Send Orders of Reprints at [email protected] 5452 Current Pharmaceutical Design, 2012, 18, 5452-5469 Oral HPV Infection: Current Strategies for Prevention and Therapy Stina Syrjänena,#, Nicoletta Termineb,*,#, Giuseppa Caprac, Carlo Padernib, Vera Panzarellab and Giuseppina Campisib aDepartment of Oral Pathology, Institute of Dentistry, University of Turku, Turku, Finland; bSector of Oral Medicine “ V. Mar- giotta”, Section of Oral Sciences, Department of Surgical and Oncological Disciplines, University of Palermo, Palermo, Italy; cSec- tion of Virology – Department of Sciences for Health Promotion, University of Palermo, Palermo, Italy Abstract: Infection with High Risk (HR) Human Papillomaviruses (HPVs) is the main aetiological agent of Cervical Squamous Cell Carcinoma (CSCC) and also associated in a subgroup of other neoplasms, including Oropharyngeal Squamous cell Carcinoma (OPSCC). HPV infection, in genital as in oral mucosa, can also be subclinical or associated with benign proliferative lesions (common warts, con- dylomas, papillomas) caused mostly by infection with Low Risk (LR)-HPVs. In the last decades, extensive research has resulted in grow- ing knowledge on HPV biology and specifically viral life cycle, biochemical properties of viral proteins and their interaction with the host proteins leading to potential new targets of prophylactic or therapeutic vaccines and therapies for HPV infection. In addition, notable progresses have been made in the field of diagnostics to detect HPV DNA or RNA. The recent epidemiological data suggest the signifi- cant changes in HPV endemic, due to the changes in sexual habits especially among young generations (i.e. early sexual debuts, multiple sexual partners, oral and anal sex); this scenario has urged on the need of adequate campaigns of primary (sexual education, vaccination programs) and secondary prevention (diagnostics of HPV-related diseases). Due to the growing interest on HPV infection and HPV re- lated cancers, the authors made a narrative review of the literature on oral HPV infection and oral-genital transmission. After this, in view of the controversies about the strategies of therapy and prevention of HPV infection, the present review focuses on the current state of art about the available tools for the therapeutic and, if any, preventive management of oral HPV infection. Keywords: Human papillomavirus, oral mucosa, oral diseases HPV-related, viral transmission, oral surgery, HPV vaccines, genital HPV infection, oral sex, head and neck cancer. 1. INTRODUCTION diseases, although the medical and especially dental community, is probably still not acquainted with these perspectives. Human Papillomavirus (HPV) infection has been recognized as a necessary cause of cervical squamous cell carcinoma (CSCC) This review focuses on oral HPV infection and oral-genital worldwide [1-3]. There is also strong evidence of its causal role for transmission and current state of art about the therapeutic and, if a subset of SCC, in the anogenital area (i.e. vulva, vagina, anus and any, preventive management of oral HPV infection. penis) and in the head-neck districts (i.e. pharynx, larynx, oral cav- ity). Genital HPV infection is one of the most frequent sexually 2. VIROLOGY AND NATURAL HISTORY OF HPV INFEC- transmitted disease, being reported with a prevalence of about 42% TION among 19-54 aged females in the US [4] and being strongly corre- 2.1. HPV Genome and Source of Infection lated to sexual behavioural risk factors [5]. Moreover, epidemiol- HPV are small, double-stranded DNA viruses with around - ogical data from last decades suggest significant changes in HPV 8000 base pairs, covered by an iso-exahedric capsid without enve- endemic, with regard not only to genital but also oral area. This has lope consisting of 72 capsomeres [13-15]. Capsid proteins are rep- been explained by risky sexual behaviour especially among young resented by a major capsid protein (L1) and a minor capsid protein generations [6, 7]. This also would explain the increased incidence (L2). HPV genome consists of ten Open Reading Frames (ORFs) of HPV-related SCC in head and neck region [8-10]. Although the sequences, with potential of transcription for specific mRNA found modes of HPV transmission in oral cavity (see below, paragraph in the same DNA strand. The HPV genome can be divided into 2.a) are still not completely elucidated, epidemiological data sug- three different regions: the first region (Early or “E” region) ex- gest that detection of HPV (i.e. HPV 16) in oral exfoliated cells tends for about 45% of the viral genome and codifies for 8 early- increased the risk more than 14-fold (OR 14.6; 95% CI: 6.3-36.6) functional proteins (E1 - E8); the second one (Late or “L” region) of oropharyngeal cancer (tonsils and base of tongue) [11] and 3.8- extends for about 40% of the viral DNA and codifies for 2 late- fold the risk of oral cancer [12]. structural proteins (L1 and L2); the third one (Long Control Region HPV-related diseases in humans have taken an increased impor- or “LCR” or Upstream Regulatory Region “URR”), containing tance over the years and are now a major concern for public health. sequences regulating gene transcription, performs exclusively regu- The current evidence suggests that the potential fallouts of the so latory functions [16-18]. After HPV inoculation, the life cycle of called “HPV endemic” on the epidemiologic, therapeutic/preventive HPV, the synthesis of viral DNA and the expression of viral genes and socio-economic viewpoints are enormous, and in the future are linked to the differentiation program of the infected epithelial there is a great opportunity to design adequate strategies of preven- host cells, with production of mature virion particles restricted to tion and management of HPV-related diseases, including SCCs of differentiated superficial cells [19]. The normal viral replication anogenital and head-neck region, similarly to other infectious cycle is a highly regulated process. Thus, the dynamic of changes in keratinocytes, from the basal layer up to the surface, predisposes to the creation of a suitable micro-environment for the productive cell *Address correspondence to this author at the Sector of Oral Medicine “ V. Margiotta”, Section of Oral Sciences, Department of Surgical and On- replication, responsible for the transformation of the epithelial cell cological Disciplines, University of Palermo, Palermo, Italy; into a permissive cell [19, 20]. Tel:/Fax: +39 091 6552236; E-mail: [email protected] Infection usually occurs through micro-wounds of the epithe- #S. Syrjänen S. and N. Termine contributed equally to the manuscript prepa- lium that expose basal and para-basal cells of the squamous epithe- ration. lium to viral access. This process it seems coordinated by specific 1873-4286/12 $58.00+.00 © 2012 Bentham Science Publishers Oral HPV Infection Current Pharmaceutical Design, 2012, Vol. 18, No. 34 5453 HPV receptors, including (cid:1)-6 integrin, extracellular laminin 5 and Regarding the natural history of cervical HPV infection, it heparan sulphate proteoglycans. After entry, HPV establishes itself would depend on the genotype involved. HPVs are traditionally in the cellular nucleus as episomes with a low, non-productive viral distinguished, on the base of specific epithelial tropism, in cutane- replication (vegetative replication) [18]. In the basal cells HPV ous and mucosal types: the firsts infect hands and feet skin, the replicates together with cellular DNA during S-phase at relative low second prefer mucosal surface of upper aero-digestive tract, ano- copy numbers (20-100 copies). This initial phase, responsible for genital district, urethra and conjunctive [35-38]. On the basis of the latent status of infection is characterized by ORFs E1 and E2 epidemiologic data regarding the behaviour of the viral lesions, expression only, codifying for replication factors. In intermediate HPV have been also divided in two groups: those at “high risk” layer during differentiation the vegetative replication occurs and oncogenic [High Risk (HR) –HPV genotypes like 16-18-31-33-35, more than 1000 copies/cell are amplified in each cell. The episomal 39,45,51,52,53, 56,58,59, 66, 67,70,73,68, 82), associated with form is a kind of reservoir of infected cells, morphologically un- potentially and overtly malignant lesions (anogenital cancers, giant distinguishable from non-infected ones; the classic viral cytopathic condyloma of Bruschke and Lowenstein) and those at “low risk” effects (acanthosis, diskeratosis, multinucleation of keratinocytes oncogenic [Low Risk (LR)–HPV genotypes like 2, 4, 27 (the skin and coilocytosis) appear when the viral replication enters into a types) and mucosal types 6, 11, 13, 32, 42), more commonly asso- productive phase. It is characterized by the complete viral DNA ciated with benign diseases (ordinary wart, condyloma, focal codification and the assemblage of several infecting virions. Par- epithelial hyperplasia, squamous cells papilloma, Bowen’s papillo- ticularly, following the epithelial squamous differentiation, starting matosis) [39-43]. After entry to host cell, HPV infection can mani- from basal and para-basal cells, where early portions of the viral fest in two clinical states: genome are more active and going up to higher epithelial layers 1) subclinical or unapparent infection, that is the silent presence (both the intermediate and the superficial) along with the formation of viral genome into inoculation site without any clinical of the complete virions (i.e. the infecting viral particles) [19]. The and/or histological and/or cytological alterations of cervical virus is expelled from the epithelial cells when they undergo des- mucosa [44]; quamation and is transmitted by direct contact (especially in pres- 2) clinical infection, expression of proliferation of infected ence of lesions, i.e. genital warts) or by indirect contact. keratinocytes and associated with clinical and histological le- The genital HPV infection is classified as a Sexually Transmit- sions of cervical mucosa. These lesions are usually benignant ted Disease (STD): the transmission of the virus in the genital area (e.g. genital condylomas or warts) when infection is sustained is mostly due to sexual contact with an infected partner and the by LR HPV [45]; conversely the HR HPV infection, especially probability of infection is directly related with a sexual risk behav- when it persists for over 18-24 months [46] and it is accompa- iour (e.g. early sexual debut, high number of partners) [21, 22]. nied by the viral DNA integration into the eucariotic DNA in Conversely, the transmission modes of oral HPV infection, the risk basal cells, may be associated with the development of poten- factors as well as the natural history is poorly studied. Sexual tially malignant and malignant epithelial lesions of the cervical transmission is by far the most common route of spreading the vi- mucosa. The latter form of infection has been recognized as rus. However, detection of HPV in virgins, infants and children the necessary but not sufficient cause of cervical SCC [46, 47]. suggest that vertical transmission also exists, but the exact rates and This biological and aetio-pathogenetical model related to cervi- routes have not been well established [23-29]. Possible non-sexual cal HPV infection has been shifted to oral infection, although with transmission modes include vertical or horizontal transmission and some important differences derived from very recent researches, auto-inoculation. Vertical transmission can be divided into three discussed below. categories, according to the assumed time of HPV transmission: 1) peri-conceptual transmission (time around fertilization), 2) pre-natal Immune response plays a very important role in HPV infection; (during pregnancy), and 3) peri-natal (during birth and immediately it is well documented, for instance, that in immunocompetent sub- thereafter). Controversial views have been presented on oral sex as jects skin warts often regress spontaneously, whereas immune defi- a main transmission mode of HPV [24, 30]. In the more recent Fin- ciency, both inherited (e.g. warts-like epidermal dysplasia) and nish Family HPV Study on HPV dynamics within family, 11% and acquired (e.g. transplants, AIDS), may favour a higher incidence 26% of the women and their husbands reported regular practice of and persistence of skin and mucosal infections induced by HPV oral sex, respectively [24, 25]. However, any significant association [48]. Nevertheless, in HPV-related diseases, immune responses are between oral sex and carriage of HPV in the oral mucosa was estab- generally poorly expressed compared to other viral infections, since lished [25]. This is contradictory to the data reported by D’Souza the viral replication cycle takes place inside maturing keratinocytes and co-workers, who studied in a case-control setting, whether sex- able to continuously remove mature virions. These viruses also ual behaviour that increased the odds of oropharyngeal cancer cause cell proliferation, rather than cell lysis; as a result, during would similarly increase the risk of oral HPV infections among infection, the local or systemic viral antigens presentation to the their control patients [30]. HPV infection was detected in 4.8% of immune system by the APC is minimal and infection generally 332 control patients (from an outpatient clinic) and in 2.9% of 210 persists for months or even years [49]. college-aged men. Among control patients, the odds of HPV- infection developing independently increased with increased life- 3. CLINIC OF HPV INFECTION time number of oral (P=0.007) or vaginal sex partners (P=0.003). 3.1. Genital HPV Infection Among college-aged men, there was an association between oral It is believed that genital HPV infection could result in two HPV and deep kisses (P=0.023, for trend) and oral sex (P=0.046, different conditions, depending largely on the genotypes involved for trend) [24]. The importance of sexual transmission is confused (Table 1). by the fact that the HPV-type concordance in oral and genital infec- Genital Normal Mucosa and HPV Infection tions seems to be poor between the sexual couples and even in the same person [26, 31-33]. HPV detection in healthy genital mucosa is a common condi- tion characterized by staying silent of the viral genoma in the site of 2.2. Natural History of HPV Infection inoculation and, therefore, without any clinical, colposcopic, cyto- The current knowledge about the natural history of oral HPV logical and histological resultants. The infection can be discovered infection come from gynaecological literature about cervical infec- only with the detection of HPV-DNA in the epithelial cells, by tion, although, as recently suggested by D’Souza et al [34], oral and means of techniques of hybridization on cytological or histological genital HPV infection seem to differ themselves about some as- samples taken from healthy subjects. pects. 5454 Current Pharmaceutical Design, 2012, Vol. 18, No. 34 Syrjänen et al. Table1. Conditions Potentially Associated with HPV Infection in Genital/Cervical Mucosa TAB.1 CERVICAL/GENITAL HPV INFECTION SUB-CLINICAL CLINICAL POTENTIALLY/OVERTLY MALIGNANT BENIGN LESIONS DISORDERS Normal mucosa Condyloma acuminatus CIN, VaIN, VuIN, AIN, PIN Warts (Verruca vulgaris) SCC of uterine cervix, vagina, vulva, anus, penis CIN: Cervical Intraepithelial Neoplasia; VaIN: Vaginal Intraepithelial Neoplasia; VuIN: Vulvar Intraepithelial Neoplasia; AIN: Anal Intraepithelial Neoplasia; PIN: Penis Intraepi- thelial Neoplasia. Subclinical infection can be further distinguished in productive, therapy; the disease worsens during therapy; the patient is immune- when it is accompanied by the expression of viral DNA and by the compromised; or warts are pigmented, indurated, fixed, and ulcer- release of mature viral particles, and latent/persistent, when the ated). No data support the use of type-specific HPV nucleic acid virus persists, for a variable time, as episome, in the deep layers tests in the routine diagnosis or management of visible genital epithelium in a completely silent condition. Persistent HPV infec- warts. tion supported by HR is seen as a necessary, but not sufficient, state In addition to the external genitalia (i.e., the penis, vulva, scro- for the occurrence of cervical cancer [46]. It is estimated that a wide tum, perineum, and perianal skin), genital warts can occur on the range of healthy and sexually active female population is a carrier uterine cervix and in the vagina, urethra, anus, and mouth; these of subclinical genital infection, contributing to the spread of the warts are sometimes symptomatic. Depending on the size and anat- virus. The prevalence of infection among worldwide population of omic location, genital warts can be painful, friable, and pruritic, healthy women was estimated to be between 2% and 44% in rela- although they are commonly asymptomatic. tion to the sensitivity of the diagnostic test applied [44, 47, 50-52]. HPV types 16, 18, 31, 33, and 35 are found occasionally in The highest prevalence (about 40%) of HPV infection was found visible genital warts and have been associated with external genital among women under the age of 25 years, progressively reduced in (i.e., vulvar, penile, and anal). Patients who have visible genital successive age groups [53]. These data support the hypothesis that warts can be infected simultaneously with multiple HPV types [5, the virus, acquired after the onset of sexual activity, leading to tran- 58]. sient infection, is eliminated in about 6-20 months [53, 54]. This time of clearance seem to be longer only for HPV-16 infection [18, Genital Potentially/Overtly Malignant Lesions HPV-related 22, 55, 56]. The genotypes isolated more frequently, both in pa- HPV persistent infection, supported by HR genotypes, is con- tients with cervical carcinoma and in healthy controls, are 16, 18, sidered a necessary condition and an important cause for the occur- 45, 31 and 58; it is also common the detection of infection with rence of cervical and anogenital (such as vaginal, vulvar, penile and multiple genotypes, with percentages prevalence in the two groups anal) potentially/overtly malignant lesions respectively [59]. Due to of 8, 1% and 13.9% respectively [55]. the close connection between HPV infection and cervical cancer, In a very recent investigation on HPV infection in U.S. female, the discussion will focus on that district. Hariri and colleagues, show a prevalence estimate for HPV equal to Cervical potentially malignant lesions are defined as circum- 42.5% [4]. This percentage drops dramatically (8.8%) if the geno- scribed areas of the mucosal lining of the cervix characterized by types considered are only those at high risk (HR 16 and 18), with varying degrees of epithelial dysplasia, burdened by a greater neo- higher prevalence among women aged 20-24 years [5]. plastic potential than the normal mucosa. Over 90% of these condi- However, as renowned by Sanders et colleagues, most of these tions are HPV-positive; in some cases, the genome is only present cross-sectional estimates of HPV prevalence fail to connect the in episomic or integrated form, in other cases, the complete virions HPV status with a chronic infection and its role in malignant trans- are visible by electronic microscope [45, 60-64]. formation of healthy mucosa. A longitudinal design is needed for From histological point of view, dysplastic lesions usually have all future research aimed to investigate the causal link between an uneven surface, sharp edges, non-uniform caliber of vessels, HPV infection and its oncogenic potential [6]. after application of acetic acid often assume pearly white bulleted Genital Benign Lesions HPV-related or "mosaic" and are iodine-negative at test of Schiller. The most frequent clinical expression of infection is the condy- According to the Bethesda classification, there are three catego- loma acuminatum, macroscopically characterized by small, sessile ries of cytological findings concerning these cervical lesions: or pedunculated multiple growths, generally asymptomatic, covered I. ASC (Atypical Squamous Cells), further divided into: with keratinized epithelium, tending to confluence and to growth I.a ASC-US (Atypical Squamous Cells of Undetermined like a “cauliflower”. It is the most common sexual transmitted viral Significance); infection (although it can also be acquired by vertical transmission or extragenital contact) associated in 95% of cases with infection by I.b ASC-H (Atypical Squamous Cells, cannot exclude H- LR-HPV 6 and 11 [22, 45, 57]. The lesion, expression of a benign SIL); polyclonal proliferation of infected keratinocytes, may regress II. L-SIL (Low-grade Squamous Intraepithelial Lesion); spontaneously in 3-4 months, may remain stable or undergo an III. H-SIL (High-grade Squamous Intraepithelial Lesion); expansion. IV. Carcinoma. Genital warts represent another common disease related to the productive infection of HPV, especially that supported by LR-HPV Currently, researchers are apt to believe that the lesions referred to as L-SIL and H-SIL are the expression of two distinct pathoge- 6 or 11. Diagnosis of genital warts can be confirmed by biopsy, netic processes, both induced by HPV. The genotypes more de- although biopsy is needed only under certain circumstances (e.g., if the diagnosis is uncertain; the lesions do not respond to standard tected are the HR, except for a portion of L-SIL, about 30%, result- ing LR-HPV- positive [22, 60]. The L-SILs represent the transient Oral HPV Infection Current Pharmaceutical Design, 2012, Vol. 18, No. 34 5455 expression of viral production, and are not accompanied by disrup- detected both in healthy oral mucosa and in several lesions, as re- tion of differentiation and maturation processes of epithelium. The ported on Table 2. H-SILs, however, are considered the true precursors of cervical Normal Oral Mucosa and HPV Infection cancer. These lesions are the result of HPV infection in metaplastic The detection rate of asymptomatic oral HPV infection in adults and immature cells, resulting in disruption of the differentiation (i.e. presence of HPV DNA) varies among different studies from process of epithelium, in the uncontrolled replication of cells and in 0% to 81%. The mean prevalence rate is approximately 11% [6, 7, the accumulation of genetic abnormalities, to the selection of a 72-81]. Kreimer and co-workers made a new meta-analysis on HPV clone of cancer cells [22]. The mean age of H-SIL onset is 28 years; detection in oral mucosa and showed that only 4.5% (95% CI: 3.9- compared with the average ages of acquisition of HPV infection 5.1) of 4070 subjects were positive for any HPV and 3.5% (95% (20-25 years) and of onset of cervical cancer (40-50 years), we CI: 3.0-4.1) of 4441 subjects had carcinogenic mucosal HPV [59]. deduce some key points: the interval of time during which the virus HPV16 was detected in 1.3% (95% CI: 1.0-1.7%) of 3977 healthy is able to induce dysplastic changes is relatively short, while the subjects accounting 28% of all HPV positive cases of the oral re- time needed within these alterations develop invasive cancer is gion [82]. Detection rate of any HPV varied widely in individual about two decades. studies from 2.6% to 20.7%, even when the same PCR primers This suggests a very long time to tumor progression, during were used [59]. The reasons for this variability would be found in which a variety of factors, in addition to HPV, play a role in pro- the lack of standardization of the parameters used in the several moting the malignant progression. investigations such as: samples size characteristics of ethno- Cervical carcinoma represents the second cause of death among geographic and socio-demographic variables of the populations women worldwide after breast cancer. The curve of incidence of examined, the nature of the specimens (histology vs. cytology), this tumor shows a gradual increase to over 55 years, with a peak sensitivity of the diagnostic test used for the viral detection (ie, around 48 years [1, 22, 37, 65, 66]. The association between genital SBH, DBH, ISH, PCR and nested variant) and inclusion criteria for HPV infection and cervical cancer, suggested in the 80s by Harold mucosal lesions based on different classification systems [59, 79]. zur Hausen [67] has been widely confirmed by numerous epidemi- The most recent systematic review of the literature on this topic ological and molecular studies carried out in the last decades. The selected 18 studies investigating HVP-DNA prevalence in 4581 DNA of HPV 16, 18 and 33 has been shown in over 90% of inva- cancer-free subjects which resulted in a 4.5% and 3.5% overall sive cervical carcinomas [1, 68-71]. The information obtained in the prevalence of any HPV and HR-HPV only, respectively [82]. These last years on the replication cycle of the virus and its interaction results are similar to those reported in a previous systematic review with the transitional epithelium of the cervix, have allowed the regarding HPV infection in OSCC and potentially malignant disor- development of a pathogenetic model of cervical cancer consists of ders [12] that registered, among control samples from healthy oral three basic stages: mucosa, percentages of HPV infection equal to 12%. Based on these results, it is believed that the oral mucosa represents a reser- i) persistent HR HPV; voir of infection, excluding a primary involvement of the virus in ii) progression of HVP-related epithelial dysplasia (from mild the process of oral carcinogenesis. to severe degree); Oral Benign Lesions HPV-related iii) local tumor invasion and distant spread. HPV has involved in a variety of benign oral lesion such as The reason why a small percentage of infections tends to persis- common wart (verruca) (Fig. 1 and 2), condyloma acuminatum tence and neoplastic evolution is still debated and, according to the (Fig. 3), squamous cell papilloma (SCP) (Fig. 4 and 5) and focal most recent and accredited acquisitions, is to search in the inter- epithelial hyperplasia (FEH) (Fig. 6). These are asymptomatic exo- individual differences in HLA (Human Histhocompatibilty Leuko- phytic growths, single or multiple, with surface smooth or warty, cyte Antigen) haplotype [46]. white or pinkish depending on the degree of epithelial keratiniza- tion; from the histological point of view are characterized by 3.2. Oral HPV Infection epithelial polyclonal proliferation, koilocytosis and dyskeratosis The natural history of viral infection in the oral cavity is not [83-85]. The lesion may affect any part of the oral surface, more clear, though having characteristics in common with those de- frequently on the tongue, the soft palate and the lips. These lesions scribed for the cervical district. In the oral cavity, HPVs have been Table 2. Conditions Potentially Associated with HPV Infection in Oral Cavity TAB. 2 ORAL/OROPHARYNGEAL HPV INFECTION SUB-CLINICAL CLINICAL POTENTIALLY/OVERTLY MALIGNANT BENIGN LESIONS DISORDES Squamous cell papilloma Oral potentially malignant disorders (OL, PVL, Normal mucosa Condyloma acuminatum OLP) Wart (verruca vulgaris) OSCC, VC, OPSCC Focal epithelia hyperplasia OL: Oral Leukoplakia;PVL: Proliferative Verrucous Leukoplakia; OLP: Oral Lichen Planus; OSCC: Oral Squamous Cell Carcinoma, VC: Verrucous Carcinoma, OPSCC: Oro- pharyngeal Squamous Cell Carcinoma 5456 Current Pharmaceutical Design, 2012, Vol. 18, No. 34 Syrjänen et al. Fig. (1). Oral wart of the hard palate. Fig. (5). Oral SCP of the tongue. Fig. (2). Oral wart of the dorsal surface of the tongue. Fig. (6). Focal Epithelial Hyperplasia (FEH) of the hard and soft palate. are in most cases clinically indistinguishable and their differential diagnosis is based on histopathology. Condyloma acuminatum is characterized by a central core connective tissue in a treelike struc- ture covered with epithelium presenting acanthosis (ie hyperplasia of the spinous layer), koilocytosis (ie presence of cells with pykno- tic and irregularly shaped nuclei surrounded by a perinuclear vacuo- lation), parakeratosis (i.e. abnormal formation of horn cells of the not keratinized epithelium caused by the persistence of nuclei, in- complete formation of keratin, and moistness and swelling of the horn cells) associated with the presence of chronic inflammatory infiltrate in the dermis. Oral SCPs are a small benign growth that begins in squamous epithelial cells. The lesion appears as a soft, Fig. (3). Condyloma acuminate of the palate. pedunculated mass with numerous finger-like projections. The pro- jections may be long and pointy or short and rounded if keratin (skin-forming protein) has built-up round the lesion. Less kerati- nized lesions are pink or red in color and resemble a raspberry, whilst heavily keratinized lesions are white and look like the head of a cauliflower. The covering squamous epithelium shows a nor- mal maturation pattern, although occasional papillomas demon- strate pronounced basilar hyperplasia and mild mitotic activity which could be mistaken for mild epithelial dysplasia may or may not be found in the superficial layers of the epithelium. The SCP differs from the oral condyloma acuminatum in that its surface projections are typically more elongated and more often pointed. It will usually have considerably more keratin on its surface and is much less likely to contain koilocytes in large numbers. The ver- ruca vulgaris is also characterized by a proliferation of hyperkera- totic stratified squamous epithelium arranged into finger-like or pointed projections, each with its connective tissue core. It differs Fig. (4). Oral squamous cell papilloma (SCP) of the palate. from SCP in that elongated rete ridges tend to converge toward the center of the lesion, producing a "cupping" effect. Also, a promi- nent granular cell layer (hypergranulosis) exhibiting coarse, Oral HPV Infection Current Pharmaceutical Design, 2012, Vol. 18, No. 34 5457 clumped keratohyaline granules is typically found and abundant koilocytes are often seen in the superficial spinous layer. Eosino- philic intranuclear viral inclusions are sometimes noted within the cells of the granular layer, a feature never found in the SCP. The HPV-related etiology of these lesions is suggested by the presence of LR HPV DNA, such as HPV -2, HPV-4 and HPV-57 (warts), HPV-6 and HPV-11 (SCPs and condylomas), HPV-32 and HPV-13 (FEH), in more than 80% of cases [28]. There are no population-based studies on the incidence or prevalence of oral papillomas or condylomas. The largest cohort comprises 20.000 Swedish citizens, of whom 0.1% had oral warty lesions [86]. SCPs are the most common benign tumors of oral epithelium. However, in some text books, papillomas are clumped together with benign epithelial neoplasia, which represent a reaction to injury rather than true tumors. SCPs are reported to be most frequent in children and adults in their 4th and 5th decades. There are also Fig. (7). Oral Leukoplakia (OL) - non homogeneous form - of the buccal some rare syndromes which are known to be associated with multi- mucosa. ple oral papillomas, e.g. focal dermal hypoplasia syndrome, ac- rodermatitis enteropatica, Cowdens´s syndrome, nevus unius later- Recently, Szarka et al reported an increase of HPV prevalence alis, Costello syndrome and Down syndromes (OMIM data base, in OPMD with increasing malignant potential: 32.8%, 40.9% and http: //www.ncbi.nlm.nih.gov/omim OMIM). 47.7% in Oral Lichen Planus, OL and OSCC respectively [101]. Oral condylomas have been traditionally associated with oral Oral proliferative verrucous leukoplakia (OPVL) (Fig. 8) is a sex, in contrast to oral SCP. However, no reliable differential diag- different and particularly aggressive form of OL that, in the past, nostics can be done between oral SCP and oral condyloma on the seemed to have the strongest relationship to HPV infection. After, basis of this socio-behavioural variable. In the literature published the association of OPVL and HPV (especially genotype 16) has by 1998, the author found a total of 481 SCPs and 284 OCs, of been supported by several reports [96, 102, 103], although with a which 50% and 75% tested HPV-positive. HPV6 and 11 were the wide range from 10% to 85%, of HPV infection [96, 102]. How- major HPV types in these lesions [43, 81, 87-90]. ever, a multi-centre study recently reported no statistically signifi- The role of HPV in other benign epithelial hyperplasia is un- cant difference, in terms of HPV-DNA detection, between OPVL known. Recently some evidence was presented that the majority of (24.1%) and common OL (25.5%) - nor any special role for HPV in white oral patches’ containing hyperplasia but not dysplasia harbor PVL onset [104]. To date, this controversy remains not to be re- low copies of HR HPV DNA [91]. solved. Oral Potentially Malignant/Overtly Disorders HPV-related HPV prevalence, in oral potentially malignant disorders (OPMD) and in oral squamous cell carcinoma (OSCC) ranges from 0% to 85% [43] with a higher prevalence of HPV 16 or 18 geno- types [92-96]. Such a wide variation is probably due to: i) demo- graphic variables, ii) different categorization of lesions investigated (sometimes not supported by histology) and different site of the mucosa examined, iii) different sampling techniques (biopsies, mouth rinsing or brushing) and different detection techniques - from the less sensitive ISH to the highly sensitive “nested” PCR [43]. To date, data on HPV prevalence in Oral Leukoplakia (OL) (Fig. 7) are still controversial. Miller and White [77], in a review of studies using ISH to identify HPV in benign OL (i.e. without dys- plasia), reported a prevalence ranging from 0% to 80%, signifi- cantly greater in fresh and frozen specimens (43.1%) than in paraf- Fig. (8). Oral Proliferative Verrucous Leukoplakia (PVL) of the buccal and fin-embedded tissues (12.2%). However, in OL with dysplasia and masticatory mucosa. using PCR for HPV-DNA detection, the prevalence ranged from 17% [97] up to 68.6% [72]. Moreover, if Uppsala criteria are con- Oral erythroplakia (OE) (Fig. 9) is a potentially malignant dis- sidered [98] and the Proliferative Verrucous Leucoplakia (PVL) is order with the highest potential for malignant transformation. It is excluded from the analysis, the overall HPV prevalence in other OL described as a “red patch clinically and pathologically un-related to dropped to 17.6% (in paraffin-embedded biopsies from non- any other disease” [105]. By 1998, only 11 OEs have been screened dysplastic and dysplastic lesions) [99]. for HPV DNA and 54.5% has tested HPV 16 positive [12]. Campisi et al examined the relationship in OL between HPV Oral Lichen Planus (OLP) (Fig. 10) is a chronic inflammatory and some molecular predictors of malignant progression such as disease of unknown aetiology and immune pathogenesis, with a apoptosis markers (bcl-2 and survivin) and proliferation markers small malignant potential. Information on HPV infection in OLP is (Proliferating Cell Nuclear Antigen; PCNA). The risk of HR-HPV scanty, but the few studies published report a high frequency [106, infection was found to be independently associated with survivin 107] from 27.3% (75), to 42.0% [108] or even 65.0% [109]. Both and PCNA expression, suggesting that these markers could be in- Gonzales-Moles [110] and Jontell [109] observed that only the volved in HPV-mediated disorders of epithelial maturation [100]. 5458 Current Pharmaceutical Design, 2012, Vol. 18, No. 34 Syrjänen et al. Fig. (12). OSCC of the alveolar ridge. Fig. (9). Oral eritroplakia of the border of the tongue. Nevertheless, the role of HPV infection in oral carcinogenesis is still unclear because conflicting results have been reported. A very wide range of viral prevalence (0%-100%), in addition to the pres- ence of HR HPV in normal oral mucosa, has been reported in litera- ture [43, 73, 77]. Apart from the differences among the patient groups examined regarding ethno-geographical characteristics and sexual behaviour, this wide range of prevalence could be related to: i) the site of lesion (oral cavity vs. oropharynx and tonsils), and ii) the sensitivity of the applied diagnostic technique, in terms of sam- pling method and HPV detection procedure. The last comprehensive meta-analysis conducted on this issue (and utilizing a strict case-control setting) showed that HPV signifi- cantly increase the risk for OSCC, as compared with the controls (OR 3.98, 95% CI: 2.62-6.02); especially when HPV 16 is investi- gated (pooled OR 3.86, 95% CI: 2.16-6.87) [12]. However, the Fig. (10). Oral Lichen Planus - reticular form- of the tongue. HPV association was found to be significant also when biopsy samples of OSCC were compared with cytological samples of the erosive variant of OLP was found to be HPV positive but this was controls. According to this findings and considering that that HPV not confirmed in another study [111]. In the recent systematic re- infection is multi-focal (but not necessarily persistent/oncogenic), view of HPV and OPMDs, the prevalence of any HPV genotypes the authors recommend the use of biopsied tissue for HPV testing in and only HPV-16 in OLP were 5.12% and 5.61 respectively [12]. future studies [12]. With almost 130,000 annual deaths worldwide, OSCC (Fig. 11 3.3. Oro-genital HPV Infection and 12) is considered a public health emergency [112]. Usually OSCC is characterized by a very aggressive behaviour and a poor Few published investigations have evaluated how frequently prognosis, with an overall 5 years survival rate of 59.4%, [9, 113]. HPV infection occurs simultaneously in the oral districts and in the female genital area. Research so far has tried to clarify the nature of Recently, HR HPV genotypes have been identified as etiologic agents for a subset of SCCs of oral cavity and oropharynx, espe- this correlation by studying social-behavioural risk factors and in cially in non-smoker and non-drinker subjects [114, 115]. The hy- particular, transference of the virus to different epithelial sites in the same patient. With regard to this aspect, various authors consider pothesis of the participation of HPV in oral and oropharyngeal car- cinogenesis was first proposed by Syrjanen et al [116] and then the two most common transmission patterns of HPV from one anat- supported by several other authors [38, 65, 117-121] on the basis of omic site to another, as being sexual transmission through oral- genital contact with an infected partner and self-inoculation. In the following evidence: a) the epitheliotropic nature of HPV; b) the widely confirmed oncogenic potential of HR gt in the pathogenesis order to study how infection is transmitted, various researchers of anogenital neoplasms; and c) the morphological similarities be- have used questionnaires designed to assess the impact of factors likely to cause genital infection, such as, early sexual exposure, a tween oropharyngeal and genital epithelia [122]. high number of partners in general and in the last year, number of pregnancies, level of education, contraceptive methods, sexual hab- its and also personal hygiene standards of the patients. On examin- ing studies, the main topic to be considered is the direct association between the sensitivity of the diagnostic test used and the percent- age of oral-cervical infection detected. Kellokoski et al, in a study carried out over ten years ago on women with current and previous genital HPV infection, detected an elevated percentage of the virus present in normal oral mucosa in relation to the diagnostic protocol adopted: respectively through Dot Blot Hybridization (DBH) on cytologic samples the prevalence was 3.3%, by means of Southern Blot Hybridization (SBH) on histologic samples, prevalence was 15.6% and lastly by means of PCR, on the same samples, there was 23.1% prevalence [123]. Badaracco et al. [124] detected simultane- ous oral-cervical infection in 5 of 29 patients (equal to 17.2%) by Fig. (11). Oral Squamous Cell Carcinoma (OSCC) of the tongue. means of a molecular technique called “strip-test” which combines PCR with Reverse Blot Hybridization. Recently, Giraldo et al. de- Oral HPV Infection Current Pharmaceutical Design, 2012, Vol. 18, No. 34 5459 tected, once again by means of PCR, the presence of the virus in the 4.1. Management of Genital HPV Infection oral mucosa in over 37% of patients with both cytological and his- None therapy is currently available and recommended for sub- tological lesions in the HPV-related genital area, a percentage clinical genital HPV infection, since this condition is not definable which drastically drops to 4.3% in women without symptoms [125]. as a disease and it is usually transitory and self-eliminating by the These results have led many authors to suggest that infection in the host immune system over 6-20 months (usually 8-10 months). It is genital mucosa represents a condition which favours the progres- imperative to carry out the follow up of the condition and the repe- sion of the viral infection of the oral mucosa, even if many studies tition of HPV DNA test after this interval to verify the elimination have not found a genotypical concordance between the infection in of the infection, especially when sustained by HR HPV, potentially the two areas. In Badaracco et al.’s study, in 3 of 5 cases of dual able to give a persistent and transforming infection [19, 37]. In infection, HPV 16 was detected in both areas. In addition, some addition adequate education and counselling of the infected patients authors have highlighted, in a sample of women suffering from is mandatory in order to control the horizontal transmission of in- SCC of uterine cervix, a significant correlation of gt with cervical- fection and/or autoinoculation at other epithelial sites of the body. vaginal infection. In 23 of 28 women with concurrent HPV genital Because HPV infection is highly contagious, current sexual partner infection, they detected the same gt found in the oral area, leading of infected subjects are likely already HPV-infected. Condom use to the hypothesis that the virus, after primary infection in the inocu- may reduce partially the risk of transmission because the virus is lation site, is able to spread to other epithelial areas, i.e. the cervical able to infect the epithelium of the entire anogenital tract. Routine mucosa, which is particularly susceptible to HPV infection and its clinical and virological examination of sexual partners is not man- oncogenic potential [126]. Smith et al [127] and Canadas et al. [33] datory [132, 133]. On the other hand, female sex partners of male instead, did not detect any genotypic oral-genital concordance in patients who have genital infection/warts should be reminded that any of the patients examined. On the basis of this point and the fact cytological screening for cervical cancer is recommended; in fact that there is no corresponding infection in the partner, Smith et al. among men, genital infection is more frequently unapparent than in believed that both the possibility of infection through self- women, where more frequently genital/cervical infection is associ- inoculation or by sexual transmission was unlikely, and therefore ated with asymptomatic lesions [134]. put forward the hypothesis of a different susceptibility to HPV in- In presence of HPV-related lesions, the treatment is based on fection in the two anatomic sites [127]. Canadas et al. were of the surgery, local or systemic administration of drugs (cytotoxic or same opinion and assessed the many different areas where HPV immunomodulatory agents) or a combination of all of them [135] infection may be present, (cervical, vaginal, vulvar, anal and oral (Table 3). The decision whether to treat HPV-related lesions is mucosa) in a sample of high risk patients and they proposed a dif- strongly influenced by the clinical manifestations, symptoms and ferent affinity between the viral types compared in the two mucosal the potential malignancy. Genital warts and condylomas are associ- sites: in the anogenital area, infection caused by HPV HR was ated with discomfort and physical/psychological dysfunctions; al- found in over 80% of cases, while in the oral cavity a predominance though eradication of infection is not feasible, treatment of these of HPV LR was detected [33]. The genotypic variability of the benign lesions is warranted in order to reduce these negative as- infection, which is commonly found even in monogamous partners, pects. However, before starting therapy, patients should be learned therefore, seems to depend on the differences relative to tissue about the limitations of the current available therapy for genital characteristics and microbial flora of each mucosal site, even if warts/condylomas, because most of these are effective in clearing other hypothesis must still be taken into consideration. In fact, some warts or temporary reducing their size, but recurrence rates are high authors have linked the diverse anatomic susceptibility to different after all treatments [131]. mechanisms of the immune system. In his study Goncalves et al. assumed that there was concordance between genital HPV infection Surgical, ablative and destructive therapy. Surgery remains the and reduced concentration of IgA in saliva secretions, a condition basic method for the treatment of the most frequent HPV-related which would explain the fall in the protective function of local anti- anogenital lesions. The excision may be performed using cold bodies and therefore would make the oral cavity more susceptible knife, electrosurgery or laser after local anaesthesia. For anogenital to colonization by the virus [128]. Other authors put forward the warts and condylomas the standard therapy is the destruction of the hypothesis that dual or multiple infections may depend on a genetic lesion with cryosurgery, cryotherapy with liquid nitrogen, laser predisposition which is probably connected in some way, as re- ablation (CO2) or electrocautery [135]. Other modalities of as- cently suggested, to some HLA haplotypes, able to make the infec- pecific destruction of external benignant lesions are based on the tion more susceptible individually and more likely to transform typical application of acids (mono-, bi- and trichloroacetic acids – HPV-related lesions into malignant ones [129, 130]. In conclusion MCA, BCA, TCA respectively – and salicylic acid), or aldehydes it is obviously necessary to carry out further research to clarify the (glutaraldehyde, formaldehyde). As for cryotherapy, the response frequency of the concomitant infection (oral and genital) in relation rate range from 50-80% and about one-fourth of patients has a re- to possible risk factors and to social-behavioural variables, paying lapse. The photodynamic therapy, based on the local application of particular attention to sexual habits which lead to an increase in photosensitizer agents (derived from porphyrins) selectively up- susceptibility to the infection. These studies presuppose a multidis- taken by tumor cells, have been tested for HPV-related lesion, es- ciplinary synergy involving diverse medical specializations (den- pecially RRP, and successively for genital lesions (CIN, VuIN), tists, gynaecologists, virologists) to establish new ways of prevent- with a response in about 50% of cases according Martin-Hirsch et ing HPV infection through effective diagnosis and treatment. al [136, 137]. Nevertheless, results of more recent studies were contrasting, also for RRP [138, 139]. 4. THERAPIES FOR HPV INFECTION AND HPV- A specific treatment has been recommended for CIN, that is the RELATED LESIONS application of the LLETZ (Large Loop Excision of Transformation For many infectious diseases, it is evident that therapy is bene- Zone), derived from the Loop Electrode Excision procedure (LEEP) ficial and that their eradication can be achieved by means of control and associated with greater safety and efficacy (95% cure rate in a and prevention of infection [131]. Conversely for HPV-related le- year) and minor recurrence rates than other surgical procedures sions, these considerations are not similarly correct. No current [135, 140]. Other potentially malignant lesions of anogenital area therapy is able to eradicate the HPV infection and the available (AIN, VaIN, VuIN, PIN) represent challenging clinical problems therapeutic tools have a limited impact in preventing lesion recur- because there are no standard surgical recommendations as for CIN rence and reducing the likelihood of viral transmission. Following lesions [141]. Noteworthy, tobacco smoking represents a well the current therapies for HPV-related lesions in genital and oral known co-carcinogenic agent for development of these HPV-related mucosa are described (see Table 3). diseases, so dedicated education and counselling for smoking cessa- 5460 Current Pharmaceutical Design, 2012, Vol. 18, No. 34 Syrjänen et al. Table 3. Management of HPV Infection/Diseases in Genital and Oral Mucosa Genital mucosa Oral mucosa Strategy Dosage/ route of drug Reported clinical Dosage/ route of Reported clinical Treatment Treatment administration indication drug administration indication Surgery Cold knife Warts Cold knife Benignant lesions Electro-surgery Warts QMR scalpel HPV-related lesions and OPMD (if ab- Laser therapy Warts sent/low grade dyspla- LLETZ CIN sia ) Electro-surgery Not recommended Laser therapy Not recommended Ablative destruc- Cryotherapy External warts Photodynamic Limited clinical data tive therapy MCA, BCA, TCA Topical 80-90% solu- External warts therapy (OL, PVL) tion (office use) Aldehydes External warts Other ablative therapies Salicylic acid External warts are not tested for oral mucosal use Photodynamic therapy CIN, VuIN Antiproliferant, Podophyllin Topical 10-25% solu- Warts citotoxic and anti- tion weekly (office viral agents use) Podophylloxotin Warts Topical 0.5% solution Drugs scarcely/not twice/day for 3 days tested for oral mucosal (self-application) use. Not specific regimes 5-FU Topical 5% cream 1-3 Warts and dosage are cur- times/week (self- rently available. application) Cidofovir Topical /intralesio-nal Warts and CIN injection Immunomodulator IFN (cid:1) and (cid:2) Topical/intralesional Warts (limited indi- Drugs scarcely/not agents injection/systemic cation) tested for oral mucosal use. Not specific regimes Imiquimod Topical 5% cream 3 and dosage are cur- times/week Warts rently available. Novel therapies Ribozymes siRNA Limited clinical data Antisense oligonu- No data are available out of standard cleotides for oral mucosa therapy Molecular E6/E7 targeted therapy tion in patients who smoke are required [19, 131]. A more detailed A significant proportion (about 50%) of patients treated in first discussion of the treatment of anogenital intraepithelial neoplasia line with surgery (especially with traditional cold knife technique or and SCC is not the aim of this review and we forward to dedicated ablative therapy) are subjected to a recurrence and need of an adju- gynaecological literature. vant medical treatment [142, 143]. Moreover ablative approaches Oral HPV Infection Current Pharmaceutical Design, 2012, Vol. 18, No. 34 5461 have significant sequelae such as bleeding, scarring, pain and in- results of reported small sized studies and lack of large series and/or flammation. In addition, when visible lesions are effectively re- RCT and have reduced the use of this agent. moved, residual viral particles or DNA in surrounding tissues may Very recently Roberts et al. [157] reported interesting findings reseed the treated site and lead to recurrence of lesion. A series of about the topical use of carrageenan to reduce susceptibility to HPV medical therapies have been proposed, as following reported. of cervical mucosa during cytology screening. The same molecule Antiproliferative, cytotoxic and antiviral agents. These drugs have been proposed as adjuvant to peptide-based vaccine for their play as metabolic inhibitors when topically applied or injected into ability to induce antigen specific immune response, especially visible lesions. Podophyllin, resin extracted from the roots of some against E7 [158]. Berberiacee species, are a cytotoxic agents able to arrest mitosis at Noteworthy, an important limitation of the immune-modulatory metaphase by binding tubulin and also to destroy HPV virions. It therapy is that the viral replicative cycle and the protein transcrip- was abandoned in favour of its derived podophyllotoxin (also tion in the infected cells entirely occur into the nucleus and the known as podofilox), associated with minor local and systemic side cytoplasm, without expression of viral protein on the cellular mem- effects, indicated for topical treatment of external genital warts brane, in this way infection happens without immune response acti- (0.5% solution) [144] (Table 3). 5-Flururoacil (5-FU) inhibit DNA vation. Moreover the viral oncoproteins E6 and E7 have specific and RNA synthesis and acts as antimetabolite when used topically mechanisms for abrogate the host immune response [159]. These as 5% cream for self-application for treatment of multifocal and two mechanisms may explain the variable efficacy of immune- extensive genital warts or VaIN/ VuIN, although its use is recently therapies in different patients in presence of specific HPV geno- restricted because frequently cause local irritation [144]. Bleomy- types. cin, able to bind DNA, is not indicated for mucosal lesions because New therapeutic strategies. The new therapeutic challenge is intralesional injection is very painful and associated with local side the development of more targeted strategies based on nucleic acids effects (swelling and eschar formation) [145]. Cidofovir is a nu- (small interfering RNA, antisense oligonucleotides, and ribozymes cleotide analogue (acylic nucleoside phosphonate) able to inhibit able to inhibit expression or activity of viral proteins) [159, 160]. DNA synthesis and, according some reports, may induce apoptosis E6 and E7 proteins demonstrated to play together to nullify host [146]. It has a broad spectrum antiviral activity against several immune responses, thus interdicting E6- and E7- mediated actions DNA viruses. However, the rational for using cidofovir for the could increase the likelihood of infection elimination by the natural treatment of HPV-related lesions is not clear since the HPV replica- immunity. tive cycle is completely different from other DNA viruses sensitive to this molecule, so the question remains if the activity of cidofovir For E6 targeting therapy, the abrogation of telomerase activity against HPV produces more antiproliferative than antiviral effects. and the interdiction of E6-p300/CBP interaction have been studied Its clinical use as topical cream or injected solution, intralesionally [159, 161-164]. or systemically, has been tested in several HPV-related lesions For E7 targeting strategy, potentially candidate for therapeutic (anogenital condylomas, skin warts, CIN, recurrent respiratory intervention is the interaction of E7 proteins with cdk inhibitors papillomatosis) with success [147], also among immunocom- (CKIs: p21CIP1 and p27KIP1) and the disruption of the E7-IRF-1 promised patients [148] and as co-adjuvant agents to ablative and/or complex and/or inhibition of HDAC activity [159, 165, 166]. surgical removal to reduce relapse [149]. However large random- In addition numerous other interaction between HPV E/L pro- ized clinical trials are still not available, thus different questions teins and host regulatory factors have been recognized are still stud- (about the exact mechanism of action, the treatment modalities, the ied for molecular targeted strategy (reviewed in [159, 167]), never- response rate and the suspected carcinogenic potential of the mole- theless the use of this approach is still a promising strategy that will cule) still remain [150]. require further exploration. Immunomodulatory drugs. While previous strategies act indis- 4.2. Management of Oral HPV Infection criminately destroying both healthy and infected tissues, approach with immunomodulatory agents consent to enhance the host im- As discussed with respect to genital HPV infection, also in oral mune response in order to organize a selective defence to HPV cavity HPV subclinical infection does not need of treatment since infected cells. This effect may be obtained directly by local applica- any tested antiviral drugs (e.g. acyclovir, ribavirin) have been dem- tion of interferon (IFN), or, indirectly, by administration of imiqui- onstrated able to eliminate the infection [135] (Table 3). The man- mod. IFN (cid:1) and (cid:2), playing both immunomodulatory and direct an- agement of this condition is based on the follow up of the patient tiviral activity, were used alone or in association with other drugs over at least 8-12 months, repetition of HPV DNA test after this for treatment of several HPV-related lesions, especially when diffi- interval and periodically until elimination of the infection occurs. cult to treat with surgery, such as RRP [141]. IFN may be used with As assumed for gynaecology field, a persistent viral status (sus- several routes of administration (topical application, intralesional tained by HR-HPV types) maintained for at least 2 years represents injection, systemic), but the variable efficacy results with frequent a necessary although not sufficient basis for HPV-related lesions rebound phenomenon at the end of therapy [151], the high costs and [46, 47, 85, 168]. Differently by gynaecologists, employing a stan- the frequent systemic side effects (i.e. leukopenia, fever, head- dard sampling method for HPV testing in uterine cervix, in oral heache, myalgia, asthenia), have progressively limited the use of medicine a uniform method of specimens collection for HPV detec- these molecules [144]. Imiquimod, a member of the class of imi- tion still does not exist. Several procedures of oral specimens col- dazquinolines, acts as a potent immunostimulator, antiviral and lection were evaluated in literature [169, 170]. However, the virus antitumoral agents in animal model. Imiquimod demonstrated able is rarely found in oral mucosa, probably due to saliva clearance. to induce the release of pro-inflammatory Th1 class cytokines from Potentially, it is more probable that tonsillar crypts and the glot- lymphocytes and macrophages (e.g. IFN-(cid:1), -(cid:2) and –(cid:3), TNF(cid:1) and tides, more susceptible to viral entry and replication [171], will IL12) and to enhance cell-mediated local immunity against viral constantly harbour virions than other mucosal oral districts, and targets, mechanisms that may facilitate viral clearance. It has been they will be detected with greater difficultly. Furthermore, in most indicated topically (5% cream) for the treatment of genital warts studies, the exact anatomic sites of the samples are not given and [131, 152, 153] recently, some case series test its efficacy in pa- thus, the origin of HPV infection (whether oral or oropharyngeal) is tients with VuIN [154], AIN [155], carcinoma in situ of the penis impossible to trace. [156]. Imiquimod is well tolerated, only mild to moderate local side The samples are taken either as mucosal scrapings, oral rinses effects (inflammation) have been reported, but the not conclusive or tissue biopsies. The biopsy remains one of the most common procedures of oral sampling, thanks to the possibility of using the