**3. HPV biology: General considerations**

risk types 16 and 18, as in the cervical and anogenital cancer, are the most common entities detected in pre-cancerous and HNSCC lesions. Miller & Johnstone [1] describe that likelihood of detecting HPV, comparing pre-cancerous lesions and HNSCC to normal epithelium, was three times higher in pre-cancerous lesions and four a five times higher in HNSCC. In despite of large number of studies, the accurate role of HPV in the HNSCC development and pro‐ gression has been controversial so as in OSCC. HPVs are a circular virion enclosed in a small capsid. The carcinogenic process occurs through the HPV-DNA integration into host cell. Usually, the viral oncoproteins codified by HPV-DNA, leads a functional alteration in p53 and pRb pathways, and consequently genomic instability. However, the oncoproteins expression alone is not sufficient to induce neoplastic transformation suggesting the requirement of supplementary genetic modifications. Increased understanding of the role of HPV antigens in neoplastic pathogenesis confirms the HPV as an etiological agent for cancers and, the knowl‐ edge of HPV cancer biology consequently will provide the development of preventive vaccines and antiviral treatment. The HPV vaccines have been formulated as a result of core technolo‐ gies implementation that is able to construct virus-like particles (VLPs) equivalent to natural virions but, at the same time, are not capable to induce an infectious process. In addition, in this chapter we will discuss the HPV relationship with the pre-cancerous lesions and OSCC. The present data summarize the knowledge regarding the epidemiology, behavior, biology,

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

malignant transformation mechanisms, and prognosis of HPV infection.

Human Papillomavirus infection was firstly identified from the embalmed body of a 12th century B.C. ancient Egyptian worker [2]. During the mummy necropsy procedure Scientists observed a wart on the sole of his foot. This evidences demonstrated that HPV infection occurred [3]. Medical literary tidings regard skin and genital warts were described in classical Greek and Roman literature [2]. However, association between viral origin of warts and sexual transmission was only confirmed in 19th century [2]. Rigoni-Stern (1842) hypothesized that cervical cancer could be promoted through sexual contacts. The Italian physician postulated this conception through the observation of high rates on cervical cancer in sexually active women, in comparison with non-sexually active women. [4]. Essays to establish relationship between cervical cancer and HPV-infections were initiated in 1972, this hypothesis was supported by a rare description of condylomata acuminate malignant transformation into squamous cell carcinomas highlighting the carcinogenic potential of hpv [4]. Harald zur Hausen, in 1975 [5], published that HPV could have a pivotal role in human cervix carcino‐ genesis. Eight year after, Hausen et al. identified the subtypes HPV16 and 18 on cervix cancer. HPV infection was well established as etiologic factor of almost 100% of cervical malignancies [6]. After this appointment, several studies have addressed to find presence and prevalence of HPV infection in different tumor sites, including skin, urethra, nasal cavity, paranasal sinus, larynx, tracheobronchial mucosa and oral cavity [7]. In 1983, a series of studies presented by Syrjänen et al. [8] highlighted the possible correlation between HPV and oral lesions (nonneoplastic, benignant and malignant lesions). At the same year, a light microcopy study

**2. A brief history of papillomavirus and cancer**

HPV represent a group of DNA viruses that was recently recognized to form their own family, Papillomaviridae that initially, together with polyomaviruses, was grouped in the Papova‐ viridae family [13]. They are an ancient family of pathogens and are known to infect epithelial tissues of amphibians, reptiles, birds and mammals [14]. The virus is formed by a nonenveloped icosahedral capsid with circular double-stranded DNA [15-16]. The genome is small, comprising to 8.000 base pairs, but it is complex, composed of three distinct regions: early region (E), late region (L) and upstream regulatory region (URR) or long region control (LCR).

The E region contains from seven to eight genes (E1, E2, E3, E4, E5, E6, E7 and E8), of which E1 is related with viral replication, E2 with viral transcription and DNA replication, E4 with maturation and alteration of extracellular matrix cell and E5, E6 and E7 are involved in cellular transformation. The E3 and E8 genes have been recently described only in a few HPV types but their function is unknown [16-18].The L region containing two genes, L1 and L2, which encode structural proteins necessary for viral capsid formation in the final stages of replication. Both E and L are coding region therefore called open reading frames (ORF), however the region URR does not fit in this description because it is a non-coding region. The URR region is found between E and L region and contain promoter and enhancer DNA sequences critical to regulate viral replication and transcription by both viral and cellular genes [19].

Based on phylogenetic analysis, the HPV is classified into genera (alpha, beta, gamma, mu and nu), species and types [15]. The classification of HPV types is based mainly on analyses of the L1 gene, which is the most conserved gene in all known papillomaviruses. When the DNA sequence of the L1 ORF differs by more than 10% from the closest known virus type, a new papillomavirus is recognized. Differences between 2% and 10% homology define a subtype and less than 2% a variant. A viral variant can differ between 2% in coding regions and 5% in non-coding regions [13,15]. Currently, approximately 150 different types are recognized and 120 HPV types are fully sequenced [18]. Types classified as members of the same species with approximately 80-90% of similarities trend to share biological properties such as the tissue tropism, disease manifestation, and pathogenicity [14].

According to their tropism, the HPV also can be classified as cutaneous and mucosal type. The cutaneous type are associated with skin lesions, being HPVs 1, 2 and 4 the most prevalent in common and plantar warts, and the types 5, 8, 9, 12, 14, 15, 17, 19-25, 36, 46 and 47 the most frequent in epidermodysplasia verruciforme. HPV-5 and -8 are associated with skin carcinoma [20-21]. HPV with mucous tropism infects the anogenital tract, upper aero digestive tract, other head and neck mucosa and are generally subdivided into high-risk and low-risk type based on their oncogenical potential. The most relevant low-risk type are HPV-6 and 11, however the types 40, 42, 43, 44, 54, 61, 70, 72 can be observed in genital benign lesions. Among the highrisk types, the HPV 16 and 18 are most common; especially type 16, which can be found in various cancers such as cervical, oropharyngeal and penile carcinomas. Types 31, 33, 35, 52, 58 and 67 belong to a category of moderate to high-risk [20,22-24].

HPV life cycle is closely linked to the differentiation program of infected epithelial cells, more specifically the keratinocytes. Infection is initiated through microlesions in the epithelium, which allow virions come in contact with the basal cell layer by direct HPV receptor connection to surface host cell ligands. The receptors involved are not fully identified, but some data revealed a role for α6 integrin and heparin sulfate. Following infection, the virus probably maintains its genome as a low copy number episome in the basal cells of the epithelium, providing a reservoir of viral DNA for further use in cell divisions. When infected basal cells begin to divide, viral DNA is distributed among the daughter cells with a massive upregulation of expression of all early genes mainly the E6 and E7. After mitosis some daughter cells may persist in the basal layers, whereas other move toward the upper layers of the epithelium and begin to differentiate. During this differentiation process there is viral DNA replication that amplifies the amount of virus at least 1000 copies per cell, and finally expression of the coat proteins L1 and L2 followed by assembly of infectious virus [25-28].

Recently, a specific correlation between HPV-positive patients and sexual behavior has been established in HNSCC [32]. This study shown, in patients with HNSCC, that high-risk HPV-16 was correlated with vaginal/oral sex partners, casual sex habits and infrequent users of barriers during vaginal/oral sex. Heck et al. [31] presents association between HNSCC subtypes and sexual behavior. In Oropharynx Cancer the prevalence of HPV infection is close to 36% [30] and this entity was associated with the number of sexual partners and lifetime oral sex partners [31]. A similar result was described by D'Souza et al. [33], which presented an association between HPV-16 measurements and presence of oral HPV-infection. The authors has also found HPV-16–positive oropharyngeal cancer correlated with oral-sex or vaginal-sex partners, engagement in casual sex, early age at first intercourse, and infrequent use of condoms. In tonsillar cancers, Hemminki et al. [34] demonstrated that women with cervical lesions present an increased risk of tonsillar cancer. In addition, increased risk was also found among husbands of women with invasive cervical malignancies. Heck et al., [31] has also found correlation among tonsillar cancers, number of oral sex partners, and earlier age at sexual debut. At the same study, another subtype of HNSCC (Cancer of the Base of the Tongue) was associated with sexual behavior; it was related with oral sex among women, number of sexual

**Figure 1.** Human Papillomavirus Life Cycle: A: Early gene expression E1, E2, E6 and E7; B: Viral genome amplification;

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C: Virion assembly and release (Adapted from Moody & Laimins 2010).

The sexual behavior has been associated with oral HPV-infection. Univariate analysis showed that oral HPV-infection was significantly increased with the lifetime number of oral/vaginal sex partners. Multivariate analysis demonstrated that oral HPV-infection was significantly elevated among individuals who reported having either 10 oral or 25 vaginal sex partners during their life [35]. In addition, a curious fact was demonstrated: the open-

partners, and among men presenting history of same-sex sexual contact.

The mechanism of viral-induced cell growth is analogous to other tumors viruses that deregulate the cell cycle. Cancer appearance in lesions with persistent HPV is related to the overexpression of E6 and E7 proteins. E6 interfere with the function of p53 whereas E7 with the function of Rb protein, leading to abnormal cells growth by promoting inhibition of apoptosis and dysregulation of cell cycle, respectively. [28]. Basically, HPV infection occurs through sexual contact, non-sexual contact and maternal contact. In healthy individuals most (around 80%) HPV infections clear spontaneously but in some cases, HPV infection persist, leading to cancer development [26,29]. A series of events allows the viral persistence: differ‐ entiation-specific organization of the virus life cycle, mechanisms to maintain genome copynumber in undifferentiated cells, angiogenesis promotion, and strategies to evade both innate and adaptive immune surveillance [28].
