**2. Penile cancer**

Penile malignancies are thought to arise from the accumulation of multiple mutations that may occur as consequence of progressive genetic instability. This intricate process of genetic instability may be caused by environmental factors, such as history of intense smoking, penile tears, phimosis, and poor genital hygienic habits. (Chaux & Cubilla 2012). In addition, a recent study conducted by Chaux et al. (2011) also described the poor education, penile chronic inflammation, genital warts and Human papillomavirus (related to number of sexual partners during lifetime) as environmental factors to malignant transformation.

There is a worldwide geographic difference in occurrence of penile malignancies that could be caused by differences in socio-economic status, cultural and religious conditions (Bleeker et al. 2009, Chaux & Cubilla, 2012). The higher incidences are frequent in tropical or subtropical regions of Latin America, Asia and Africa but have uncommon incidence rates in Europe, Japan, USA and Israel (Cubilla 2009). Recently, were reported higher incidence rates in underdeveloped regions such as Africa, South America, and Asia (2-4/100000 inhabitants) as compared with North America (United States) and Europe (0.3-1/100000 inhabitants) (Chaux & Cubilla, 2012). Pow-Sang et al. (2010) also described the penile malignancies prevalence rate among different populations. Prevalence rates in developed countries as Israel (0.1/100 000) and United States (0.3-1.8/100 000) and interestingly, compared with underdeveloped coun‐ tries such as Uganda (2.8/100 000) and Brazil (1.5-3.7/100 000). Once again confirming the disease geographical difference and the influence of country development.

Squamous cell Carcinoma represents vast majority of histological subtype of primary penile malignancies with heterogeneous features due to differences in morphology pathogenesis and prognosis (Hakenberg & Protzel, 2012; Stankiewicz et al., 2012; Syed et al., 2012). Knowledge of origin and progression of penile squamous cell carcinoma depends on an intricate relation between anatomy and histopathology.

cell cycle checkpoints and to entrance in S1 phase of cell cycle, leading to disruption of normal

222 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Following cell division, infected cells leave the basal layer, migrate towards the suprabasal regions and begin to differentiate. Increased understanding of cervical pathogenesis has led to confirmation of HPV as an etiological agent for several cancers and consequently to the development of preventive vaccines targeting HPV antigens for the control of HPV-related cancers. HPVvaccinewasdevelopedas a result ofthe achievement of core technologies,that are able to produce virus-like particles (VLPs), which, in turn, are able to mimic the natural virus andelicithigh-titersofvirusneutralizingantibodies.Withtheprogressthroughadvancedstages of clinical trials and further exploration of combinatorial strategies, there is a great promise for significantadvancesalsointhefieldoftherapeuticHPVvaccinedevelopment,notonlytocervical cancer, but other several malignancies related to HPV infection. Moreover, in this chapter we discuss the current status of HPV vaccines as well as the most common associated factors that might interfere on establishment of strategies that could control the HPV infections and the

Penile malignancies are thought to arise from the accumulation of multiple mutations that may occur as consequence of progressive genetic instability. This intricate process of genetic instability may be caused by environmental factors, such as history of intense smoking, penile tears, phimosis, and poor genital hygienic habits. (Chaux & Cubilla 2012). In addition, a recent study conducted by Chaux et al. (2011) also described the poor education, penile chronic inflammation, genital warts and Human papillomavirus (related to number of sexual partners

There is a worldwide geographic difference in occurrence of penile malignancies that could be caused by differences in socio-economic status, cultural and religious conditions (Bleeker et al. 2009, Chaux & Cubilla, 2012). The higher incidences are frequent in tropical or subtropical regions of Latin America, Asia and Africa but have uncommon incidence rates in Europe, Japan, USA and Israel (Cubilla 2009). Recently, were reported higher incidence rates in underdeveloped regions such as Africa, South America, and Asia (2-4/100000 inhabitants) as compared with North America (United States) and Europe (0.3-1/100000 inhabitants) (Chaux & Cubilla, 2012). Pow-Sang et al. (2010) also described the penile malignancies prevalence rate among different populations. Prevalence rates in developed countries as Israel (0.1/100 000) and United States (0.3-1.8/100 000) and interestingly, compared with underdeveloped coun‐ tries such as Uganda (2.8/100 000) and Brazil (1.5-3.7/100 000). Once again confirming the

Squamous cell Carcinoma represents vast majority of histological subtype of primary penile malignancies with heterogeneous features due to differences in morphology pathogenesis and prognosis (Hakenberg & Protzel, 2012; Stankiewicz et al., 2012; Syed et al., 2012). Knowledge

development of penile carcinoma associated to this infection.

during lifetime) as environmental factors to malignant transformation.

disease geographical difference and the influence of country development.

cell cycle controls.

**2. Penile cancer**

The anatomy of the penis is complex and has important implications to define predictive risk model and delineate the prognostic factors (Chaux & Cubilla, 2012). The same authors described 3 anatomical compartments in the penis (Glans, Foreskin and Coronal Sulcus) where the malignant neoplasms may be originated (Fig 1). However, the penile malignant neoplasms have a predilection to originate first on the Gland followed by Foreskin inner mucosa and lastly the Coronal Sulcus is rarely affected by neoplastic entity.

**Figure 1.** Paraurethral longitudinal section presenting anatomical levels of the Penis. **CC:** Corpus Cavernosum; **CS:** Cor‐ pus Spongiosum; **LP:** Lamina Propria; **SF:** Skim of the Foreskim and **TA:** Tunica Albuginea. (Adapted from Chaux & Cu‐ billa 2012).

Recently, Hernandez et al. (2008) performed an epidemiological study with 4967 United States men with the diagnosis of penile squamous cell carcinoma. Thirty four percent of patients (1712) presented neoplasms arising in gland, 13.2% in prepuce, 5.3% in penis shaft, 4.5%, in overlapping of penis, and 42,5% in unspecified site. Lesions generally initiate on the glans and slowly extend to involve completely the glans and shaft of the penis. During the neoplasm progress Buck's fascia act as a natural barrier to local tumor invasion defending the corporal bodies from tumoral expansion (Pow-Sang et al. 2010). This assessment is schematically illustrated in figure 2.

The anatomy of the penis presents a pivotal role in tumor invasion and prognosis of cancer. Moreover, the TNM staging system is based, at least partially, on the commitment of these anatomical levels (Velazquez et al. 2010). The glans can be divided in 4 levels: squamous epithelium, lamina propria, corpus spongiosum, and corpus cavernosum (corpus spongio‐ sum, and corpus cavernosum are subdivided by the tunica albugínea). Anatomical levels in the foreskin, like in glans, are divided in squamous epithelium, lamina propria, dartos muscle, and outer skin (Chaux & Cubilla, 2012).

**Figure 2.** Natural history of penile cancer (Adapted from Pow-Sang et al. 2010).

A previous study suggests that different tumor histological features could be based on anatomical site. This hypothesis is sustained by the histological differences among the urethral segments and their corresponding neoplasms (Velasquez et al. 2005). As discussed previously, Squamous Cell Carcinoma of Usual Type (SCCUT) is the most frequent histopathologic diagnoses in penile malignancies (Chaux & Cubilla, 2012; Stankiewicz et al. 2012) affecting around 48%-65% of all type of penile carcinoma. Additionally, Epstein et al. (2011) reported 11 more subtypes of SCCUT (Table 1). Macroscopic features of SCCUT range from endophytic to irregular exophytic masses, presenting white-to-gray coloration. However, the reddish pigmentation also can be observed (Chaux et al. 2010). Microscopilly, the SCCUT is similar to oral, vulvar and cervical Squamous Cell Carcinomas (Cubilla et al. 2001). SCCUT may vary from well-differentiated tumors to anaplastic entities. Other presented feature is keratiniza‐ tion, ranging from highly keratinized, presented in well-differentiated tumors, until scarce or minimal keratinization observed in anaplastic neoplasm. Chaux et al. (2010)

Lopes et al. (2002) performed a study that aimed to investigate the p53 in Brazilian patients with PSCC to establish a new prognostic factor for lymph node metastasis and its possible influence on prognosis. This study observed the nodal stage as a factor that influenced survival (independent risk factors) in the univariate and multivariate analyses. Gunia et al. (2012) shown that p16INK4a is a good prognostic marker for penile squamous cell carcinomas, surpassing the prognostic impact of histologically confirmed koilocytosis. In their study, p16INK4a expression predicted better cancer specific survival rates. Furthermore, p16INK4a can be useful in differentiate subtypes of PSCC. According Chaux & Cubilla (2012), warty carcinomas tend to be p16INK4a positive, whereas giant condylomas and papillary and

**Table 1.** Classification of Squamous Cell Carcinomas (SCCs) of the penis (Adapted from Chaux & Cubilla 2012)

**Classification of SCCs of the penis**

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**Subtype Frequency (%)**

Usual SCC 48-65 Basaloid carcinoma 4-10 Warty carcinoma 7-10 Verrucous carcinoma 3-8 Papillary carcinoma 5-15 Sarcomatoid carcinoma 1-3 Mixed carcinomas 9-10 Adenosquamous carcinoma 1-2 Pseudohyperplastic carcinoma <1 Carcinoma cuniculatum <1 Pseudoglandular carcinoma <1 Warty-basaloid carcinoma 9-14

Medical record analysis of 145 men with penile squamous cell carcinomas was performed to identify prognostic factors for lymph node involvement (Lopes et al. (1996)). The authors found that lymph node metastasis presents correlation with tumor thickness, lymphatic and vascular embolization. Interestingly, univariate analysis did not reached statistically significant values to pathologic stage of primary tumor, clinical lymph node stage (cN), and histological grade. However, histological grade may be considered an important prognostic factor in penile squamous cell carcinoma. In accord with Cubilla AL. 2009 these prognostic factors are

Currently, different methods are employed to grade Penile Squamous Cell Carcinomas. For instance, Akhter et al. (2011) uses the Broder's system as histological grade system in Squamous Cell Carcinoma. In the Broder's system Penile Squamous Cell Carcinomas is stratified in 4 grades levels based only in differentiation of the cells: Grade I (well differentiated) presenting <25% undifferentiated cells; Grade II (Moderately differentiated) presenting <50% undiffer‐

predictive to the nodal spread, metastasis and tumoral dissemination.

verrucous carcinomas are consistently negative.

Evidences from pertinent literature indicate the involvement of groin lymph nodes as the most relevant and unfavorable prognostic factors predicting cancer-specific survival in patients with penile squamous cell carcinoma. Numerically, in the same review, the 5-year cancerspecific survival rate for those presenting cN0 tumors were between 75% and 93%, compared with a 5-year cancer-specific survival rate for those presenting cN3 tumors ranging between 20% and 34%. There is a substantial decrease in the survival rates with N progression (Novara et al. 2007).


**Table 1.** Classification of Squamous Cell Carcinomas (SCCs) of the penis (Adapted from Chaux & Cubilla 2012)

A previous study suggests that different tumor histological features could be based on anatomical site. This hypothesis is sustained by the histological differences among the urethral segments and their corresponding neoplasms (Velasquez et al. 2005). As discussed previously, Squamous Cell Carcinoma of Usual Type (SCCUT) is the most frequent histopathologic diagnoses in penile malignancies (Chaux & Cubilla, 2012; Stankiewicz et al. 2012) affecting around 48%-65% of all type of penile carcinoma. Additionally, Epstein et al. (2011) reported 11 more subtypes of SCCUT (Table 1). Macroscopic features of SCCUT range from endophytic to irregular exophytic masses, presenting white-to-gray coloration. However, the reddish pigmentation also can be observed (Chaux et al. 2010). Microscopilly, the SCCUT is similar to oral, vulvar and cervical Squamous Cell Carcinomas (Cubilla et al. 2001). SCCUT may vary from well-differentiated tumors to anaplastic entities. Other presented feature is keratiniza‐ tion, ranging from highly keratinized, presented in well-differentiated tumors, until scarce or

224 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Evidences from pertinent literature indicate the involvement of groin lymph nodes as the most relevant and unfavorable prognostic factors predicting cancer-specific survival in patients with penile squamous cell carcinoma. Numerically, in the same review, the 5-year cancerspecific survival rate for those presenting cN0 tumors were between 75% and 93%, compared with a 5-year cancer-specific survival rate for those presenting cN3 tumors ranging between 20% and 34%. There is a substantial decrease in the survival rates with N progression (Novara

minimal keratinization observed in anaplastic neoplasm. Chaux et al. (2010)

**Figure 2.** Natural history of penile cancer (Adapted from Pow-Sang et al. 2010).

et al. 2007).

Lopes et al. (2002) performed a study that aimed to investigate the p53 in Brazilian patients with PSCC to establish a new prognostic factor for lymph node metastasis and its possible influence on prognosis. This study observed the nodal stage as a factor that influenced survival (independent risk factors) in the univariate and multivariate analyses. Gunia et al. (2012) shown that p16INK4a is a good prognostic marker for penile squamous cell carcinomas, surpassing the prognostic impact of histologically confirmed koilocytosis. In their study, p16INK4a expression predicted better cancer specific survival rates. Furthermore, p16INK4a can be useful in differentiate subtypes of PSCC. According Chaux & Cubilla (2012), warty carcinomas tend to be p16INK4a positive, whereas giant condylomas and papillary and verrucous carcinomas are consistently negative.

Medical record analysis of 145 men with penile squamous cell carcinomas was performed to identify prognostic factors for lymph node involvement (Lopes et al. (1996)). The authors found that lymph node metastasis presents correlation with tumor thickness, lymphatic and vascular embolization. Interestingly, univariate analysis did not reached statistically significant values to pathologic stage of primary tumor, clinical lymph node stage (cN), and histological grade. However, histological grade may be considered an important prognostic factor in penile squamous cell carcinoma. In accord with Cubilla AL. 2009 these prognostic factors are predictive to the nodal spread, metastasis and tumoral dissemination.

Currently, different methods are employed to grade Penile Squamous Cell Carcinomas. For instance, Akhter et al. (2011) uses the Broder's system as histological grade system in Squamous Cell Carcinoma. In the Broder's system Penile Squamous Cell Carcinomas is stratified in 4 grades levels based only in differentiation of the cells: Grade I (well differentiated) presenting <25% undifferentiated cells; Grade II (Moderately differentiated) presenting <50% undiffer‐ entiated cells; grade III (Poorly differentiated) presenting <75% undifferentiated cells and grade IV (Anaplastic/Pleomorphic) >75% undifferentiated cells. Cell anaplasia degrees are also pointed as common approach to determine Penile Squamous Cell Carcinomas grading (Mikuz et al. 2004; Slaton et al. 2001), absence of anaplasia (well differentiated cells), grade 1; grade 2, moderately differentiated (<50% anaplastic cells); and grade 3, poorly differentiated (>50% anaplastic cells). Cubilla et al. (2009) reported a method to grade Penile Squamous Cell Carcinomas. Carcinomas with a minimal deviation from normal/hyperplastic morphology of squamous epithelium were considered Grade 1 (extremely well-differentiated). Grade 3 are tumors showing any proportion of anaplastic cells, identified as solid sheets or irregular small aggregates, cords or nests of cells with little or no keratinization, high nuclear cytoplasmic ratio, thick nuclear membrane, nuclear pleomorphism, clumped chromatin, prominent nucleoli and numerous mitosis. Grade 2 is composed by remainder tumors. Grading both extremes of the spectrum is simple and reproducible.

established. Specifically, more than a dozen protein-protein interactions between E6 and cellular proteins have been shown (Villa et al., 2002). Taken into a carcinogenic point of view, E6 and E7 ORF are considered to play the most important roles, encoding for oncoproteins that allow viral replication and the immortalization and transformation of the epithelial cell

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Proving the importance of p53 and pRb in cell cycle progression, the repression of HPV 16 E6 and E7 expression by dual shRNA transfection has been shown to be capable of restoring the p53 and pRb tumor suppressor pathways and activating apoptosis (Psyrri et al., 2009, Rampias et al., 2009). Thus, the demonstration of this tumor suppressor inactivation by the E6 and E7 HPV oncoproteins has provided a basic explanation for how the high-risk HPV types exert their oncogenic effects on cervical cells, and this explanation are under investigation to be related with other sites of HPV-infection. This is particularly important in penile cancerassociated HPV infection, whereas HPV16 seems to develop a pivotal role, and accounts for

Different from other viruses, HPV does not infect or replicate in antigen-presenting cells (APCs) of the epithelium nor induce cell lysis, which is a key escape mechanism to avoid that APC recognize and produce antigens derived from the virion, and alerts immune system. About more than 50% of infections present seroconversion in the patients, but the production of antibodies usually occurs only months after the initial infection (Vidal & Gillison, 2008). The life cycle of papillomaviruses is closely tied to the epithelial differentiation process. Infection occurs exclusively in squamous epithelial cells with a preference for the keratinocyte stem cell as the initial target of HPV infection, which will allow the maintenance of viral replication (Vidal & Gillison, 2008). The route of entry for HPV infection is microtraumas or small wounds in the skin or mucosal surface, which are particularly important in penile HPV-infection. These breaks in the epithelial surface allow the virus to access and persist in the nuclei of infected basal layer cells of the epithelium. Until now, no single receptor has been definitively identified and established as being responsible for HPV entry, although is believed that receptors closely related to wound healing might be preferential targets for HPV infection, such as α6 integrin

As most viruses, HPV uses the host cell DNA machinery to maintain the production of viral progeny. This mechanism of viral-induced cell growth is very well known and is analogous to other viruses that disrupt the control of cell growth (Hebner & Laimins, 2006). Following cell division, as the basal cells divide into squamous epithelial cells, HPV establishes its DNA genome in the host cell nuclei, replicates and reaches a high copy number. Infected cells then leave the basal layer, migrate toward the suprabasal regions and begin to differentiate. In the basal layer phase, the HPV genome is maintained at a low copy number, providing a type of stock of viral DNA for further use in cell divisions. At the same time, 'early' viral genes (E5, E6 and E7) are expressed, resulting in enhanced proliferation of the infected cells and their

that host the HPV DNA (Doorbar et al., 1991).

more than 60% of HPV-related tumors.

**4. HPV impact in squamous cell homeostasis**

and glycosamioglycan heparin (Vidal & Gillison, 2008).

In summary, Penile Squamous Cell Carcinomas represents an aggressive locoregional malignancy with dissemination process may occurring 20-40% (Guimarães et al. 2009), a lethal disorder that often presents after significant delay (Barocas & Chang 2010). Usually, death occurs within 2 years after initial diagnosis (Guimarães et al. 2009). Accurate evaluation of clinical stage, anatomical site, regional lymph nodes, and metastatic disease presents a pivotal role in treatment planning to predict the survival outcome (Barocas & Chang, 2010).
