) [55, 56].

Chronic UV irradiation causes p53 mutation and loss of Fas–Fas ligand interaction with consequent apoptosis deregulation.

p53 mutations seem to be an early genetic event in the development of UV-induced skin cancers. In fact it has been described in approximately 53 % of premalignant actinic keratosis (AK) lesions [57]. In addition, 40 % of Bowen’s disease carry p53 mutations [58]. Many reports have recorded p53-mutant cell clones in nonneoplastic cells of sun-exposed skin [57–62]. However, p53 mutations in cSCC and in their respective precursors seem to be different from those in nonneoplastic skin areas [61]. This suggests that not all UV-induced p53mutations confer a malignant phenotype to neoplastic cells and that there is a latency from the occurrence of p53 mutation and cancer development. Some in vivo experiments have demonstrated that the occurrence of the most critical p53 mutations is recorded from 17 to 80 daily UVB exposures and skin tumors around 80 to 30 weeks later [63, 64]. Analyzing both C  →  T and CC  →  TT transitions in p53 gene, it has been observed that mutations are much earlier, being present at the first week from the beginning of chronic UV irradiation, with higher frequency at 4–8 weeks [65].

Therefore, selective expansion of p53-mutant cells occurs in the context of epidermis, due to both the resistance to UV-induced apoptosis and the proliferative advantage over normal keratinocytes in response to UV irradiation [64, 66, 67] (Fig. 3.1). Regression of a part of precancerous p53-positive clones can be observed after discontinuation from UV irradiation [64, 68] although tumor occurrence is only delayed [69].

The epidermis is continuously regenerated by mitotically active keratinocytes in the inner basal layer which, following detachment from the basement membrane, migrates to the outer cornified layer (terminally differentiated compartment): a process called cornification. The epidermis is continuously renewed by a regulated balance between proliferation and differentiation. The renewal capacity is due to the presence of epidermal stem cells and transient amplifying (TA) cells in the basal layer of the interfollicular epidermis and in the bulge of the hair follicle. The importance of the transcription factor p63 for the formation of the epidermis and other stratified epithelia arises from two lines of evidence. First, p63 is strongly expressed in the innermost basal layer where epithelial cells with high clonogenic and proliferative capacity reside. Second, mice lacking all p63 isoforms have no epidermis or squamous epithelia (prostate, urothelium), probably due to a premature proliferative rundown of the stem and TA cells. Mice knockout for p63 (p63^−/−) also show lack of epithelial appendages, such as mammary, salivary, and lachrymal glands, hair follicles, and teeth, and have truncated limbs and abnormal craniofacial development. This is most likely due to failure to maintain or differentiate the apical ectodermal ridge, a structure that is important for coordination of epithelial–mesenchymal interactions and is required for limb outgrowth and palatal and facial structure formation. p63 is expressed from two different promoters that generate two classes of proteins, TAp63, which contains the N-terminal transactivation(TA) domain, and the N-terminal truncated (DNp63) isoform, which lacks the transactivation domain. In addition, alternative splicing at the 3′ end of the transcripts generates three different C-termini: α, β, and γ. However, information about the protein expression levels of splice variants is not currently available due to the lack of isoform-specific antibodies, thus hampering the identification of specific functions.

It is emerging that p63 is involved in tumorigenesis and in controlling chemosensitivity.

The notion that the transcription factor p63 is essential for the formation of the epidermis and other stratified epithelia arises from the fact that mice lacking p63 show profound defects information of the epidermis. Remarkably, genetic mutations of p63 in man are causatively linked to ectodermal dysplastic syndromes. The involvement of p63 in the generation of skin cancer is related to its ability to control apoptosis through both extrinsic and intrinsic pathway. The loss of p63 in cancer can favor the survival of mutated cell clones that in the presence of p63 have not the possibility to develop towards cancer. Moreover, p63 is involved in the control of cell proliferation through the regulation of both the expression and activity of several growth factor receptors. In fact, p63 can affect the fibroblast growth factor receptor 2b (FGFR2b) receptor and its associated signaling, since it has been shown that there is a reduction in keratinocyte proliferation in the epidermis of Fgfr2b^−/− embryos. In addition, the proliferative capacity of p63^−/− mice keratinocytes could be also reduced as a consequence of increased expression of p21, a direct transcriptional repression target of DNp63. p63 functions as a selective modulator of Notch1-dependent transcription and function and also directly transactivates the Notch ligand, JAGG-1. Thus, a complex crosstalk between Notch and p63 could regulate the balance between keratinocyte self-renewal and differentiation. Moreover, a microarray analysis for p63 target genes, which was then validated in the genetically complemented mice, indicates that the function of p63 in epithelial development is at least in part mediated by IKKα (IκB kinase-α) and GATA-3 [70].

After the initial upregulation of Fas and Fas ligand expression induced by the acute exposure to UV, transcriptional inhibition of Fas ligand expression has been observed after 1 week of continuous UV irradiation in mice, with corresponding decrease in the number of apoptotic cells [65, 71]. Deregulation of Fas–Fas ligand-mediated signaling favors accumulation of p53 mutations, since Fas ligand-deficient mice with prolonged UV irradiation develop more p53 mutations than wild-type mice [54].

The tyrosine kinases human epidermal receptor (HER) family (epidermal growth factor receptor (EGFR), HER-2, HER-3, and HER-4) are transmembrane glycoproteins related to cell proliferation, differentiation, and apoptosis. Altered expression of the HER family is associated with several epithelial tumors such as breast carcinoma and esophageal squamous cell carcinoma. Small studies have also shown altered HER expression in localized squamous cell carcinoma when compared to normal skin. Given the paucity of unresectable or metastatic cSCC, reliable information on the frequency of EGFR expression is limited. One study of 13 metastatic specimens by IHC demonstrated that all had strong membranous expression of EGFR. Another study of locally advanced and nodally metastatic cutaneous SCC demonstrated EGFR expression above background in only 9 of 21 (43 %) specimens using a quantitative Western blotting technique. Another study using IHC and fluorescence in situ hybridization demonstrated higher levels of EGFR protein expression in cutaneous SCC than in the precursor actinic keratoses and an association between this elevated protein expression and higher EGFR gene copy number. One study, examining the role of EGFR in cSCC arising in the head and neck, demonstrated that primary lesions associated with subsequent metastases were more likely to overexpress EGFR (79 %) than those not associated with subsequent metastases (36 %). Interestingly, metastatic nodal disease exhibited only a 47 % rate of EGFR overexpression; EGFR overexpression in that study was not associated with EGFR gene amplification. Recently, a study on 55 primary tumors and 22 lymph node metastases derived from cSCC of the trunk and extremities has demonstrated that EGFR had no influence on prognosis. It is possible that altered EGFR expression may be associated with local recurrence, which is more frequently life threatening at other sites. HER-2 was negative in all samples and may play little part in CSCC progression as found in squamous cell carcinomas from other sites. High HER-4 expression in lymph node metastases was associated with poor prognosis suggesting a role in progression of cSCC of the trunk and extremities. It is possible that altered HER-4 expression occurs late and is present only in metastases. The altered coexpression of the HER family may play a role (i.e., EGFR/HER-4, HER-3/HER-4, and EGFR/HER-3/HER-4), but the small number of cases in this study meant this could not be analyzed.

Podoplanin, originally detected on the surface of podocytes, belongs to the family of type-1 transmembrane sialomucin-like glycoproteins. Although specific for lymphatic vascular (LV) endothelium, podoplanin is expressed in a wide variety of normal and tumor cells. Podoplanin plays an important role in preventing cellular adhesion and is involved in the regulation of the shape of podocyte foot processes and in the maintenance of glomerular permeability. Moreover, podoplanin is involved in LV formation and does not influence formation of blood vessels.

The expression of podoplanin is induced by the homeobox gene Prox-1, and a specific endogenous receptor was identified on platelets. Immuno histochemical detection of podoplanin/D2-40 in LECs was used in many studies to evaluate the LV microvascular density (LVMD) in peritumoral and tumoral areas and to correlate LVMD with lymph node status and prognosis. Podoplanin significantly increases the detection of lymphovascular invasion in different types of malignant tumors. Podoplanin expression was found in tumor cells of various types of cancer, such as vascular tumors, malignant mesothelioma, tumors of the central nervous system (CNS), germ cell tumors, and squamous cell carcinomas. This expression in tumor cells is useful for pathological diagnosis, and podoplanin seems to be expressed by aggressive tumors, with higher invasive and metastatic potential [72]. Recently, it was demonstrated that podoplanin expression was not associated with the presence of lymph node metastasis, but was a prognosticator of reduced survival indicating a locally aggressive tumor, with survival impact in cSCC. Altered expression of podoplanin is associated with mesothelioma, squamous cell carcinoma of oral mucosa, and germ cell tumors, suggesting that podoplanin may influence invasive and proliferative activity. As cSCC metastases occur preferentially via lymphatic vessels, podoplanin expression may be associated with disease progression. Hyperexpression has been related to undifferentiated skin tumors, but its impact on prognosis and metastasis has not been established. Podoplanin can be suggested as a possible target for the development of novel therapies, and its expression has to be studied in other settings to completely understand its role in cSCC development and progression.

The members of ras family, H-ras, K-ras, and N-ras, encode 21 kDa guanosine triphosphate (GTP)-binding proteins placed on the inner surface of the cell membrane [73]. They are functional in the transduction of signals to the nucleus in order to activate proliferation machinery, when growth factors are activated through intrinsic GTPase. Different human cancers show ras mutations in codons 12, 13, and 61 [74] with consequent continuous activation of ras-mediated signal transduction. Also human skin cancers contain mutations in all three members of the ras family [75].

Activated c-H-ras oncogenes derived from UV-exposed cSCC are tumorigenic for NIH 3T3 cells [76]. Therefore, the transfected NIH 3T3 cells induce tumors at the subcutaneous site of injection and spontaneous lung metastases in nude mice [77]. Also ras family mutations rely at pyrimidine-rich sequences, suggesting a direct role of UV irradiation [78]. Besides point mutations, deregulation of ras genes could be due to amplification and rearrangement of specific gene sequences [79, 80].

Mutations in INK4a/ARF tumor suppressor gene have been described in a subset of cSCCs. This locus encodes for two independent growth inhibitors: the cyclin-dependent kinase (CDK) inhibitor p16INK4a and the p53 activator p14ARF (mouse p19Arf). p16INK4a inactivates CDK4/6, favoring sequestration of the transcription factor E2F1 by the Rb tumor suppressor protein [81]. p14ARF binds human double-minute protein 2 (HDM2, mouse Mdm2), thereby blocking HDM2-induced translational silencing and degradation of p53 [82]. Also in these cases gene alterations were induced by UV. Recently, in a series of cSCC, all the alterations of the INK4a/ARF locus have been analyzed. Point mutations were less common (10 %) and loss of heterozygosity (32.5 %) and promoter hypermethylation of p16INK4a and p14ARF (36 %) more common [83].

Epigenetic alterations described in cancer progression include DNA methylation and histone modifications, the latter consisting in methylation, acetylation, phosphorylation, ubiquitination, and sumoylation, chromatin remodelling and different microRNA profile [84–86]. Gene specific methylation responsible of transcriptional control is described in cSCC development and progression [84–86]. Promoter methylation of FOXE1, a transcriptional factor involved in regulation of many organ development and located on chromosome 9q22, a region frequently lost in cSCC, is highly methylated in cSCC [87]. microRNAs (MiRNAs) appear to play an important role in the epigenetic regulation of cSCC at posttranscriptional level [88]. In fact, a recent study has demonstrated that a distinct microRNA profile is specifically induced by UV radiation [89]. Finally, mitochondrial DNA mutations, affecting displacement loop (D-loop) and other regions, are described in cSCC [90–92].

It is commonly considered a cSCC precursor lesion. It is related to solar exposure; therefore, it develops in sun-exposed skin areas, including face, neck, dorsal hands, and forearms, upper chest, back, and scalp [4, 5, 15, 101]. Due to its relation to exposure to sun, it generally affects middle-aged or older individuals. It can occur in younger individuals, but always in relation to longtime sun exposure or concomitant sensibility to UV damage, such as immunosuppression [15, 24]. AK spontaneously regresses and evolves to invasive cSCC in only 5–10 % of cases [102]. Different AK variants have been described including hypertrophic, atrophic, acantholytic, pigmented, proliferative, and bowenoid subtypes. Hypertrophic and proliferative variants are associated to a more aggressive biologic behavior [4, 5, 103, 104].

It is characterized by aggregates of atypical, pleomorphic keratinocytes that show nuclear atypia, dyskeratosis, and loss of polarity, extending variously through the epithelium till to cover all the thickness in the bowenoid variant, similar to Bowen’s disease [5, 105, 106] (Fig. 3.3a). The profile of dermo-epidermal junction in AKs shows small round epithelial buds protruding slightly into the upper papillary dermis. Solar elastosis in the dermis is always observed.

The two definitions are synonymous. Most of them occur in sun-exposed skin areas, such as the head, neck, and hands [107]. Moreover, mucosal surfaces and nail could be frequently involved [108]. Bowen’s disease in genital areas is also called erythroplasia of Queyrat. Clinical presentation is generally represented by erythematous, scaly patch or plaque [108]. Only 3–5 % of cases progress towards invasive cSCC, and this rate is higher for erythroplasia of Queyrat [109]. The invasive cSCC developed from Bowen’s disease seems to be more aggressive, since 20 % of these cases develop metastatic disease [110]. Histopatho logically, the epidermis is characterized by atypical cells with loss of polarity, high mitotic activity, pleomorphism, and greatly enlarged nuclei through the entire thickness (Fig. 3.3b). Multinucleated cells could be also observed [107]. Atypical cells may involve adjacent follicular epithelium and adnexal structures [5].

For low-risk, local lesions, the usual treatment is surgical excision, electrodessication and curettage, or cryosurgery. Destructive treatment methods leave no tissue to analyze for marginal control. Nevertheless, using these methods, the 5-year control rate in patients with low-risk primary lesions can be as high as 96 %.

For higher-risk tumors, the primary treatment is surgical excision. The key factor in determining the cure rate is the ability to achieve negative surgical margins. Surgery may be conventional or microscopically controlled, the latter procedure referred to as “Mohs’ surgery,” after its originator. In this procedure, the targeted lesion is excised and the circumferential margins are assessed microscopically for residual tumor. Margins remaining involved undergo repeated excisions, followed by histological assessment, until negative margins are obtained. Mohs’ surgery yields local control rates of 92–100 %, versus 38–87 % for standard surgical excision. Mohs’ surgery cure rates decrease as tumor grade increases, with a 45.2 % cure rate for grade 4 cSCC.