Penile cancer is a relatively rare disease in devel- oped countries. The incidence in Europe is 1 per 100,000 men per year with a mean age at diag- nosis of 60 years. Higher incidence rates have been reported in Africa, Asia, and in parts of South America [1–3]. Approximately 95% of all penile cancers in developed countries are squa- mous cell carcinomas (SCCs); the remaining 5%
are nonsquamous primary neoplasms such as sarcomas, melanomas, basal cell carcinomas, and lymphomas. Penile carcinomas include several different histological subtypes. The majority are well-differentiated keratinizing SCCs; the second most common subtype is verrucous carcinoma, and less prevalent types include basaloid carcinomas and warty carcinomas. A further subtype is the giant condyloma of Buschke-Löwenstein (GCBL). There is confusion in the literature with regard to this tumor, be- cause some investigators believe that GCBL is simply a clinical variant of verrucous carcinoma;
in contrast, others regard these lesions as dis- tinct entities. Although the GCBL tumor shows none of the histological criteria for malignancy, it behaves like a carcinoma, with a tendency to compress and displace deeper tissues by down- ward growth rather than by infiltration or metas- tasis. Invasive penile cancer initially occurs on the glans (48%), the prepuce (25%), the glans and prepuce (9%), the coronal sulcus (6%) and the penile shaft (2%) [4].
Three preneoplastic lesions of the penis have been described: erythroplasia of Queyrat, Bowen’s disease, and bowenoid papulosis. These
lesions have also been referred to as high-grade penile intraepithelial neoplasia (PIN), dysplasia, and carcinoma in situ. However, the minor his- tological differences between bowenoid papulo- sis and Bowen’s disease and erythroplasia of Queyrat do not allow for an accurate diagnosis on the basis of histological findings alone. Essen- tially, they are distinguished on the basis of clin- ical features. Bowenoid papulosis and Bowen’s disease both occur on the penile shaft, whereas erythroplasia is found on the glans or pre- puce. Characteristic features include papules in bowenoid papulosis, crusted and scaly plaques in Bowen’s disease, and erythematous plaques in erythroplasia of Queyrat. Bowenoid papulosis usually presents in men aged 20 to 40 years, Bowen’s disease at 30 to 50 years, and erythro- plasia of Queyrat at 40 to 60 years. The incidence of progression to invasive SCC is more common for erythroplasia of Queyrat than for Bowen’s disease, with an incidence varying from 10% to 33% [5,6]. Patients with carcinoma in situ of the penis are usually uncircumcised.
Etiology
The development of penile cancer is most likely a stepwise chain of events over a period of years, from preneoplastic lesions to SCC. The etiology of penile SCC is probably multifactorial [7]; poor personal hygiene associated with smegma reten- tion, and phimosis are the most commonly incriminated. Other factors that have been asso-
A Scientific Understanding of the Development of Penile Tumors
T.R. Leyshon Griffiths and J. Kilian Mellon
275
ciated with this tumor include balanitis xerotica obliterans (BXO), a history of smoking, and human papillomavirus (HPV) infection. Men exposed to psoralens and ultraviolet irradiation for psoriasis are also at higher risk of develop- ing penile cancer.
Personal Hygiene and Circumcision
Although there are data suggesting that circum- cision at birth provides excellent protection against penile cancer, equally low incidence rates can be achieved in uncircumcised males who practice good hygiene. Indeed, the incidence of penile cancer has been reported to be falling in uncircumcised men. In one study, a decreasing penile cancer rate of 0.82/100,000 was found in Denmark (where circumcision is uncommon) compared with an incidence of 1/100,000 in the United States [8].
Phimosis and Balanitis Xerotica Obliterans
Phimosis has been reported to be present in 44%
to 85% of men with penile SCC [7]. Balanitis xerotica obliterans (BXO) of the penis is a well- recognized inflammatory dermatosis that causes atrophic and sclerotic changes; this may lead to secondary problems with phimosis and meatal strictures. The association of SCC arising on a background of BXO has not been examined as fully in males as has lichen sclerosus for vulvar SCC in females. It is known that about 40% of
penile SCC also have histological changes of BXO [9,10]. However, the pathogenesis of penile SCC developing in BXO has never been described. One study described 86 patients with penile BXO, three of whom (3%) subsequently developed SCC [11]. However, phimosis often accompanies BXO. It therefore remains unclear whether phimosis is a more important etiologi- cal factor than BXO itself.
Smoking
A consistent association has been found be- tween penile cancer and smoking that is dose dependent [7]. In a multivariate analysis of risk factors in 503 patients with penile cancer and age-matched controls, phimosis (odds ratio 7.2), smoking (odds ratio 1.7), chewing tobacco (odds ratio 4.1), or the use of snuff (odds ratio 4.2) were shown to be independent variables [12].
The Human Papillomavirus
Prevalence in Penile Cancer (Table 23.1)
The HPV has been detected in 15% to 80% of penile carcinoma specimens, depending on the sensitivity of the detection method and the selection of the tumor type [13–20]. In studies of more than 100 patients and utilizing polymerase chain reaction (PCR)-based techniques, HPV prevalence in penile carcinoma is 22% to 63%
[15,19,20]. In the largest multicenter study of patients with penile cancer to date, of 142
Table 23.1. Prevalence of human papillomavirus (HPV) DNA in penile lesions in Europe and the United States
Overall HPV-DNA High-risk HPV-DNA Genital lesion positivity (%) positivity (%)
Penile SCC 40 80–90
Verrucous 20 30
Keratinizing 30 80
Basaloid 80 100
Warty 80 100
Penile carcinoma in situ 90 80
Anogenital condylomas 100 5–10
Low-risk HPVs: -6, -11.
High-risk HPVs: -16, -18, -31, -33, -39, -42, -51, -52, -53, -54.
patients from the United States and Paraguay, HPV-DNA was detected in 40 (42%) patients [19]. DNA amplification was performed using a novel, sensitive, broad-spectrum HPV PCR assay. There was no significant difference between HPV prevalence in tumors from Paraguay and the United States.
A comparison of HPV prevalence rates in penile, cervical and vulvar carcinoma indicate that the etiology and the pathogenetic pathways of penile SCC may parallel the pathogenetic pathways of vulvar, but not cervical, carcinoma.
Indeed, the overall prevalence of HPV-DNA in penile carcinoma is lower than in cervical carci- noma (approximately 100%) [21,22] and similar to that reported for vulvar carcinoma (approxi- mately 50%) [23]. Moreover, the correlation between HPV-DNA detection and histological tumor subtypes is similar in vulvar and penile carcinoma. In one study, the basaloid subtype of penile carcinoma was HPV-DNA–positive in 80% (12 of 15) cases and was associated with HPV-16 [19]; in another study, HPV-16 was detected in 82% (nine of 11) cases of the basa- loid subtype [24]. With regard to the warty subtype of penile carcinoma, overall HPV- DNA positivity was 100% (five of five) [19]
and HPV-16 positivity was 60% (three of five) [24].
The HPV status of verrucous carcinomas of the penis has been assessed in several reports; of 26 cases, only three have been found to be posi- tive for HPV-DNA (12%), and all were positive for low-risk HPVs [13,15,25–28]. Some authors advocate that screening for HPV may be a useful adjunct in differentiating GCBL from verrucous carcinoma. Indeed, published reports suggest that GCBL is always associated with HPV infec- tion. However, in view of our current under- standing, the presence or absence of specific HPV types cannot be used to predict malignant transformation.
Prevalence in Carcinoma in Situ
Approximately 90% of carcinoma in situ are HPV-positive, of which around 80% are high- risk HPVs; HPV-16 is the most frequently detected [13,19]. These findings suggest that carcinoma in situ may be a precursor lesion to only a subset of invasive carcinomas, which would include the basaloid and warty subtypes.
Oncogenic Effect of Human Papillomavirus
High-Risk and Low-Risk Human Papillomavirus
The ability of genes to extend the life span of cells in culture indefinitely is termed immortal- ization. By contrast, transformation requires the acquisition of at least some of the properties characteristic of malignant cells, in particular autonomous proliferation. So-called low-risk HPV subtypes 6 and 11 have a strong tendency to induce anogenital condylomata, but are rarely associated with genital cancer. They do not have immortalizing or transforming properties. In contrast, so-called high-risk HPV subtypes 16, 18, 31, 33, 39, 42, 51, and 54 are linked with genital carcinoma. Immortalizing activities of HPV-16 and HPV-18 DNA have been demonstrated in cultures of primary human keratinocytes, cells that resemble the normal target of the virus [29].
High-risk HPVs exert their oncogenic effect by expressing the oncoproteins E6 and E7, which bind to and inactivate the p53 and retinoblas- toma (Rb) tumor-suppressor products, respec- tively. These activities obviate the need for (epi)genetic alterations, leading to disturbance of the p14ARF/MDM2/p53 and p16INK4A/cyclin D/Rb pathways. Alteration of these pathways is among the most common alterations seen in human carcinomas, and it has been firmly estab- lished that inactivation of these pathways is essential for the genesis of the great majority of human malignancies [30].
HPV-E7 Viral Oncoproteins and p16INK4A/Cyclin D/Rb Pathway
The protein product of the Rb tumor-suppressor gene and other Rb family members, including p107 and p130, can block cell cycle progression from the G1 to the S phase. In its active state, Rb is hypophosphorylated and binds to a number of transcription factors, most notably members of the E2F family. Cyclin-dependent kinase (CDK) phosphorylation of Rb inactivates Rb. As a con- sequence, transcription factors are released from Rb, allowing them to mediate transcriptional activation of S-phase genes. The binding of
HPV-E7 proteins to Rb and Rb-related proteins equates with functional inactivation of Rb;
higher affinity binding can be detected for E7 proteins of high-risk HPVs than for those of low-risk HPVs such as HPV-6 and HPV-11.
Additionally, the E7-induced ubiquitin-mediated degradation of Rb appears to be essential to efficiently overcome cell cycle arrest. E7 may also degrade Rb family members [31].
There is substantial genetic evidence that only one component in the p16INK4A/cyclinD/Rb path- way needs to be inactivated for neoplastic clonal expansion [32,33]. Evidence from studies in lung cancer suggests that there are important phenotypic differences between cells that have inactive Rb but active p16 function, as compared to cells with active Rb and inactive p16 [32,34].
For example, Rb undergoes mutational inactiva- tion in the genesis of 90% of small cell lung cancers, whereas in non–small-cell lung cancer, the preferential target is p16. In many SCCs that do not reveal viral involvement, the p16INK4A/cyclinD/Rb pathway is commonly dis- rupted through mutation, deletion, or hyperme- thylation of the p16 gene, resulting in reduced or absent p16 expression [35,36]. Data from func- tional studies in mice suggest that overexpres- sion of the polycomb group (PcG) gene BMI-1 can provide a further alternative mechanism to downregulate p16 [37]. In contrast, where the Rb protein is functionally inactive either as a consequence of gene mutation or binding of high-risk HPV-E7 proteins, p16 is released from negative feedback by Rb, and is expressed at enhanced levels in these tumors [38]. P16 is a negative regulator of the cell cycle; its primary action is to inhibit interaction between CDKs 4/6 with cyclin D1. It also simultaneously releases free p27Kip1, which can now transfer to form inhibitory CDK2/cyclinE/p27 complexes.
HPV E6 Viral Oncoproteins and p14ARF/MDM2/p53
The p53 tumor-suppressor gene is located on chromosome 17p13.1 and functions as a nega- tive regulator of cell growth. In response to DNA damage, it can induce G1 arrest or apoptosis. It is known that the p53 protein is a transcription factor that blocks cell proliferation and mediates G1 arrest via the induction of the p21 gene (WAF-1). The 21-kd protein product of this gene
encodes for an inhibitor of the cyclin-dependent kinases, CDK2 and CDK4; consequently, via hypophosphorylation of Rb and subsequent E2F binding, the cell cycle is prolonged in the G1 phase.
Mutations in the p53 gene are very common in almost all solid tumors, with the exception of anogenital carcinomas [39]. This suggests that HPV targeting of p53 protein is an alternative to p53 gene mutation as a mechanism for p53 inac- tivation. The carcinogenic effect of HPV may be explained, in part, by the transforming viral protein E6, which binds to and induces the degradation of p53 protein through the ubiqui- tin pathway [40]. It has been proposed that the existence of a common polymorphism of the p53 gene at codon 72, which results in trans- lation to either proline (p53Pro) or arginine (p53Arg), could play a critical role in the devel- opment of mucous and cutaneous SCC [41]. One study has shown that the protein E6 from HPV- 16 and HPV-18 is more effective at degrading p53Arg than p53Pro in vivo; HPV-11 E6 is less active toward p53Arg and inactive with p53Pro [42].
High-Risk HPV-E6 Viral Oncoprotein and p53-Independent Activities
Activation of Telomerase
The E6 protein has been implicated in the acti- vation of the enzyme telomerase—a potential mechanism for HPV-induced immortalization [43]. Mammalian telomeres are structures at the chromosomal tips consisting of multiple repeats of TTAGG, which shorten as a function of divi- sion in vivo as a consequence of an intrinsic inability to replicate the 3’ end of DNA. Telom- erase replaces the telomeric repeats and is not normally expressed in somatic cells. Activation of telomerase enables cells to escape from the senescence signaled by telomeric shortening.
Regulation of telomerase activity has been shown to occur primarily through the level of expression of the human telomerase reverse transcriptase (hTert) gene, encoding the cat- alytic subunit. The precise mechanism of telom- erase activation by E6 protein is unknown; the current favored mechanism is transcriptional activation of the promoter of the hTert gene [44].
Interaction with PDZ Domain-Containing Proteins
High-risk E6 proteins interact with several PDZ domain-containing proteins like hD1g [45], MUPP1 [46], and MAG-1, -2, -3 [47], resulting in their ubiquitin-mediated degradation. PDZ domains consist of approximately 90 amino acid long protein–protein interaction units located at areas of cell–cell contact, such as synaptic junc- tions in neurons and tight junctions in epithelial cells. It is suggested that PDZ proteins act as molecular scaffolds.
p14ARF/MDM2/p53 and Pathways in Human
Squamous Cell Carcinoma of the Penis
In the largest study to date, the frequency of p53 gene mutations was 33% (seven of 21) in SCC of the penis [48]. This included only 22% (two of nine) HPV-positive tumors, and 42% (five of 12) HPV-negative tumors. In a study of 45 French men with penile SCC, the p53 Arg/Arg genotype was not a risk factor for the development of SCC, and no correlation was found between p53 poly- morphism at codon 72 and the presence of HPV- DNA [49].
Nuclear immunopositivity for p53 has been detected in 26% to 41% of cases [48,50,51]. This difference could be attributable to the different antibodies used in these series. The simultane- ous presence of p53 protein accumulation and DNA of high-risk HPV types appears to be a common finding in cervical and penile lesions.
In one study, p53 immunopositivity was detected in 40% (17 of 42) penile carcinomas, most of these being also HPV-DNA positive [50]. P53 immunoreactivity is an independent prognostic factor for lymph node metastasis in penile car- cinoma [51].
In contrast, two studies have compared the clinical outcomes of patients with HPV-positive versus HPV-negative penile carcinomas. Both re- ported no difference in lymph node metastasis rates or survival [52,53].
p16INK4A/Cyclin D/Rb Pathways in Human Squamous Cell Carcinoma of the Penis
In a recent study, alterations pointing to a dis- turbed p16INK4A/cyclin D1/Rb pathway are com- monly present in penile carcinomas [54]. Three alternative mechanisms of disruption were identified (Fig. 23.1); activity of high-risk HPV and the resulting increase in p16INK4Aexpression was the most frequently detected mechanism, followed by p16INK4Ahypermethylation and BMI- 1 overexpression. Strong p16 immunostaining was found in 65% of the tumors containing HPV-DNA; this frequency increased to 81%
when only the high-risk HPV-positive cases were considered, and 92% when the analysis was restricted to the HPV-16–positive cases with E6/E7 expression. In contrast, in tumors without HPV-DNA, only 6% stained strongly for p16. The
CDK4&6
p16INK4a (secondary to p16 hypermethylation or overexpression of BMI-1) Cyclin D
Rb-E2F1-3
complex Phospho-Rb
Cell cycle progression
+ -
E2F1-3
X
A
CDK4&6
p16INK4a Cyclin D
Rb-E2F1-3
complex Rb-E7
complex
Cell cycle progression
+ - +
E2F1-3 E7
B
Fig. 23.1. Simplified models depicting effect of alterations in the p16INK4A/cyclin D/Rb pathway on cell cycle progression and p16 protein expression. A: Effect of p16 gene alteration. B: Effect of HPV-E7 oncoprotein binding to Rb.
frequency of p16INK4Apromoter methylation was higher in HPV-DNA–negative tumors (21%) than in HPV-DNA–positive cases (10%). The suggestion again is that penile carcinoma is eti- ologically heterogeneous, with only a proportion of cases attributable to HPV infection.
Conclusion
The great majority of penile carcinomas diag- nosed in Europe and the United States are of a nonbasaloid, nonwarty histological type. At present, an etiological relationship with HPV seems most plausible for penile carcinomas of the basaloid or warty subtypes; the precursor lesion is likely to be carcinoma in situ. In con- trast, the precursor lesion for keratinizing SCC or verrucous carcinoma of the penis is not well established. Moreover, a further assessment of BXO in the absence of phimosis, or after cir- cumcision, is needed to determine its role as an independent risk factor for the development of invasive penile cancer. It is also becoming clear that the mere presence of HPV is insufficient to have prognostic implications. Demonstration of HPV-mediated alterations in signaling pathways appears to be necessary. Currently, detection of elevated p16 expression appears to be a poten- tial biomarker of HPV-mediated Rb inactivation in penile SCC.
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