Epidemiology of BCC

Basal cell carcinoma of the skin (BCC) is the most prevalent cancer type in the world, affecting primarily Caucasian individuals (Roewert-Huber et al., 2007). It was first described in 1827 by Sir Arthur Jacob (Jacob, 1827), an Irish surgeon and ophthalmologist, who provided a detailed clinical description of three patients with the disease.

Because cancer registries do not collect data on non-melanoma skin cancers (BCC and squamous cell carcinoma, SCC) (Siegel et al., 2015, Cancer-Council-Australia, 2008, Torre et al., 2015), detailed statistics of BCC do not exist. However, it is estimated that there are approximately between one and 2.6 million new cases of BCC every year in the USA (Rubin et al., 2005, Rogers et al., 2010) and that the prevalence of BCC in Australia, the country with the highest incidence of this cancer type, more than doubles this number, with 884 cases per 100,000 individuals (Cancer-Council-Australia, 2008). Based on world statistics from Lomas et al. (2012) , it is possible to calculate a world incidence of BCC of 7 million new cases per year worldwide. BCC represents more than 80% of all non-melanoma skin cancers (Rubin et al., 2005) and, although relatively easy to treat when detected on time, it might affect quality of life by causing disfiguration after surgical management (Cancer-Council-Australia, 2008). Because of the high number of affected individuals, BCC also poses a very important economic burden in health systems worldwide (Miller and Weinstock, 1994, Cancer-Council-Australia, 2008).

Over the last few decades, the incidence of BCC, SCC and melanoma has increased at a rate of 4% per year worldwide and about 50% of individuals over 60 years of age with light skin tones are very likely to develop skin cancer (Chinem and Miot, 2011). BCC usually appears in individuals over 40 years of age and younger patients are rarely affected, unless they are high risk individuals (Crowson, 2006).

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Box1. Risk factors for the development of BCC (Modified from Rubin et al., 2005 with information from Crowson, 2006.

Exposure to ultraviolet (UV) light is the major risk factor for BCC (Rubin et al., 2005, Kasper et al., 2012, Iwasaki et al., 2012), and intermittent and intense exposure seem to confer greater risk for BCC than the overall amount of sun exposure (Rubin et al., 2005), which is perhaps why the number of cases

has increased not only in the elderly but also in younger individuals that expose themselves to high amounts of UV light due to recreational activities (Chinem and Miot, 2011, Cancer-Council-Australia, 2008).

Besides UV light exposure, other specific physical traits, contact to other carcinogens and diseases affecting the skin, have also been linked to BCC and can be found in Box 1.

After having one BCC tumor, the incidence of subsequent tumors increases by a factor of 10 as compared to the general population. Superficial and truncal tumors, along with age greater than 60 years and being a male, are all associated with an increased number of BCCs (Rubin et al., 2005).

2.3. Clinical and histological features

BCC usually occur in sun-exposed areas of the body, in 80% of cases in the head or neck (Rubin et al., 2005). Macroscopically, these tumors tend to appear as pearl-colored or eritematous papules with telangiectasia, and can present scaling or ulcerations. Based on their clinical features, BCCs can be classified as nodular, superficial or morpheaform BCC (Crowson, 2006).

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Figure 9. Basal cell carcinoma clinical subtypes. a. Nodular BCC in the face. Reproduced from Crawson, 2006. b. Recurrent superficial BCC on the abdomen. Reproduced from Rubin et al, 2005. c. Morpheaform BCC of the lip. Reproduced from Rubin et al., 2005.

Nodular BCC is the classical and most common form of BCC and its clinical presentation is typically the one described above (Figure 9a). It can also look like an enlarged pore in the central region of the face. Superficial BCC is usually observed as an eritematous plaque, but it can also resemble a scar and present either depigmentation or hyperpigmentation, or be multifocal (Figure 9b). Morpheaform BCC, also known as sclerosing, fibrosing or infiltrative BCC, is typically an indurated plaque resembling a scar with diffuse margins (Figure 9c).

The tissue architecture and growth pattern of BCC has proven to be relevant when predicting biological behavior. The histological classification extends beyond the clinical one to include more tissue characteristics. The histological features of a BCC can hint to its behavior. For example, nodular and superficial BCCs tend to grow slower, be less aggressive and easier to resect surgically and therefore have a better prognosis than mixed, morpheaform, metatypical, micronodular or infiltrative BCC histological subtypes (Crowson, 2006, Richter and Sexton, 2009).

Nodular BCC is the most common BCC subtype and it is histologically characterized by basaloid cell nests forming a well-defined mass, with borders in palisade and surrounded by a loose and mucinous stroma (Calonje E., 2012) (Figure 10a). Micronodular BCC is similar to the nodular subtype but the nests are small and multiple and the palisade

a b c

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borders are usually absent. Micronodular BCC can infiltrate the dermis and deeper layers of the skin, making its resection difficult and are usually surrounded by collagenized stroma (Calonje E., 2012) (Figure 10b). Superficial BCCs are frequently multifocal and are characterized by basaloid nodules appearing in the border of the epidermis that can be interconnected and are often pigmented (Calonje E., 2012, Crowson, 2006) (Figure 10c).

Morpheaform infiltrative or sclerosing BCC presents with small nests or columns of cells inside a collagenized and fibrotic stroma; the tumor is not well circumscribed and it commonly invades the reticular dermis and can infiltrate subcutaneous tissues (Crowson, 2006, Calonje E., 2012) (Figure 10d).

Finally, metatypical or basosquamous BCC is histologically characterized by a superficial or nodular tumor coexisting with infiltrating and elongated groups of cells with an irregular palisade border and cytoplasmic keratinization. It is correlated with a more aggressive Figure 10. Representative hematoxylin-eosin stainings of skin basal cell carcinoma histological subtypes. a. Nodular BCC. Reproduced from www.pathologyoutlines.com b.

Micronodular BCC. Reproduced from Wikibooks.org, Atlas histopathology of skin tumors c.

Superficial BCC. Reproduced from emedicine.medscape.com/article/276624-workup#c8 d.

Morpheaform BCC. Reproduced from www.pathologyoutlines.com e. Metatypical BCC. Reproduced from www.epathologies.com/pcoll/

a b

c d e

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disease and poorer outlook. It can be confused with SCC and it shows histological components typical of both tumor types. Whether metatypical BCC is an independent tumor type or a co-existence of two different carcinomas, is not clear and still under debate among different groups (Crowson, 2006, Calonje E., 2012, Tarallo et al., 2008, Tchernev et al., 2014) (Figure 10e).

Most BCCs are of mixed histology, while nodular BCC represents the 21% of the tumors, superficial BCCs the 17.4%, micronodular the 14.5%, morpheaform or infiltrative the 8.5%

and metatypical ~2.7% out of a retrospective study of 1039 consecutive BCCs (Sexton et al., 1990) and as reported in a metatypical BCC study by Allen et al. (2014) (Figure 11).

BCC’s histological subtype can also be used to classify these tumors in high or low

recurrence risk groups. According to the National Comprehensive Cancer Network (NCCN) of the U.S.A and the Australian Cancer Network clinical practice guidelines

(http://www.nccn.org/professionals/physician_gls/pdf/nmsc.pdf last accessed 15.11.2015 Figure 11. Basal cell carcinoma frequency per histological subtype. Each of the subtypes listed on the right is represented in the pie chart and corresponds to the frequency reported in Sexton et al., 1990 except for the metatypical subtype that corresponds to Allen et al., 2014.

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Figure 12. Interfollicular epidermis. The four principal layers of the epidermis are represented and labeled on the left. The basal layer contains a population of progenitor cells that divide to replicate themselves and the rest of the cells of the epidermis. The cell colored in dark red represents a cell carrying mutations that when dividing could give rise to BCC.

Redrawn with modifications fromhttp://www.servier.com/Powerpoint-image-bank.

and http://www.cancer.org.au/content/pdf/HealthProfessionals/

ClinicalGuidelines/Basal_cell_carcinoma_Squamous_cell_carcinoma_Guide_Nov_2008-Final_with_Corrigendums.pdf last accessed 15.11.2015), nodular and superficial BCCs have a low recurrence risk while metatypical, morphean and mixed types have a high recurrence risk.

2.4. Pathogenesis of BCC

The cells giving origin to BCC are progenitor cells found in the basal layer of the interfollicular epidermis (Figure 12) and in the upper infundibulum of hair follicles that accumulate mutations (Youssef et al., 2010, Ikram et al., 2004) and, as previously discussed, this mutational processes are usually the consequence of UV light induced mutagenesis.

Although most BCCs occur sporadically and as isolated cases, patients with Gorlin syndrome (GS) have a high susceptibility to develop BCCs. GS is a rare, autosomal dominant genetic disease (OMIM 109400) with high penetrance but variable expressivity characterized by a few to thousands of BCCs, most commonly appearing in the neck, face, chest and back at around 25 years of age. Other common clinical signs of Gorlin syndrome include odontogenic keratocysts, lamellar calcification of the falx, palmar and plantar pits,

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bifid ribs, kyphosis, macrocephaly, protruding forehead, facial milia, eye abnormalities, mesenteric cysts, heart and ovary fibromas, and an increased incidence of other tumors such as medulloblastoma and meningioma (Gorlin, 2004). GS incidence has been estimated to be of 1:57,000 individuals (Lo Muzio et al., 2011).

Through the screening for allelic losses with restriction fragment length polymorphisms (RFLP) in sporadic BCC tumors and due to the identification of relevant haplotypes in linkage studies of families with Gailani et al. (1992) narrowed down the causative gene to chromosome 9q13 and hypothesized said gene acted as a tumor suppressor.

Four years later, the first BCC gene was simultaneously reported by Johnson et al. (1996) and Hahn et al. (1996) as PTCH1, the homolog of a well-known gene in Drosophila melanogaster. PTCH1 had already been identified as a Hedgehog (Hh) signaling pathway inhibitor in flies and it was afterwards proven that this function was also relevant in mammals (Epstein, 2008).

The great majority of sporadic BCCs (80%) harbor inactivating mutations in at least one of their PTCH1 alleles (Athar et al., 2014), and most of the remaining tumors are caused by mutations in other members of the Hh signaling pathway.

2.4.1. The hedgehog signaling pathway

There are a handful of fundamental, highly conserved signaling pathways that coordinate important process during the development of organisms. One of these pathways is the Hedgehog signaling pathway (Ingham and McMahon, 2001), first recognized in 1980 when the Hh ligand was identified as an important regulator of body patterning while studying the process in Drosophila melanogaster larvae (Nusslein-Volhard and Wieschaus, 1980).

This pathway has been shown to be highly conserved all the way to mammals (Huangfu and Anderson, 2006) and it is key in development as it not only controls patterning but also induces cell proliferation in several tissues such as skin, central nervous system, limbs and skeleton. In 1993, the Drosophila Hh three mammalian homologs were identified and called Desert (DHH), Sonic (SHH) and Indian hedgehog (IHH). DHH is very similar to the Drosophila Hh, it is mostly expressed in the gonads and is therefore mostly involved in spermatogenesis. IHH is expressed in primitive endoderm, gut and bone metaphysis. SHH is

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the most broadly expressed one. In early embryogenesis it is expressed in several midline tissues and controls the patterning of the embryo while during organogenesis it is expressed in epithelial tissues. All three Hh proteins act together during development and can have complementary or redundant functions (Huangfu and Anderson, 2006, Varjosalo and Taipale, 2008, McMillan and Matsui, 2012). For a comprehensive description of all the mammalian developmental processes the Hh pathway is involved in, see Table 1 from Ingham and McMahon (2001) and McMahon et al. (2003).

Mammalian Hh proteins bind to the Patched transmembrane receptors (PTCH1 and PTCH2) after being secreted (Taipale and Beachy, 2001). PTCH receptors inhibit the Hh pathway in the absence of Hh, but when it binds to PTCH1, the G-protein-coupled receptor-like protein Smoothened (SMO), a second transmembrane protein and the pathway’s key signal transducer, is released (Ingham and McMahon, 2001).

SMO activates transcription and nuclear localization of the glioblastoma-associated proteins GLI2 and GLI1, the main effectors of the pathway, by releasing them from their inhibitory partners, the serine/threonine kinase Fused and suppressor of fused (SUFU). It also promotes the degradation of the repressor form of GLI (mainly GLI3). Once in the nucleus, GLI transcription factors induce the expression of multiple genes; among them MYC, BCL2, SNAIL and CCND, as well as Hh pathway genes such as GLI1, PTCH1 and HIP1 (Figure 13)(Pasca di Magliano and Hebrok, 2003, Amakye et al., 2013, Ng and Curran, 2011).

During normal mammalian maturation, the Hh pathway controls bone growth while in adults, the role of Hh is drastically diminished in most tissues and it is only reactivated for tissue repair as response to injury. An important exception is germ cells, where Hh regulates spermatogenesis and ovarian follicle maturation throughout adult life (Varjosalo and Taipale, 2008, McMillan and Matsui, 2012). When Hh signaling is altered during development, embryonic death or diseases involving abnormal development of musculo-skeletal tissues can arise (Varjosalo and Taipale, 2008, McMahon et al., 2003).

However, when the Hh pathway is abnormally activated in adults, it can lead to the development of several types of cancer, the most common and first described being BCC.

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Figure 13. Simplified hedgehog signaling pathway. A. When Hh is not present, SMO is inhibited by PTCH. Without SMO signaling, the complex formed by SUFU and Fused represses GLI and prevents its translocation to the nucleus and activation of the target genes. B. In the presence of Hh, PTCH releases SMO that prevents the inhibitory complex from repression GLI, which is translocated into the nucleus and activates the transcription of the Hh target genes. Redrawn with modifications from Pasca di Magliano and Hebrok, 2003.

a b

2.4.2. Hedgehog genes and their role in BCC

Aberrant upregulation of the Hh signaling pathway is the primary cause of BCC in both Gorlin syndrome individuals and sporadic tumors and most of the time (about 75-80% of cases), the initiation event can be traced to homozygous inactivation of the Hh pathway tumor suppressor gene PTCH1 (Epstein, 2008).

PTCH1 gene

The PTCH1 gene has 23 exons, spans 47 kb of coding sequence, and encodes a 1447 amino acid, twelve-transmembrane protein (uniprot.org/uniprot/ Last accessed 4.11.2015).

Although the originally identified oncogenic events in PTCH1 were point mutations, it is now known that loss of heterozygosity (LOH) of the 9q22.32 region containing PTCH1 can occur. While studying chromosomal imbalances in BCC, Teh and collaborators (Teh et al.,

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2005) found that 93% of the studied tumors had LOH of the 9q region and that the great majority (62%) carried an inactivating mutation in the remaining PTCH1 allele. Most interestingly, a significant fraction of the tumors with LOH (38%) did not have a change in copy number. This was considered to be possibly caused by a somatic recombination event causing “uniparental disomy” for the 9q region. We now know this is indeed what happens, and this mechanism is called copy neutral loss of heterozygosity (cnLOH). Both LOHs and cnLOHs of the PTCH1 gene are important oncogenic driver events in BCCs (Pan et al., 2010, Epstein, 2008, Jayaraman et al., 2014).

Gorlin syndrome patients carry a predisposing germline mutation and therefore, acquired somatic events provide the “second hit” for the inactivation of the remaining wild type (wt) PTCH1 gene and the eventual development of BCC(for example, (Tate et al., 2014)). On the other hand, for a sporadic BCC to arise, the tumor initiating cells require homozygous inactivation of PTCH1 that can be the consequence of LOH and persistence of a mutated allele, of a cnLOH event, or of the co-occurrence of two pathogenic point mutations (Pan et al., 2010). No matter what the final mechanism is, the ultimate inactivation of the tumor suppressor PTCH1 leads to a continuing loss of inhibition over SMO that is then able to activate the Hh signaling pathway in the absence of Hh ligands.

The study of the distribution of disease-relevant point mutations along the PTCH1 gene and its protein product has shown that some regions tend to harbor mutations more than others. Lindstrom et al., (2006) carried out an evaluation of 284 mutations and 48 SNPs submitted to a carefully curated PTCH1 mutation database. The study of the distribution of mutations along the PTCH1 coding sequence shows that overall, damaging mutations tend to cluster in the regions encoding the two large extracellular and the intracellular loops, when compared to the distribution of frequent SNPs, much more common in the second half of the protein and the sterol-sensing domain (SSD, Figure 14).

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Figure 14. Distribution of mutations in the PTCH1 protein. a. Mutations in 86 sporadic BCCs.

b. Distribution of 31 exonic SNPs reported to the PTCH mutation database. Black circles represent splice mutations, blue triangles nonsense mutations, red triangles missense events, yellow triangles silent events. The thicker line corresponds to the SSD. Figures reproduced with minor modifications from Lindström et al., 2006.

It is also possible to evaluate the PTCH1 mutations that have been submitted to cancer mutation databases. For example, the COSMIC database (Forbes et al., 2015, Cosmic Database) currently contains mutation information for 32,184 tumors of different types and tissue of origin. The data available for BCC is based on candidate gene or mutation scan and not on exome or whole genome sequencing studies, and COSMIC contains limited mutation information for 632 BCCs. The mutational status of PTCH1 is available for 550 of them and 27% (147 samples) have a PTCH1 mutation predicted to be pathogenic (Table 3) (www.cancer.sanger.ac.uk Last accessed 4.11.2015).

SMO gene

Smothened (SMO), a G-protein-coupled receptor-like protein, is the second most common Hh gene mutated in BCCs, and it can drive tumor progression in the absence of PTCH1 mutations (Reifenberger et al., 2005). The SMO gene is located in chromosome 7q32.1 and contains 12 exons in 24 kb of genomic DNA (uniprot.org/uniprot/ Last accessed 4.11.2015). It encodes a seven-pace transmembrane protein of 787 amino acids length.

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Activating missense mutations in SMO were first described in 1998 (Xie et al., 1998). Xie and collaborators were looking for additional mechanisms of activation of the Hh pathway besides PTCH1 mutations and scanned the full coding sequence of SMO with PCR of the exons and subsequent single strand conformation polymorphism (SSCP) tests, for activating mutations. In 47 tested sporadic BCC tumors, they identified two different mutations (p.R562G and p.W535L) in three tumors in the absence of PTCH1 mutations or

Table 3. Pathogenic mutations in BCC reported in the COSMIC database and Reifenberger et al., 2005. Data from Reifenberger et al., 2005 and COSMIC database, last accessed on 4.11.2015. *Pathogenicity evaluation automatically provided by COSMIC and performed with FATHMM: Functional Analysis through Hidden Markov Models (cancer.sanger.ac.uk, fathmm.biocompute.org.uk). N/A: mutational status not available.

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LOHs. The SMO variants were absent from the blood of the individuals and a control set of 100 samples, confirming their somatic status. When mutant SMO protein was transfected into mouse embryonic fibroblasts (MEF), density-dependent growth inhibition was impaired and cells lost their normal morphology when compared to control cells transfected with wild type SMO.

The authors suggest that these mutations may not impair the interaction of SMO with PTCH1 but instead cause its constitutive activation, since the p.W535L mutation occurs in a pocket conserved among several G-protein coupled receptors that keeps them in a latent state when unaltered (Xie et al., 1998). The relevance of the p.W535L mutation was reinforced by a later report of a collection of 97 sporadic BCCs where the mutation was found in 21% of cases, although the PTCH1 mutational status was not assessed (Lam et al., 1999).

Since the description of these two activating mutations, additional ones have been identified and have been shown to activate Hh signaling as well (Mao et al., 2006, Reifenberger et al., 1998). The mutational status of SMO in BCCs has been evaluated in 157 samples reported in COSMIC. 14% of the tumors have a SMO mutation and around half of them are predicted to be pathogenic (Table 3, www.cancer.sanger.ac.uk Last accessed

Since the description of these two activating mutations, additional ones have been identified and have been shown to activate Hh signaling as well (Mao et al., 2006, Reifenberger et al., 1998). The mutational status of SMO in BCCs has been evaluated in 157 samples reported in COSMIC. 14% of the tumors have a SMO mutation and around half of them are predicted to be pathogenic (Table 3, www.cancer.sanger.ac.uk Last accessed