Important notice: even though the proportion of patients in which the HPV DNA was tested at The Pathology center was relatively low (12 and 15%, respectively), the application of a complex approach Figure 9 proven the prevalence of HPV DNA presence by CIN III/HSIL.



\* Department of Clinical Microbiology, Alpha medical Ltd, Ružomberok, suitable to recognize the genotypes.

\*\* HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.

**Table 12.** Results of HPV DNA testing by means of the Cobas 4800 equipment\* .

**Figure 9.** A simple algorithm allowing to assess the precise diagnosis of dysplasia and to estimate its prognosis (all the basic information shown should be at disposal for exact decision making).

## **5. HPV DNA detection in association with cytological and histological examinations**

In countries where cytological diagnostic is widely used for screening, the most useful option is to use HPV DNA testing especially for getting more reliable information allowing to lengthen safely the next smear examination interval [86]. In general, the DNA tests are based either on DNA/DNA hybridization (using labeled complementary DNA probes), or on amplification of vDNA by polymerase chain reaction (PCR) as well as on DNA/RNA hybrid‐ ization (using complementary RNA probes) followed by visualization of the labeled hybrid signal. Early approaches utilized various modifications of the vDNA to DNA probe hybridi‐ zation tests, such as in situ hybridization and/or various blotting techniques [87]. To improve the PCR method, multiple primers for L1 and/or E1 gene amplifications have been introduced, aiming to identify the most frequent genotypes (i.e., HPV 6, 11, 16, and 18) in a single-tube reaction [88]. The principle of multiple genotype amplification was further modified using general primers (GPs), which flank the strongly conserved regions located either on L1 and/or E1 ORFs, enabling detection of a wide spectrum of genotypes [89]. The GPs annealed not only to the ORFs of genotypes, which they had been designed for, but also to some another which sequence was not known at that time (later on these were identified as HPV 13, 30, 31, 45, and 51). The consensus or general primer GP5+/GP6+ based procedure became widely used, since it enabled the differentiation between several HR and LR HPV genotypes in a single assay [90, 91]. The GP-PCR technique became further improved in order to detect more genotypes (at least 14 HR HPV along with the 6 frequent LR HPV genotypes). In addition, it was modified in order to visualize the reaction product by enzyme linked immunosorbent assay, that is ELISA [92]. The high-risk types being assessed this way were, as a rule, HPV 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 66, and 68, and the low-risk ones were at least HPV 6, 11, 40, 42, 43, and 44. Further improvements of GPs allowed their annealing to the DNA of additional genotypes, such as HPV 26, 30, 53, 70, 73, 82, and 83, in order to increase the number of routinely detectable genotypes to 27 out of the 40 possible mucosal human papillomavirus types [93]. Additional consensus primers, having been introduced for L1 ORF amplification, were further modified to avoid synthesis of irreproducible fragments [94]. The latter primer set (PGMY07/11) increased the number of multiple HPV genotype infections detected by adding the rare genotypes such as HPV 26, 35, 42, 45, 52, 54, 59, 66, and 73. Any routine HR versus LR HPV testing may be influenced by the DNA extraction technic, namely depending whether a recommended manual extraction procedure or an automated extraction protocol supplied by the manufacturer of given equipment was used [95]. To avoid the methodic variations, automated vDNA extraction was recommended for HPV genotyping by both, the classical PCR (GP5/GP6 primers) as well as for the real-time PCR-based quantitative TaqMan assay. Further modification of HPV detection in the direction of immunochemistry has resulted into an assay omitting vDNA amplification, while introducing the labeled signal amplification instead. In a latter assay, the denatured vDNA was hybridized under high stringency condi‐ tions to single-stranded RNA probes either for LR genotypes (at least 6, 11, 42, 43, and 44) and/ or for HR genotypes (at least 16, 18, 31, 33, 35, 45, 51, 52, and 56). The RNA/DNA hybrid complex was then bound to microplates (or tubes) coated with an alkaline phosphatase conjugated monoclonal antibody, able to capture the specific RNA/DNA hybrid [96]. The reaction is then visualized by addition of the chemilumiscent substrate, in which emission light is amplified and measured in a luminometer; the results are expressed in relative light units (RLU). This method is referred to as hybrid capture (HC). At its beginning HC showed lower sensitivity, as compared to as few as 10–100 vDNA copies (about 100 fg HPV DNA) were detectable per 1 ml sample when using the classical PCR. The recent HC2-based high-risk HPV DNA test (Qiagen), which was previously used in the Alpha medical laboratory as well, detects 13 HR genotypes (HPV 16, 18, 31, 33, 35, 35, 39, 45, 51, 52, 56, 58, and 68) at a sensitivity of 1– 2 pg/ml (i.e., about 100,000 HPV DNA copies/ml). Another variation of this assay can be also used for detection of at least 5 LR genotypes (HPV 6, 11, 42, 43, and 44). Recio et al. [97] used the first generation HC test for investigating the HPV DNA presence in patients with ASCUS, LSIL, HSIL, and carcinoma *in situ* smears, the latter being used as a relevant standard. Altogether 44% of patients were tested positive, mainly for HR HPV genotypes. The authors

**Figure 9.** A simple algorithm allowing to assess the precise diagnosis of dysplasia and to estimate its prognosis (all the

**5. HPV DNA detection in association with cytological and histological**

In countries where cytological diagnostic is widely used for screening, the most useful option is to use HPV DNA testing especially for getting more reliable information allowing to lengthen safely the next smear examination interval [86]. In general, the DNA tests are based either on DNA/DNA hybridization (using labeled complementary DNA probes), or on amplification of vDNA by polymerase chain reaction (PCR) as well as on DNA/RNA hybrid‐ ization (using complementary RNA probes) followed by visualization of the labeled hybrid signal. Early approaches utilized various modifications of the vDNA to DNA probe hybridi‐ zation tests, such as in situ hybridization and/or various blotting techniques [87]. To improve the PCR method, multiple primers for L1 and/or E1 gene amplifications have been introduced, aiming to identify the most frequent genotypes (i.e., HPV 6, 11, 16, and 18) in a single-tube

basic information shown should be at disposal for exact decision making).

136 Human Papillomavirus - Research in a Global Perspective

**examinations**

concluded that testing of HPV DNA by the HC method is helpful for clinical diagnostic. Monsenogo et al. [90] reported that the HC2 test as compared in 470 patients with the PCRbased Roche Amplicor HPV test, reached an agreement of 96.2%; only 18 cases were found discordant. It should be mentioned that the Amplicor HPV test identifies the PCR-amplified vDNA by means of 13 HR-HPV genotype probes and that the vDNA is being obtained from cervical cells collected into a transport medium [98]. In patients revealing ASCUS smears, both tests for HPV DNA showed a positive rate of 42.3%, while in patients showing LSIL smears, the HPV DNA positive rate was 66.3 and/or 66.8%, respectively, depending on the test performed (the PCR-based test seemed in this case even less sensitive). In patients with HSIL smears, the DNA was positive in both tests at the highest rate of 92.8%.

The HC2 high risk HPV DNA Test takes advantage of the Hybrid Capture 2 (HC2) technology; it is a nucleic acid hybridization assay with signal amplification that utilizes microplate chemiluminescent detection for the qualitative detection of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) in cervical specimens. Specimens containing the target DNA hybridize with a specific HPV RNA probe. The resultant RNA:DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA:DNA hybrids and detected with chemiluminescent substrate. Several alkaline phosphatase molecules are conjugated to each antibody. Multiple conjugated antibodies bind to each captured hybrid resulting in substantial signal amplification. In contrast to the HC technology, the Cobas® 4800 Human Papillomavirus (HPV) Test is a qualitative *in vitro* test for the detection of human papillomavirus in cervical specimens. The test utilizes amplification of target DNA by the polymerase chain reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk HPV types in a single analysis. The test specifically identifies HPV16 and HPV18 while concurrently detecting the other high-risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) at clinically relevant infection levels. The Cobas® 4800 HPV Test primers define a sequence of approximately 200 nucleotides within the polymorphic L1 region of the HPV genome. An additional primer pair would target the human β-globin gene (330 bp amplicon) to provide a process control. A pool of HPV primers present in the Master Mix is designed to amplify HPV DNA from 14 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The detection of amplified DNA is performed during thermal cycling using oligonucleotide probes labeled with four different fluorescent dyes. The amplified signal from twelve high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), is detected using the same fluorescent dye, while HPV16, HPV18, and β-globin signals are each detected with their own dedicated fluorescent dye. Fluorescent oligonucleotide probes bind to polymorphic regions HPV and human β-globin gene within the sequence defined by primers.

Summing up, the DNAs can be distinguished according to (1) the ability to identify a pool of high-risk HPV types, with or without genotypization of the most common high-risk viruses (i.e., HPV16 and 18) or (2) to detect a broad spectrum of oncogenic and non-oncogenic HPVs along with individual genotyping. While the assays of the first group are mainly used in screening programs, where there is no clinical benefit from the knowledge of specific HPV types, the assays of the second group are primarily used in HPV surveillance studies and to monitor the eventual spreading of particular viral types in vaccinated women [99]. The HPV16 and HPV18 genotyping, for its high specificity, have been included in the US guidelines for the triage of HPV positive and cytology negative women [100]. In general, the probability of developing precancerous (HSIL) lesions was high in women who were LSIL as well as DNA positive; a lower, but still medium probability for developing cancer was found in HPV DNA positive, but by cytology negative women. The lowest probability was noted in HPV negative but ASCUS/LSIL positive cases. Similar findings were observed in the Microbiology Depart‐ ment of Alpha medical (**Table 12**) showing that the positive rate of HR genotypes was the highest in HSIL cases, the closet in cases with not precisely determined diagnosis. As stressed by Mandelblatt et al. [101], screening of HPV plus patients with LSIL tests within 2 years appears to save lives and is more reasonable than performing the cytology test alone. Another large cohort study in US (performed from 2003 to 2005) found that women aged less than 30 who have ASCUS-grade smears showed HPV positive rate at 53% [102]. On the other hand, women older than 30 years with NIL PAP test were HPV (HC2) positive at a rate of 9%. Since ASCUS smear diagnosis is a clinical and prognostic challenge, it should be combined with HPV testing and repeatedly investigated to show whether or not the transition to HSIL occurs [103]. Also in this follow up, a relatively low proportion (6.7%) of ASCUS positive patients developed HSIL or cancer. Among the patients with HSIL smears, up to 98% was found HPV 565 DNA positive; theoretically such women harbor the integrated incomplete genome in the cervical tissue. It was concluded that the residual specimens collected from routine cervical cytology in ASCUS cases could provide additional information about the HPV DNA status that is of substantial help by identifying those patients, who are likely to develop HSIL, especially if they test positive for the HPV DNA presence.

## **Acknowledgements**

concluded that testing of HPV DNA by the HC method is helpful for clinical diagnostic. Monsenogo et al. [90] reported that the HC2 test as compared in 470 patients with the PCRbased Roche Amplicor HPV test, reached an agreement of 96.2%; only 18 cases were found discordant. It should be mentioned that the Amplicor HPV test identifies the PCR-amplified vDNA by means of 13 HR-HPV genotype probes and that the vDNA is being obtained from cervical cells collected into a transport medium [98]. In patients revealing ASCUS smears, both tests for HPV DNA showed a positive rate of 42.3%, while in patients showing LSIL smears, the HPV DNA positive rate was 66.3 and/or 66.8%, respectively, depending on the test performed (the PCR-based test seemed in this case even less sensitive). In patients with HSIL

The HC2 high risk HPV DNA Test takes advantage of the Hybrid Capture 2 (HC2) technology; it is a nucleic acid hybridization assay with signal amplification that utilizes microplate chemiluminescent detection for the qualitative detection of 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) in cervical specimens. Specimens containing the target DNA hybridize with a specific HPV RNA probe. The resultant RNA:DNA hybrids are captured onto the surface of a microplate well coated with antibodies specific for RNA:DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA:DNA hybrids and detected with chemiluminescent substrate. Several alkaline phosphatase molecules are conjugated to each antibody. Multiple conjugated antibodies bind to each captured hybrid resulting in substantial signal amplification. In contrast to the HC technology, the Cobas® 4800 Human Papillomavirus (HPV) Test is a qualitative *in vitro* test for the detection of human papillomavirus in cervical specimens. The test utilizes amplification of target DNA by the polymerase chain reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk HPV types in a single analysis. The test specifically identifies HPV16 and HPV18 while concurrently detecting the other high-risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) at clinically relevant infection levels. The Cobas® 4800 HPV Test primers define a sequence of approximately 200 nucleotides within the polymorphic L1 region of the HPV genome. An additional primer pair would target the human β-globin gene (330 bp amplicon) to provide a process control. A pool of HPV primers present in the Master Mix is designed to amplify HPV DNA from 14 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The detection of amplified DNA is performed during thermal cycling using oligonucleotide probes labeled with four different fluorescent dyes. The amplified signal from twelve high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), is detected using the same fluorescent dye, while HPV16, HPV18, and β-globin signals are each detected with their own dedicated fluorescent dye. Fluorescent oligonucleotide probes bind to polymorphic regions HPV and human β-globin gene within the sequence defined by

Summing up, the DNAs can be distinguished according to (1) the ability to identify a pool of high-risk HPV types, with or without genotypization of the most common high-risk viruses (i.e., HPV16 and 18) or (2) to detect a broad spectrum of oncogenic and non-oncogenic HPVs along with individual genotyping. While the assays of the first group are mainly used in screening programs, where there is no clinical benefit from the knowledge of specific HPV

smears, the DNA was positive in both tests at the highest rate of 92.8%.

138 Human Papillomavirus - Research in a Global Perspective

primers.

The authors thank Dr. J. Hybenová the Head of the Department of Clinical Microbiology, Alpha medical Ltd, Ružomberok (for her kind advice), as well as Mrs. J. Papanová and Mrs. S. Drahošová from the Pathology Diagnostic Center, Martin, Alpha medical, Slovakia, for their helpful assistance.

## **Author details**

J. Rajčáni1\*, K. Kajo2,3, O. el Hassoun1 , M. Adamkov1,4 and M. Benčat<sup>1</sup>

\*Address all correspondence to: viruraj@savba.sk

1 Pathology Ltd, Diagnostic Center, Alpha Medical Ltd, Záborského 2, 03601 Martin, Slovak Republic

2 Pathology Ltd, Diagnostic Laboratory, Alpha medical Ltd, Banská Bystrica, Slovak Repub‐ lic

3 Department of Pathology, St. Elisabeth Cancer Institute, Bratislava, Slovak Republic

4 Department of Histology, Jessenius Medical Faculty, Comenius University, Martin, Slovak Republic

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2 Pathology Ltd, Diagnostic Laboratory, Alpha medical Ltd, Banská Bystrica, Slovak Repub‐

4 Department of Histology, Jessenius Medical Faculty, Comenius University, Martin, Slovak

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## **Diagnosis and Prevalence of High-Risk Human Papillomavirus Infection in Heterosexual Men**

Elena López-Díez, Sonia Pérez and Amparo Iñarrea

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62948

### **Abstract**

A better understanding of human papillomavirus (HPV) infection in men is an essen‐ tial component of prevention programs aimed to reduce cervical cancer and other HPVrelated diseases. A screening test capable of detecting asymptomatic/subclinical genital HPV infection in men at a reasonable price and causing minimal discomfort to the patient would be very valuable. The following chapter focuses on acetowhite test usefulness in the detection of asymptomatic/subclinical genital high-risk (HR) HPV infection in highrisk men populations, HR-HPV prevalence in sexually active healthy male partners of women diagnosed of high-grade cervical intraepithelial neoplasia and genotypespecific concordance between partners, addressing the preventive strategies that would reduce HPV infection in men. We present data from 125 men, sexual partners of women with preneoplastic cervical lesions. Prevalence of HR-HPV infection in male was high (50, 24% HPV16) and genotype concordance within the 60 infected couples was remarkable (62% shared at least one genotype). Acetowhite (AW) test was positive in 27% patients, showing low sensitivity for the identification of HR-HPV infection but allowed the diagnosis of subclinical HPV-related lesions in more than 10%. Current smoking and genital warts were associated with an increased risk of HR-HPV infection in men (OR: 2.4 and 5.6, respectively).

**Keywords:** human papillomavirus DNA test, prevention, prevalence, cervical intrae‐ pithelial neoplasia, male, mass screening, genital warts, diagnosis

## **1. Introduction**

Human papillomavirus (HPV) infections are one of the most common sexually transmitted infections worldwide [1], representing a significant health problem due to their high preva‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lence and transmissibility. HPVs are a very large family of double-stranded DNA viruses (dsDNA), very resistant that can survive in the environment without a host and is able to infect humans. These viruses are not classified as serotypes, but as genotypes on the basis of DNA sequence. Currently, over 120 genotypes have been identified and about 40 genotypes (the alpha genus) can be transmitted through sexual contact and infect the anogenital region. HPV genotypes have been classified into low-risk genotypes, associated with anogenital warts, lowgrade cervical lesions and recurrent respiratory papillomatosis, and high-risk genotypes (HR-HPV) [1] (**Table 1**), which eventually can lead to malignant transformation. HR-HPV are strongly associated with cancer and high-grade neoplasia of the anogenital tract, including the anus (AIN), penis (PeIN), uterine cervix (CIN), and vulva (VIN), and also a proportion of orophar‐ yngeal cancer [2]. Although these infections are typically transient and asymptomatic, some of them will result in anogenital warts, and dysplastic and/or neoplastic lesions, which cause a substantial disease burden in both sexes and generate a considerable economic distress within society [3].


Classification of oncogenic HPV genotypes detected in this work. IARC, International Agency for Research on Cancer; HR-HPV, high-risk HPV genotypes; pHR, probable/possible highrisk genotypes.

**Table 1.** Oncogenic HPV genotypes.

The virus may remain inactive for a long time and produce asymptomatic infection of the skin. It can be transmitted from one individual to another directly (by sexual contact) or indirectly. The dynamics of heterosexual transmission of HPV are still being investigated [4].

About one-third to one-quarter of invasive penile cancers (Alemany et al.) and nearly 99.7% of cervical cancer worldwide and in 96.8% of cervical preneoplastic and neoplastic lesions in our community (Perez et al.) may be related to HPV according to the retrospective studies. Although rare, penile cancer is associated with a high morbidity and mortality. The carcino‐ genesis of penile cancer is thought to involve two pathways: one related to inflammation and other dermatological conditions of the penis, and other related to HPV infection (López-Romero et al.). HPV DNA prevalence in invasive penile cancer varied geographically, with the highest prevalence in Oceania (55.6%), North America (48.7), Africa (36.8%), South America (39.7%), and Europe (45.9%), being the most common HR-HPV types: HPV16 (30.8%) and HPV18 (6.6%) [5]. So that, it is important to be cautious and not to consider overall prevalence as universal because the role of HPV in penile cancer etiology could be strongly influenced by histologic distribution and geographic region as it is also true for other HPV malignancies such as vulvar and head and neck cancers [6].

Genital warts (GWs) represent a significant public health problem associated with clinical symptoms (burning, bleeding, and pain) and psychosocial problems (embarrassment, anxiety, and decreased self-esteem). Several studies have suggested that the occurrence of genital warts has been increasing over time [7]. Approximately 65% of people who have sex with an infected partner will develop warts themselves [8].

There has been immense progress in understanding the natural history of HPV infection in women disease. HPV is the primary cause of cervical cancers. Recently, there has been an interest in understanding the relationship between HPV infection and disease in men [9]. The male sexual partner`s role and in his partner's genital warts or high-grade cervical intraepi‐ thelial neoplasia (CIN II, CIN III-Ca in situ) lesions is also undefined. The diagnosis of most cutaneous and external genital wart (GW) can be made on clinical examination or with AW test and biopsy. In case of genital intraepithelial neoplasia, determining the extent of diseases is essential.

## **2. HR-HPV transmission among sexual partners**

lence and transmissibility. HPVs are a very large family of double-stranded DNA viruses (dsDNA), very resistant that can survive in the environment without a host and is able to infect humans. These viruses are not classified as serotypes, but as genotypes on the basis of DNA sequence. Currently, over 120 genotypes have been identified and about 40 genotypes (the alpha genus) can be transmitted through sexual contact and infect the anogenital region. HPV genotypes have been classified into low-risk genotypes, associated with anogenital warts, lowgrade cervical lesions and recurrent respiratory papillomatosis, and high-risk genotypes (HR-HPV) [1] (**Table 1**), which eventually can lead to malignant transformation. HR-HPV are strongly associated with cancer and high-grade neoplasia of the anogenital tract, including the anus (AIN), penis (PeIN), uterine cervix (CIN), and vulva (VIN), and also a proportion of orophar‐ yngeal cancer [2]. Although these infections are typically transient and asymptomatic, some of them will result in anogenital warts, and dysplastic and/or neoplastic lesions, which cause a substantial disease burden in both sexes and generate a considerable economic distress within

IARC, International Agency for Research on Cancer; HR-HPV, high-risk HPV genotypes; pHR, probable/possible high-

The virus may remain inactive for a long time and produce asymptomatic infection of the skin. It can be transmitted from one individual to another directly (by sexual contact) or indirectly.

About one-third to one-quarter of invasive penile cancers (Alemany et al.) and nearly 99.7% of cervical cancer worldwide and in 96.8% of cervical preneoplastic and neoplastic lesions in our community (Perez et al.) may be related to HPV according to the retrospective studies. Although rare, penile cancer is associated with a high morbidity and mortality. The carcino‐ genesis of penile cancer is thought to involve two pathways: one related to inflammation and other dermatological conditions of the penis, and other related to HPV infection (López-Romero et al.). HPV DNA prevalence in invasive penile cancer varied geographically, with the highest prevalence in Oceania (55.6%), North America (48.7), Africa (36.8%), South America (39.7%), and Europe (45.9%), being the most common HR-HPV types: HPV16 (30.8%) and HPV18 (6.6%) [5]. So that, it is important to be cautious and not to consider overall prevalence as universal because the role of HPV in penile cancer etiology could be strongly influenced by histologic distribution and geographic region as it is also true for other HPV malignancies such

Genital warts (GWs) represent a significant public health problem associated with clinical symptoms (burning, bleeding, and pain) and psychosocial problems (embarrassment, anxiety,

The dynamics of heterosexual transmission of HPV are still being investigated [4].

society [3].

risk genotypes.

**Table 1.** Oncogenic HPV genotypes.

as vulvar and head and neck cancers [6].

**IARC classification HPV genotypes**

150 Human Papillomavirus - Research in a Global Perspective

Classification of oncogenic HPV genotypes detected in this work.

HR-HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 pHR-HPV 26, 34, 53, 66, 67, 68, 69, 70, 73, 82

Epidemiological studies show that the HR-HPV infection is necessarily the sexual transmitted cause of invasive cervical cancer in women and its precursor lesion, cervical intraepithelial neoplasia (CIN) [10].

Direct genital mucosa contact during sexual intercourse is the principal route of HPV trans‐ mission [11]. About 80% of newly sexual couples will develop HPV-related lesions within 3 years after commencing sexual activity, most of whom will spontaneously regress within 1–2 years or until the age of 30–35 years [12]. The biology and dynamics of HPV transmission among sexual partners is still a cause for debate and has not already been completely estab‐ lished. Models have shown that HPV transmissibility is substantially higher than that of other viral sexually transmitted pathogens [13], but data on the natural history of HPV transmission between heterosexual partners are limited. Many studies [14–17] analyzed the prevalence and genotypes of high-risk infections of the foreskin before first sexual intercourse found asymp‐ tomatic infection in 12–83.3% [14, 16], speculating that non-sexual routes play significant roles in HPV transmission. In this regard, HPV transmission may occur upon contact with infected towels or other objects. In contrast to these findings, Pilatz et al. [15] did not find HPV in the foreskin of boys.

Despite the recommendation of the guidelines on sexually transmitted diseases, investigation of the presence of HPV in men who are sexual partners of infected woman has not been agreed. Previous studies suggested that the cancer of the penis and cervix may share the same etiological factor(s), because significant numbers of invasive cervical cancer were detected in partners of patients with penile cancer [18, 19]. It was assessed the contribution of the males' genital HPV DNA status to the risk of development of cervical neoplasia in their sexual partners, confirming that men could be vectors of HPV types typically observed in cervical cancer [20]. However, another studies did not confirm the findings of these investigators [21]. As the process of HPV infection can take more than 15 years, the current partner could not be necessarily the source of infection.

## **3. HR-HPV prevalence in heterosexual men populations**

HPV infection causes substantial morbidity and its incidence is similar in both genders. The ongoing HPV in men study (HIM) provides the most current data on HPV infection and lesion development in men [9, 22–24]. Assessing HPV prevalence in men and investigating the sources of variation are essential for understanding the epidemiology of HPV infection.

The pooled HPV in the general population is significantly higher (20.4–36.3%) [25, 26] in studies published after 2000 (8.8%) [27]. The lower pooled prevalence in earlier publications might therefore be due to the detection method used and potentially not to a change in HPV prevalence over time. Age-specific prevalence curves among men are flatter [19, 28, 29] in contrast to the pattern observed in women [30]. The prevalence of genital infection in men does not differ significantly among age groups as it does in females [30]. In general population, HPV infection has a consistently higher prevalence within the penile epithelium of asymptomatic men than within the cervix of women with normal cytological testing [29].

Several factors have been suggested to influence HPV prevalence, varying substantially between sampling sites, techniques [31, 32], and different populations [33]. HPV prevalence is higher when samples are collected from a greater number of anatomic sites [29]. Hebnes et al. [27] in meta-analysis of studies examining HPV prevalence among men found a wide hetero‐ geneity between general and high-risk populations. HIV-positive men, men with sexually transmitted infection and male sexual partners of women with HPV, CIN, CIS, or invasive cervical cancer are considered a high-risk population [34, 35]. Number of types tested for varies between articles. In studies reporting prevalence estimates for more than one HPV type, the commonest detected types were HPV16 [20, 24, 26, 27, 36, 37] and HPV18 [27].

From a socio-epidemiological standpoint, it is important to note that HPV-infected men play a key role in the transmission of the HPV virus to their female sexual partners. The range reported in other studies for sexual partners of women with CIN was 30–68% [19, 24, 26, 36, 38]. Geographical region, anatomical sampling site, or HPV detection methods have not explained the wide heterogeneity of results [27]. In contrast, Franceschi et al. [39] showed the strongest variation by countries, with a higher prevalence of HPV infection among Brazilian sexual partners of woman with CIN compared with those detected in other countries (Colom‐ bia, Mexico, Spain).

The natural course of disease in men by establishing rates of acquisition and time to clearance of HPV infection has not been investigated properly. Although fewer data of infection duration have been reported in men, findings suggest that HPV infection clear more quickly for men than for women and that men have similar duration of infection for oncogenic and nononcogenic types [7, 28]. Mean clearance time, defined as time to elimination of 50% of all infections, was estimated to be 5.9 months (patridge JM). HPV infections in women tend to have a longer duration and are estimated to clear at average of 12.2 months [40].

## **4. Concordance between sexual partners**

**3. HR-HPV prevalence in heterosexual men populations**

152 Human Papillomavirus - Research in a Global Perspective

men than within the cervix of women with normal cytological testing [29].

commonest detected types were HPV16 [20, 24, 26, 27, 36, 37] and HPV18 [27].

bia, Mexico, Spain).

HPV infection causes substantial morbidity and its incidence is similar in both genders. The ongoing HPV in men study (HIM) provides the most current data on HPV infection and lesion development in men [9, 22–24]. Assessing HPV prevalence in men and investigating the sources of variation are essential for understanding the epidemiology of HPV infection.

The pooled HPV in the general population is significantly higher (20.4–36.3%) [25, 26] in studies published after 2000 (8.8%) [27]. The lower pooled prevalence in earlier publications might therefore be due to the detection method used and potentially not to a change in HPV prevalence over time. Age-specific prevalence curves among men are flatter [19, 28, 29] in contrast to the pattern observed in women [30]. The prevalence of genital infection in men does not differ significantly among age groups as it does in females [30]. In general population, HPV infection has a consistently higher prevalence within the penile epithelium of asymptomatic

Several factors have been suggested to influence HPV prevalence, varying substantially between sampling sites, techniques [31, 32], and different populations [33]. HPV prevalence is higher when samples are collected from a greater number of anatomic sites [29]. Hebnes et al. [27] in meta-analysis of studies examining HPV prevalence among men found a wide hetero‐ geneity between general and high-risk populations. HIV-positive men, men with sexually transmitted infection and male sexual partners of women with HPV, CIN, CIS, or invasive cervical cancer are considered a high-risk population [34, 35]. Number of types tested for varies between articles. In studies reporting prevalence estimates for more than one HPV type, the

From a socio-epidemiological standpoint, it is important to note that HPV-infected men play a key role in the transmission of the HPV virus to their female sexual partners. The range reported in other studies for sexual partners of women with CIN was 30–68% [19, 24, 26, 36, 38]. Geographical region, anatomical sampling site, or HPV detection methods have not explained the wide heterogeneity of results [27]. In contrast, Franceschi et al. [39] showed the strongest variation by countries, with a higher prevalence of HPV infection among Brazilian sexual partners of woman with CIN compared with those detected in other countries (Colom‐

The natural course of disease in men by establishing rates of acquisition and time to clearance of HPV infection has not been investigated properly. Although fewer data of infection duration have been reported in men, findings suggest that HPV infection clear more quickly for men than for women and that men have similar duration of infection for oncogenic and nononcogenic types [7, 28]. Mean clearance time, defined as time to elimination of 50% of all infections, was estimated to be 5.9 months (patridge JM). HPV infections in women tend to

have a longer duration and are estimated to clear at average of 12.2 months [40].

Positive concordance is defined as both partners having the HPV outcome of interest. HPV concordance in heterosexual couples has important clinical and public health implications. In terms of HR-HPV detection, the percentage of couples harboring HR-HPV was 32–65% [28, 36, 37, 41]. In couples where both members were HPV positive, more than 60% were infected with one or more of the same HPV types. This level of concordance was observed independ‐ ently of HPV prevalence and is consistent with the high transmissibility of HPV [25, 28, 36, 38, 41]. Studies over the past 20 years evaluating HPV infection concordance among heterosexual partners have shown many inconsistencies, reporting concordances of type-specific infection between 2 and 87% [20, 42–44]. Such heterogeneous findings may be due to diverse laboratory DNA detection techniques, methods for study population selection and different anatomical sites sampling, among other factors [25].

Positive concordance was usually higher for female partners of men with HPV infection than for male partners of women with HPV infection. Men with HPV-positive female partners had one or more of the same HPV types more often in studies that recruited men with HPV-related diseases compared with studies without this inclusion criterion for men (65.8 vs. 27.2%) [28]. These findings suggest that the epithelial cells of the penile skin are more resistant to HPV infection than the cervical epithelium and the duration of HPV infection is shorter in men than in women [28, 38].

## **5. Acetowhite test versus molecular detection of HR-HPV infection**

Infection with one or more of the 40 HPV detected at the genitals is common among men aged 18–70 years. Only 5% of these HPV infections progressed to an external genital lesions during follow-up. There were observed substantially higher rates of progression for certain HPV types [45].

Most genital infections in men are asymptomatic, detectable only by viral DNA testing and become undetectable over time. Subclinical lesions, including those related with HR-HPV types, are more than 10 times common than clinical (apparent) infection and are identified on examination after application of acetic acid solution, a procedure known as acetowhite test (AW test, peniscopy). Since the American Society for Colposcopy and Cervical Pathology recommended the use of HPV DNA testing for the triage and management of women with atypical squamous cells of undetermined significance result of Pap test, an increasing number of female patients are requesting HPV DNA testing for their partners. Although the current gold standard for HPV genotyping is a genetic sequencing targeting the product of gene amplification (Heidegger), a screening test capable of detecting asymptomatic and subclinical genital HPV infection in men at a reasonable price and causing minimal discomfort to the patient would be very valuable.

To date, economic data have primarily focused on the more common HPV-related cervical cancer and its precursor lesions, as well as the benign, very common condition of genital warts. Nevertheless, available data indicate that HPV-related disease is associated with a significant economic burden in males. Specifically, in men, the total direct cost of HPV infection acquired through the age of 24 years was estimated at 62 million dollars per year, the comparable figure for women being 2.8 billion [46].

Studies of the psychosocial effects of HPV-related disease in males are lacking. However, there is a significant psychosocial burden reported in women being screened for, or diagnosed, with HPV-related disease [47].

The currently available methods for evaluating HPV infection in male are HPV DNA test and AW test [12]. This is full description of our study procedures: The entire penis and scrotum of the patient were examined under magnification, and the presence of genital warts was recorded. After this examination, we sprayed them with 5% acetic acid solution. After 5 min, we enhanced the visualization of the skin by a colposcope under fourfold and sevenfold magnification, respectively. AW lesions were classified as typical for the presence of welldemarcated lesions with a slightly elevated border and the occurrence centrally of punctuated capillaries with or without an associated epithelial depression (Groove) and non-typical for the presence of lesions exhibiting a ragged border and lacking punctuated capillaries. Regard‐ less of AW test result, the specimen for HPV DNA detection was obtained. Samples were taken with three cytobrushes from the preputial cavity (the inner part of the foreskin, the glans and the sulcus coronarius, scrotum, and urethral meatus) rotated 360 grades and suspended together into one single vial containing TE buffer pH 8.0 Molecular Biology grade (AppliChem GmbH, Darmstadt, Germany). Samples were maintained at 2–8°C and processed within 24– 72 h after collection. The brushings were collected without spraying the genital region with saline solution. DNA was isolated using QIAamp MinElute Media Kit (Qiagen, Hilden, Germany). Extracted nucleic acids were stored at −20°C. An aliquot of the original sample was also stored at −20°C. Amplification and detection were carried out using the Linear Array HPV Genotyping Test (Linear Array. Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. We described the distribution of 22 HPV genotypes classified as HR (HR-HPV, IARC Group 1 carcinogens) or probable/possible HR (pHR-HPV, IARC Group 2A/B carcinogens) by the International Agency for Research on Cancer Monograph Working Group (**Table 1**). This test also detects human beta-globin in order to test the adequate sample cellularity and absence of inhibitors. Linear Array does not have individual probe for HPV52 but uses a probe that simultaneously detects HPV52, HPV33, HPV35 and HPV58. Additional specific PCR was performed in case of HPV33, HPV35 and/or HPV58 infection in order to properly detect confections of these three genotypes with HPV52 [48].

In our study, around 30% of positive AW results were not related with HR-HPV infection [49– 51]. False-positive results may be due to low-risk HPV infection or inflammatory conditions, common in patients with sexually transmitted diseases [52]. Nevertheless, the need for detecting subclinical genital HPV infection, associated with detectable AW lesions [53], has been emphasized and these population would need follow-up or biopsy. Afonso et al. [37] found that 50% of sexual partners of women with CIN harbored HPV in lesions and these were predominantly subclinical. The diagnosis and treatment of acetowhite lesions in men do not seem to alter or improve the progress of the squamous intraepithelial lesions in their female partners [54]. Nevertheless, these acetowhite lesions on male genitalia are in fact squamous intraepithelial alterations and should not be left due to the risk of their further development [37] as Sudenga et al. [45]. have presented the first estimates of genital HPV infection progres‐ sion to PeIN. They are the first authors that follow these HPV infections and their progress to lesion in men. We encourage the importance of the clinical follow-up of this men and perhaps of taking a biopsy afterwards, in case of HPV infection persistence.

Nevertheless, available data indicate that HPV-related disease is associated with a significant economic burden in males. Specifically, in men, the total direct cost of HPV infection acquired through the age of 24 years was estimated at 62 million dollars per year, the comparable figure

Studies of the psychosocial effects of HPV-related disease in males are lacking. However, there is a significant psychosocial burden reported in women being screened for, or diagnosed, with

The currently available methods for evaluating HPV infection in male are HPV DNA test and AW test [12]. This is full description of our study procedures: The entire penis and scrotum of the patient were examined under magnification, and the presence of genital warts was recorded. After this examination, we sprayed them with 5% acetic acid solution. After 5 min, we enhanced the visualization of the skin by a colposcope under fourfold and sevenfold magnification, respectively. AW lesions were classified as typical for the presence of welldemarcated lesions with a slightly elevated border and the occurrence centrally of punctuated capillaries with or without an associated epithelial depression (Groove) and non-typical for the presence of lesions exhibiting a ragged border and lacking punctuated capillaries. Regard‐ less of AW test result, the specimen for HPV DNA detection was obtained. Samples were taken with three cytobrushes from the preputial cavity (the inner part of the foreskin, the glans and the sulcus coronarius, scrotum, and urethral meatus) rotated 360 grades and suspended together into one single vial containing TE buffer pH 8.0 Molecular Biology grade (AppliChem GmbH, Darmstadt, Germany). Samples were maintained at 2–8°C and processed within 24– 72 h after collection. The brushings were collected without spraying the genital region with saline solution. DNA was isolated using QIAamp MinElute Media Kit (Qiagen, Hilden, Germany). Extracted nucleic acids were stored at −20°C. An aliquot of the original sample was also stored at −20°C. Amplification and detection were carried out using the Linear Array HPV Genotyping Test (Linear Array. Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. We described the distribution of 22 HPV genotypes classified as HR (HR-HPV, IARC Group 1 carcinogens) or probable/possible HR (pHR-HPV, IARC Group 2A/B carcinogens) by the International Agency for Research on Cancer Monograph Working Group (**Table 1**). This test also detects human beta-globin in order to test the adequate sample cellularity and absence of inhibitors. Linear Array does not have individual probe for HPV52 but uses a probe that simultaneously detects HPV52, HPV33, HPV35 and HPV58. Additional specific PCR was performed in case of HPV33, HPV35 and/or HPV58 infection in order to

properly detect confections of these three genotypes with HPV52 [48].

In our study, around 30% of positive AW results were not related with HR-HPV infection [49– 51]. False-positive results may be due to low-risk HPV infection or inflammatory conditions, common in patients with sexually transmitted diseases [52]. Nevertheless, the need for detecting subclinical genital HPV infection, associated with detectable AW lesions [53], has been emphasized and these population would need follow-up or biopsy. Afonso et al. [37] found that 50% of sexual partners of women with CIN harbored HPV in lesions and these were predominantly subclinical. The diagnosis and treatment of acetowhite lesions in men do not seem to alter or improve the progress of the squamous intraepithelial lesions in their female

for women being 2.8 billion [46].

154 Human Papillomavirus - Research in a Global Perspective

HPV-related disease [47].

Problems associated with screening techniques in men include inadequate collection of cells for the detection of HPV DNA by use of swabs and brushes, poor specificity, and patient discomfort during peniscopy. When lesions are not visible, sampling at multiple penile sites could increase the sensitivity of the HPV [41, 55]. In addition, the use of acetic acid and a colposcope requires specific training, clinical experience, and significant costs associated with the procedure and training. Polymerase chain reaction (PCR) has emerged as the most sensitive available method for the detection of latent HPV infection. The infectious diseases literature supports the lack of the US Food and Drug Administration (FDA) approval of HPV tests for HPV detection in men and the absence of adequate therapy for established HPV infection in this population.

## **6. Our results of HPV prevalence in a high-risk population of heterosexual men and concordance between heterosexual partners**

A cross-sectional study was conducted by the Urology Department of the University Hospital of Vigo, Spain, from January 2013 to June 2015 (López Díez et al., Enf Infecc Microbiol Clin, 2016 *in press*). We recruited 125 asymptomatic men, aged 18 years, whose SP (sexual partner, regular sexual intercourse for more than 1 year) had presented high-grade squamous cervical lesions (CIN grade 2 or CIN grade 3-carcinoma in situ) in the previous 6 months. Prevalence of HR-HPV infection in men was 50.4% (63/125). Multiple HR-HPV infections were detected in 30.4% (38/125) of this population. Data of HPV genotype were available in 120 women. HPV16 was the most frequent genotype, detected in 47.6% (30/63) of infected men and 67.5% (81/120) women (**Table 2**). HR-HPV infection was detected in both partners in 50% (60/120). Among these infected couples, 62% (37/60) harbored at least one genotype in common. The HPV16-specific concordance was as follows: 41.7% (25/60) couples were concordantly HPV16 positive and 18.3% (11/60) were concordantly HPV16 negative (Kappa value: 0.21).

The proportion of women with the same genotype as their male partner was 58.7% (37/63). The proportion of men sharing the same genotype as their female partner was 30.8% (37/120), *p* < 0.0001.

AW procedure was positive in 34/125 (27.2%) patients. AW procedure showed 25.4% (95% CI 13.8–36.9) sensitivity, 71.0% (95% CI 58.9–83.1) specificity, 47.1% (95% CI 28.8–65.3) positive predictive value and 48.3% (95% CI 37.5–59.2) negative predictive value for the identification of HR-HPV infection (**Table 3**). AW lesions and HR-HPV were detected at the same time in 16/125 (12.8%) males.


HR-HPV, high-risk HPV genotypes; pHR, probable/possible high-risk genotypes; IARC, International Agency for Research on Cancer.

Crude HPV prevalence calculated in 63 HPV-positive patients: 25 single and 38 multiple infections (López Díez et al., Enf Infecc Microbiol Clin, 2016 *in press*).

**Table 2.** Type-specific HPV prevalence in men.


Genital lesions detected by peniscopy in asymptomatic sexual partners of women with high-grade cervical lesions, according to the presence of HR-HPV.

HR-HPV DNA, high-risk HPV; AW, acetowhite test; OR, odd ratio; 95% CI, confidence interval. Statistically significant, *p <* 0.05 (chi-square test).

**Table 3.** Acetowhite lesions according to HR-HPV DNA detection.

Genital warts were present in 17/125 (13.6%) patients. AW procedure showed sensitivity 82.3 (95% CI 55.8–95.3), specificity 81.4% (95% CI 72.6–88.6), positive predictive value 41.1% (95% CI 25.1–59.1) and negative predictive value 96.7% (95% CI 89.9–99.1) for genital warts' detection (**Table 4**).


AW: Acetowhite test, OR: odd ratio, 95% CI: confidence interval. Statistically significant, \**p <* 0.05 (chi-square test).

**IARC classification Genotype Infected**

156 Human Papillomavirus - Research in a Global Perspective

Research on Cancer.

Enf Infecc Microbiol Clin, 2016 *in press*).

according to the presence of HR-HPV.

Statistically significant, *p <* 0.05 (chi-square test).

**Table 3.** Acetowhite lesions according to HR-HPV DNA detection.

**Table 2.** Type-specific HPV prevalence in men.

**men (n)**

HR-HPV HPV16 30 24.0 47.6

pHR-HPV HPV53 13 10.4 20.6

**HR-HPV DNA detection**

**Global prevalence (N = 125) %**

HPV18 4 3.2 6.3 HPV31 9 7.2 14.3 HPV33 2 1.6 3.2 HPV39 6 4.8 9.5 HPV45 5 4.0 7.9 HPV51 11 8.8 17.5 HPV52 12 9.6 19.0 HPV56 8 6.4 12.7 HPV58 3 2.4 4.8 HPV59 6 4.8 9.5

HPV66 10 8.0 15.9 HPV67 1 0.8 1.6 HPV68 2 1.6 3.2 HPV69 1 0.8 1.6 HPV70 4 3.2 6.3 HPV73 3 2.4 4.8

HR-HPV, high-risk HPV genotypes; pHR, probable/possible high-risk genotypes; IARC, International Agency for

AW lesion Yes 16 (25.4) 18 (29.0) 0.648 0.83 (0.38–1.83) No 47 (74.6) 44 (71.0) – – Genital lesions detected by peniscopy in asymptomatic sexual partners of women with high-grade cervical lesions,

HR-HPV DNA, high-risk HPV; AW, acetowhite test; OR, odd ratio; 95% CI, confidence interval.

Crude HPV prevalence calculated in 63 HPV-positive patients: 25 single and 38 multiple infections (López Díez et al.,

**n (%) n (%)** *p* **OR (95% CI)**

**Yes No**

**Prevalence in HPV-positive men (N = 63) %**

**Table 4.** Acetowhite lesions according to genital warts' detection.

HR-HPV prevalence was 6/15 (40.0%) in circumcised men and 57/110 (51.8%) in not circum‐ cised men (*p* > 0.05).

## **7. Risk factors for HR-HPV prevalence in men**

Coexistence of non-oncogenic and oncogenic HPV-types is frequent [56, 57], which may itself predispose to cancer. A Danish study of 50,000 people with GW found an elevated risk of HPVassociated cancers in people with GW compared with the general population [56]. Although test for the presence of HPV are not recommended for the diagnosis of GW [58] in our study, the AW test was sensitive and specific for genital warts' detection, showing a high negative predictive value. This procedure could avoid missing small clinical lesions. They are generally regarded as a benign condition not associated with mortality, but they can be difficult to treat and recurrence is often observed. Visible warts represent only the tip of the iceberg, and lowand high-risk HPV infections contribute to the genital lesion burden in men [24]. Healthcare providers should have a higher suspicion for HPV-associated cancers in immunocompromised patients with GW. AW test can be helpful in the diagnosis of GW. In particular, soaking acetic acid into suspicious lesions can enhance the degree of suspicion in lesions without classic features. Taking a biopsy might also be indicated if diagnosis is uncertain, the lesions do not respond to standard therapy or the disease worsens during therapy [58].

Limited data exist on the association between HPV infection and smoking in men. In this study, current smoking could increase 2.3-fold the risk of HPV-prevalent infection in males, as found in the HIM study. At present, it is unclear how smoking may influence HPV infection in men, but many possible mechanisms exist. Smoking could potentially increase viral load by weakening the cellular immune response [59].

Sexual behavior has been strongly associated with HPV infection and seropositivity in men [60]. Features previously associated with HR-HPV were as follows: young age at first sexual intercourse (FSI), a higher number of lifetime sexual partners (LSP) and a higher number of recent SP. Contradictory results about the influence of lifetime number of SP were reported [26, 41, 55, 61, 62]. This data could be attributable not only to the range of birth year of men but also to geographical characteristics [27, 33]. In Western population, the numbers of lifetime sexual partners in men and women are both relatively high, and little gender difference could be observed. Burchell et al. reported that the proportion of ≥5 lifetime sexual partners was 64.4% for men, in line with our results (55.2% in men).

The risk of having one or more SP in the preceding year was has been poorly evaluated. The risk of HPV re-infection between a monogamous couple is still a matter of debate [63]. In contrast, Rombaldi et al. [64] and Parada [25] found a high association between both variables.

In the National Questionnaire of Sexual Health, published by Spanish Government in 2009, it was found that the mean age of FSI was 17–18 years (29.3%) for Spanish men. In our study, younger age at FSI was not a risk factor for HPV infection as other authors have previously reported [27, 64]. There are contradictory data that could be attributable not only to the range of birth year of men but also to geographical characteristics [55, 60].

Similar to other studies [55, 65], we did not find the expected protective effect of circumcision on HPV acquisition. Circumcision seems to be associated with reduced persistence in men [66] even though the mechanism of protection is unclear. Removal of the foreskin could minimize the chance of acquisition of new infections or could result in an increased clearance of preexisting infections [28, 67]. Our different results could be due to the fact that circumcision is not very common in our geographical area and that analysis could not assess specific associations in the glans penis, the area expected to be most likely protected by removal of the foreskin [68].

## **8. HR-HPV risk factors found in our study**

Epidemiological characteristics of the studied high-risk population are shown in **Table 5**. Current smoking status was associated with an increased risk of HR-HPV infection in men: 38.2% (21/55) versus 60% (42/70), OR 2.3 (95% CI 1.1–4.7), *p* = 0.016.



FSI, first sexual intercourse;, SP, sexual partners; CIN, cervical intraepithelial neoplasia; CIS: carcinoma *in situ*. Age was expressed as mean ± standard deviation.

*\* p* < 0.05, statistically significant.

Sexual behavior has been strongly associated with HPV infection and seropositivity in men [60]. Features previously associated with HR-HPV were as follows: young age at first sexual intercourse (FSI), a higher number of lifetime sexual partners (LSP) and a higher number of recent SP. Contradictory results about the influence of lifetime number of SP were reported [26, 41, 55, 61, 62]. This data could be attributable not only to the range of birth year of men but also to geographical characteristics [27, 33]. In Western population, the numbers of lifetime sexual partners in men and women are both relatively high, and little gender difference could be observed. Burchell et al. reported that the proportion of ≥5 lifetime sexual partners was

The risk of having one or more SP in the preceding year was has been poorly evaluated. The risk of HPV re-infection between a monogamous couple is still a matter of debate [63]. In contrast, Rombaldi et al. [64] and Parada [25] found a high association between both variables.

In the National Questionnaire of Sexual Health, published by Spanish Government in 2009, it was found that the mean age of FSI was 17–18 years (29.3%) for Spanish men. In our study, younger age at FSI was not a risk factor for HPV infection as other authors have previously reported [27, 64]. There are contradictory data that could be attributable not only to the range

Similar to other studies [55, 65], we did not find the expected protective effect of circumcision on HPV acquisition. Circumcision seems to be associated with reduced persistence in men [66] even though the mechanism of protection is unclear. Removal of the foreskin could minimize the chance of acquisition of new infections or could result in an increased clearance of preexisting infections [28, 67]. Our different results could be due to the fact that circumcision is not very common in our geographical area and that analysis could not assess specific associations in the glans penis, the area expected to be most likely protected by removal of the

Epidemiological characteristics of the studied high-risk population are shown in **Table 5**. Current smoking status was associated with an increased risk of HR-HPV infection in men:

**Positive Negative Bivariate analysis Multivariate analysis**

64.4% for men, in line with our results (55.2% in men).

158 Human Papillomavirus - Research in a Global Perspective

**8. HR-HPV risk factors found in our study**

**Variable HPV detection (n = 125)** *p***-value**

Age at FSI 16.9 ± 2.7 17.4 ± 2.4 0.382

1–5 SP 10 (34.5%) 19 (65.5%) 0.050

>5 SP 53 (55.2%) 43 (44.8%)

38.2% (21/55) versus 60% (42/70), OR 2.3 (95% CI 1.1–4.7), *p* = 0.016.

foreskin [68].

Lifetime SP

of birth year of men but also to geographical characteristics [55, 60].

**Table 5.** HPV detection in men according to epidemiological characteristics.

Prevalence of HR-HPV infection was 14/17 (82.4%) in patients with genital warts versus 49/108 (45.4%) in patients without genital warts (OR 5.6, 95% CI 1.5–20.7, *p* = 0.008) (**Figure 1**).

**Figure 1.** Statistically significant, \**p<* 0.05 (chi-square test).

## **9. Prevention of HPV infection in men**

Until recently, no highly effective primary prevention strategy to reduce the risk of HPV acquisition existed. However, research has demonstrated that nonavalent, quadrivalent and bivalent HPV vaccines stimulate immunogenicity in males and females [69]. On October 16, 2009, the FDA approved the use of quadrivalent vaccine in males aged 9–26 years for the prevention of genital warts. Subsequently, the Advisory Committee on immunization Practices (ACID) declined to recommend the quadrivalent vaccine for routine immunization in men [70], providing a permissive recommendation in this age range for HPV vaccination. Most European countries offer HPV vaccination for girls, but vaccine recommendations for boys are warranted. HPV vaccination of girls will in theory also benefit the male population through herd immunity.

Uninfected sexual partners may be an important target population for HPV vaccination [71]. Potential interventions such as a therapeutic HPV vaccine may avert new HPV infections. Moreover, vaccinating boys would reduce HPV-related diseases in both sexes to a greater extent than herd immunity, which depends on high vaccination rates among females.

The benefits of vaccination to individuals seronegative to HPV types included in the vaccine are clear, and emerging studies suggest that HPV vaccine may also help people who previously had and cleared an infection [72] although additional researches in this population are needed. While prophylactic HPV vaccine does not have substantial impact on established infection, it may have cross-protection against non-vaccine genotypes [73]. However, if these vaccines could also be successful in lowering the HPV load, they may also assist in lowering transmis‐ sion [13].

There is no direct evidence for protection by HPV vaccines against penile cancer because penile cancer is so rare that there could never be a clinical trial large enough to measure the effect [74]. HPV vaccines have not been around long enough to measure the population impact on penile cancer. However, the observed HPV type distribution reinforces the potential benefit of current and new vaccines in reduction in HPV-related penile neoplasia lesions [6].

Future trials of HPV vaccines in men should take into account not only the presence of penile HPV infection but also the presence of penile subclinical lesions as an outcome measure for the efficacy of a vaccine. More complex study designs would also allow researchers to better understand first transmission, reinfection and back and forth passage within couples, con‐ cordance in couples in which one partner has received HPV vaccine and concordance after treatment for HPV-related lesions is an essential component of prevention programs aimed to reduce cervical cancer and other HPV-related diseases in men and women.

### **10. Final considerations**

HPV causes cancer in both men and women. The HPV-related cancer burden remains higher in women than men, even in countries that have effective cervical cancer screening programs. It is clear that males have poor knowledge of HPV infection, morbidity, transmission, and prevention. Moreover, several issues are controversial and should be addressed by adopting a multidisciplinary and multiprofessional approach. Regardless of vaccination strategies adopted, efforts should be made to educate males about HPV and its health implications.

Currently, there is no licensed test for HPV detection in men and there are no recommendations for male screening. Although routine HPV testing is not necessary for men in general popu‐ lation, findings from emerging research in high-risk population suggest that HPV infection is pervasive and persistent in these groups, warranting the adoption of additional screening and prevention policies. Our findings suggest the need for greater attention to sexual partners of HPV-infected individuals. Male sexual partners of female with high-grade lesions should be referred for evaluation and combined peniscopy, and HPV DNA test will ensure accurate detection of HPV status among males. Female partners of men with HPV-related diseases should be encouraged to get screened for HPV-related disease given that they have a high likelihood of concomitant infection and that most infection in couples are of the same viral type. Screening may also benefit male partners of HPV-infected women. Interventions that study the true prevalence of HPV infection in asymptomatic men and try to reduce HPVassociated penile lesions could be important to both men and women.

Further prospective and controlled studies in different populations are needed to provide adequate counseling to men that demand to know whether they are infected by HR-HPV. Long-term follow-up will contribute to the knowledge about the influence of persistent HPV infection in male and the potential recurrence of his sexual partner after treatment. We assume that the faster way to achieve greatest protection for cervical cancer and its precursors is to vaccinate males as well as female because both genders contribute to the transmission of HPV infection.

The prevention, diagnosis, and treatment of HPV-associated diseases in men will reduce the disease burden not only in males, but also in females, and help destigmatize the focus of the HPV-related disease on women.

## **Acknowledgements**

**9. Prevention of HPV infection in men**

160 Human Papillomavirus - Research in a Global Perspective

through herd immunity.

**10. Final considerations**

sion [13].

Until recently, no highly effective primary prevention strategy to reduce the risk of HPV acquisition existed. However, research has demonstrated that nonavalent, quadrivalent and bivalent HPV vaccines stimulate immunogenicity in males and females [69]. On October 16, 2009, the FDA approved the use of quadrivalent vaccine in males aged 9–26 years for the prevention of genital warts. Subsequently, the Advisory Committee on immunization Practices (ACID) declined to recommend the quadrivalent vaccine for routine immunization in men [70], providing a permissive recommendation in this age range for HPV vaccination. Most European countries offer HPV vaccination for girls, but vaccine recommendations for boys are warranted. HPV vaccination of girls will in theory also benefit the male population

Uninfected sexual partners may be an important target population for HPV vaccination [71]. Potential interventions such as a therapeutic HPV vaccine may avert new HPV infections. Moreover, vaccinating boys would reduce HPV-related diseases in both sexes to a greater

The benefits of vaccination to individuals seronegative to HPV types included in the vaccine are clear, and emerging studies suggest that HPV vaccine may also help people who previously had and cleared an infection [72] although additional researches in this population are needed. While prophylactic HPV vaccine does not have substantial impact on established infection, it may have cross-protection against non-vaccine genotypes [73]. However, if these vaccines could also be successful in lowering the HPV load, they may also assist in lowering transmis‐

There is no direct evidence for protection by HPV vaccines against penile cancer because penile cancer is so rare that there could never be a clinical trial large enough to measure the effect [74]. HPV vaccines have not been around long enough to measure the population impact on penile cancer. However, the observed HPV type distribution reinforces the potential benefit of current

Future trials of HPV vaccines in men should take into account not only the presence of penile HPV infection but also the presence of penile subclinical lesions as an outcome measure for the efficacy of a vaccine. More complex study designs would also allow researchers to better understand first transmission, reinfection and back and forth passage within couples, con‐ cordance in couples in which one partner has received HPV vaccine and concordance after treatment for HPV-related lesions is an essential component of prevention programs aimed to

HPV causes cancer in both men and women. The HPV-related cancer burden remains higher in women than men, even in countries that have effective cervical cancer screening programs.

and new vaccines in reduction in HPV-related penile neoplasia lesions [6].

reduce cervical cancer and other HPV-related diseases in men and women.

extent than herd immunity, which depends on high vaccination rates among females.

We thank nursery team, especially Carmen Garcia from the Urology Department of the University Hospital of Vigo, for their excellent help in this research, collecting patients. We thank M. Consuelo Reboredo from the Gynecology Department and the laboratory technicians of the Microbiology Department of the University Hospital of Vigo, for their support of the study. We are also grateful to Angel de la Orden for the revision of the manuscript.

## **Author details**

Elena López-Díez1 , Sonia Pérez2\* and Amparo Iñarrea3 \*Address all correspondence to: elena.lopez.diez@sergas.es


### **References**


[11] Liu M, He Z, Zhang C, Liu F, Liu Y, Li J, et al. Transmission of genital human papillo‐ mavirus infection in couples: a population-based cohort study in rural China. Sci Rep. 2015;5:10986.

\*Address all correspondence to: elena.lopez.diez@sergas.es

162 Human Papillomavirus - Research in a Global Perspective

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2013;89(2):148–55.

1 Department of Urology, University Hospital of Vigo, Vigo, Spain

2 Department of Microbiology, University Hospital of Vigo, Vigo, Spain

3 Department of Obstetrics and Gynecology, University Hospital of Vigo, Vigo, Spain

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## **High-Risk Human Papillomavirus and Colorectal Carcinogenesis**

Ala-Eddin Al Moustafa, Noor Al-Antary and Amber Yasmeen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63295

### **Abstract**

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2013;31(Suppl 8):I1–31.

168 Human Papillomavirus - Research in a Global Perspective

Colorectal, colon and rectal, cancer is the third most common malignancy in both men and women worldwide. Colorectal carcinogenesis is a complex, multistep process implicating environmental and lifestyle factors in addition to gene mutation and viral infections. On the other hand, it is well established that human papillomaviruses (HPVs) infection play a crucial role in certain types of human carcinomas including cervical and head and neck (HN); as roughly 96% and 30% of these cancers are positive for high-risk HPVs, respectively. Moreover, it has been reported that the presence of high-risk HPVs is associated with vascular invasion, lymph node metastases, and tumor size in cervical and HN cancers. Recently, several investigations pointed-out that high-risk HPVs are present in around 70% of human colorectal cancers. Likewise, our group has demonstrated that E6/E7 oncoproteins of HPV type 16 convert noninva‐ sive and nonmetastatic human cancer cells to invasive and metastatic form. Accord‐ ingly, it is evident that high-risk HPVs are present in human colorectal cancers where they could play an important role in the development of these malignancies. In this chapter, we will discuss the presence and role of high-risk HPVs in human colorectal carcinogenesis and metastasis; particularly, the interaction between E5 and E6/E7 oncoproteins of high-risk HPVs in colorectal malignancies, which has been linked with the initiation and progression of these tumors.

**Keywords:** colorectal cancer, high-risk HPVs, E5 & E6/E7 oncoproteins, cancer initia‐ tion, cancer progression

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Colorectal cancers (CRCs) colon and rectal, are the most common malignancies, accounting for approximately 1.36 million new cases worldwide every year [1]. These cancers are character‐ ized by a marked propensity for local invasion and lymph node metastases. Thus, the overall 5-year-survival rate for patients diagnosed with colorectal cancers is approximately 60% worldwide and has not significantly improved over the past decade [2]. Colorectal carcinogen‐ esis is a complex, multistep process involving environmental, demographic, and lifestyle factors in addition to gene alterations and viral infections. The highest incidence of CRCs is observed in Western Europe, North America, Australia as well as in some Middle-Eastern countries [3, 4]. It is notable also that although the rate of this disease is relatively lower in sub-Saharan African communities, South America, and Asia; however, CRCs are gradually increasing due to assimilating life style and dietary habits of Western countries [3–5]. Additionally, around twothirds of CRC patients will develop distant metastases during the course of their illness, which is the main cause of cancer-related death of this disease [6].

Although, human papillomaviruses (HPVs) have been established as etiological agents of invasive cervical cancer, as generally 96% of these cancers are positive for high-risk HPVs [7– 9]. However, persistent infection with high-risk HPVs is necessary but not sufficient for the development of malignant lesions [10, 11]. Furthermore, it was pointed-out that high-risk HPVs have carcinogenic effects at several other anatomical sites in women and men such as head and neck (HN) as well as colorectal [12–15]. These studies and others showed that highrisk HPVs are present in roughly 30% and 70% of HN and colorectal cancers, respectively, especially in their invasive form [14, 15]. Accordingly, we recently investigated the incidence of high-risk HPVs in CRCs in the Syrian population; our data revealed that 54% of human CRCs in Syria are positive for high-risk-HPVs; this was accompanied by an expression/ overexpression of Fascin, Id-1, and P-cadherin genes [16], which are major regulators of cell invasion and metastasis [17–19]. Meanwhile, we revealed that E5 and E6/E7 oncoproteins of high-risk HPVs could cooperate together to enhance cancer progression through the deregu‐ lation of several key controller genes of the epithelial–mesenchymal transition (EMT) event [7, 20, 21]. It is clear that CRCs and especially their invasive forms are major health problems wherein high-risk HPVs infection can play important roles in the development of these malignancies as well as their metastasis via EMT. In this chapter, we will overview the presence and contributions of high-risk HPVs in CRC initiation and progression.

### **2. Colorectal cancers**

CRCs are the most prevalent cancers worldwide, along with lung and breast cancers, they are one of the deadliest diseases today [22]. For instance, in the United States, CRCs are the third leading cause of cancer death in both sexes and the second overall in men and women combined [23, 24]. At current rates, approximately 5–6% of individuals will develop colon or rectum cancer within their lifetime [23]. These malignances are most common in Europe with 432,000 new cases reported annually in men and women combined, and the second most common cause of cancer deaths in Europe [22, 25]. In general, it is the second leading cause of cancer-related mortality worldwide and the third most commonly diagnosed malignant disease [26].

**1. Introduction**

170 Human Papillomavirus - Research in a Global Perspective

Colorectal cancers (CRCs) colon and rectal, are the most common malignancies, accounting for approximately 1.36 million new cases worldwide every year [1]. These cancers are character‐ ized by a marked propensity for local invasion and lymph node metastases. Thus, the overall 5-year-survival rate for patients diagnosed with colorectal cancers is approximately 60% worldwide and has not significantly improved over the past decade [2]. Colorectal carcinogen‐ esis is a complex, multistep process involving environmental, demographic, and lifestyle factors in addition to gene alterations and viral infections. The highest incidence of CRCs is observed in Western Europe, North America, Australia as well as in some Middle-Eastern countries [3, 4]. It is notable also that although the rate of this disease is relatively lower in sub-Saharan African communities, South America, and Asia; however, CRCs are gradually increasing due to assimilating life style and dietary habits of Western countries [3–5]. Additionally, around twothirds of CRC patients will develop distant metastases during the course of their illness, which

Although, human papillomaviruses (HPVs) have been established as etiological agents of invasive cervical cancer, as generally 96% of these cancers are positive for high-risk HPVs [7– 9]. However, persistent infection with high-risk HPVs is necessary but not sufficient for the development of malignant lesions [10, 11]. Furthermore, it was pointed-out that high-risk HPVs have carcinogenic effects at several other anatomical sites in women and men such as head and neck (HN) as well as colorectal [12–15]. These studies and others showed that highrisk HPVs are present in roughly 30% and 70% of HN and colorectal cancers, respectively, especially in their invasive form [14, 15]. Accordingly, we recently investigated the incidence of high-risk HPVs in CRCs in the Syrian population; our data revealed that 54% of human CRCs in Syria are positive for high-risk-HPVs; this was accompanied by an expression/ overexpression of Fascin, Id-1, and P-cadherin genes [16], which are major regulators of cell invasion and metastasis [17–19]. Meanwhile, we revealed that E5 and E6/E7 oncoproteins of high-risk HPVs could cooperate together to enhance cancer progression through the deregu‐ lation of several key controller genes of the epithelial–mesenchymal transition (EMT) event [7, 20, 21]. It is clear that CRCs and especially their invasive forms are major health problems wherein high-risk HPVs infection can play important roles in the development of these malignancies as well as their metastasis via EMT. In this chapter, we will overview the presence

CRCs are the most prevalent cancers worldwide, along with lung and breast cancers, they are one of the deadliest diseases today [22]. For instance, in the United States, CRCs are the third leading cause of cancer death in both sexes and the second overall in men and women combined [23, 24]. At current rates, approximately 5–6% of individuals will develop colon or rectum cancer within their lifetime [23]. These malignances are most common in Europe with

is the main cause of cancer-related death of this disease [6].

and contributions of high-risk HPVs in CRC initiation and progression.

**2. Colorectal cancers**

The prognosis of patients with colorectal cancer has slowly but steadily improved during the past decades in many countries. A 5-year relative survival has reached almost 65% in highincome countries, such as Australia, Canada, the USA, and several European countries, but has remained less than 50% in low-income countries [27–29]. Relative survival decreases with age, and at young ages, it is slightly higher for women than for men [30]; taking into consid‐ eration that the stage at diagnosis is the most important prognostic factor.

Colorectal carcinogenesis is common in the elderly; as approximately 90% of new colorectal cancers are diagnosed in patients over 50 years with the median age of diagnosis being 69 years. Furthermore, the incidence of CRCs dramatically rises as one ages, regardless of sex and racial background [26]. Although, it is well-known that patients with colorectal cancer may have a range of symptoms that include occult blood loss, rectal bleeding, change in stool caliber, unintentional weight loss, or have signs of bowel obstruction or perforation.

There are many risk factors for the development of colorectal cancer, one of which is colonic polyps. Pathologic entities include tubular adenoma, tubulovillous adenoma, villous adeno‐ ma, hyperplastic polyp, sessile serrated adenoma, sessile serrated polyp, and traditional serrated adenoma. In addition, some hamartomatous polyps are considered premalignant lesions [31]. Among precancerous polyps adenomatous and advanced adenomatous polyps that have polyp size >10 mm, in addition to villous/tubulovillous histological features, or having high-grade dysplasia (HGD), are found to have an increased prevalence and incidence in the elderly [26], and have a potential to progress to invasive adenocarcinomas [26, 32]. HGD is associated with larger size, villous morphology, *TP53* mutation, and deletion of a region of chromosome 18q. Chromosomal instability can be demonstrated in late precursor adenomas. In this sequence, *APC* mutation is a common early event, while the serrated lesions commonly have *BRAF* or *KRAS* mutation [31]. Other risk factors include diet and lifestyle (such as consumption of red meat, smoking, excessive alcohol, weight gain, etc.) as well as advancing age [23, 26].

For the most part, colorectal cancer arises sporadically, however, few cases are associated with inherited syndromes such as familial adenomatous polyposis (FAP; <1% of CRC) where patients exhibit germline mutations in one allele of the adenomatous polyposis (*APC*) tumor suppressor gene, MUTYH-associated polyposis (MAP; rare recessive condition, carrier estimated at ~1%), and Lynch syndrome/hereditary nonpolyposis colon cancer (LS/HNPCC; 2–4% of CRCs) [7, 33].

The usual malignant tumor of the large bowel is a well-to-moderately differentiated adeno‐ carcinoma secreting variable amounts of mucin [34]. In World Health Organization (WHO) classification, a number of histologic variants of this tumor are listed, such as mucinous adenocarcinoma, signet ring cell, medullary, micropapillary, serrated, cribriform comedotype, adenosquamous, spindle cell, and undifferentiated. The most widely used immunohis‐ tochemical markers for colorectal adenocarcinoma are cytokeratin (CK) 20, CK7, and CDX2. The most common immunophenotype of colorectal adenocarcinoma is positivity for CK20 and negativity for CK7 [35]. The CRCs are divided in to four grades. G1 are well-differentiated tumors (usually adenocarcinomas) that have more than 95% glandular structures. Further, G2 are designated as moderately differentiated tumors with 50–95% gland formation. G3 are poorly differentiated tumors with 5–50% gland formation; whereas G4 are highly aggressive and undifferentiated tumors with less than 5% gland formation. Recently, WHO also suggests dividing CRCs into low grade (G1 and G2) and high grade (G3 and G4) categories. The diagnosis of G3 and G4 is relatively consistent, but differentiation between G1 and G2 is associated with a significant degree of inter-observer variability [36, 37] .

As we mentioned above, CRCs are characterized by a marked propensity for invasion and metastasis. About 20% of patients with newly diagnosed colorectal cancer present with distant metastases [38, 39]. The most common location is the liver [38, 40]; however, investigators identified lung metastases in 2·1% of patients newly diagnosed with CRC in a large cancer registry in France [41]. Frequency was nearly three times higher for patients with rectal cancer than for patients with colon cancer. Smaller studies [42–44] have shown isolated lung meta‐ stases in 9–18% of patients with rectal cancer; although distant metastases can be identified in other organs including the bone and the brain [38].

As we cited above, lifetime risk of CRCs is estimated to be 5–6% in the general population of Western countries [45, 46]. Although hereditary forms of CRC have been well established; however, most cases are sporadic [47]. Numerous epidemiological studies have identified lifestyle and environmental factors contributing to the occurrence of CRCs [48, 49]. In the past decades, *Helicobacter pylori* and Epstein Barr virus infections have been identified as potential causal factors of gastric cancer [50, 51] and personal communication. A number of studies aimed to assess the possible role of viral infections, such as infection with high-risk HPVs, human polyomaviruses, and human herpesviruses in colorectal carcinogenesis [7, 45, 52, 53]. Thus, in the next paragraph the presence and role of high-risk HPVs in human CRCs will be reviewed.

## **3. Human papillomaviruses (HPVs)**

Papillomaviruses were first identified in rabbits in 1933, and they were found to be involved in transmissible growth of benign papillomas [54]. HPVs were first identified in 1956, and they were associated with a variety of benign growths in humans [55]. However, it was later observed that HPVs, a highly prevalent sexually transmitted infection, have potentially serious health consequences in males and females. HPV infections have received considerable attention in recent years. So far, more than 150 HPV types have been isolated and characterized. While the involvement of HPV in causing benign warts was already known, the first evidence of the association between human cancer and certain HPV types was proposed more than thirty years ago by zur Hausen and his colleagues [56].

The common mode of transmission and acquisition of HPV is by horizontal transmission consequent to sexual activity. Occasionally, HPV may be transmitted through modes other than sexual activity [57–61]. Thus, prevalence sites of HPVs include the epithelium of the vagina, vulva, penis, anal canal, cervix, perianal region, crypts of the tonsils, and oropharynx. Persistent HPV infection is essential for the development of cervical precancerous lesions and cancer. However, this may take a long time, usually a decade or more after the initial infection [62].

tochemical markers for colorectal adenocarcinoma are cytokeratin (CK) 20, CK7, and CDX2. The most common immunophenotype of colorectal adenocarcinoma is positivity for CK20 and negativity for CK7 [35]. The CRCs are divided in to four grades. G1 are well-differentiated tumors (usually adenocarcinomas) that have more than 95% glandular structures. Further, G2 are designated as moderately differentiated tumors with 50–95% gland formation. G3 are poorly differentiated tumors with 5–50% gland formation; whereas G4 are highly aggressive and undifferentiated tumors with less than 5% gland formation. Recently, WHO also suggests dividing CRCs into low grade (G1 and G2) and high grade (G3 and G4) categories. The diagnosis of G3 and G4 is relatively consistent, but differentiation between G1 and G2 is

As we mentioned above, CRCs are characterized by a marked propensity for invasion and metastasis. About 20% of patients with newly diagnosed colorectal cancer present with distant metastases [38, 39]. The most common location is the liver [38, 40]; however, investigators identified lung metastases in 2·1% of patients newly diagnosed with CRC in a large cancer registry in France [41]. Frequency was nearly three times higher for patients with rectal cancer than for patients with colon cancer. Smaller studies [42–44] have shown isolated lung meta‐ stases in 9–18% of patients with rectal cancer; although distant metastases can be identified in

As we cited above, lifetime risk of CRCs is estimated to be 5–6% in the general population of Western countries [45, 46]. Although hereditary forms of CRC have been well established; however, most cases are sporadic [47]. Numerous epidemiological studies have identified lifestyle and environmental factors contributing to the occurrence of CRCs [48, 49]. In the past decades, *Helicobacter pylori* and Epstein Barr virus infections have been identified as potential causal factors of gastric cancer [50, 51] and personal communication. A number of studies aimed to assess the possible role of viral infections, such as infection with high-risk HPVs, human polyomaviruses, and human herpesviruses in colorectal carcinogenesis [7, 45, 52, 53]. Thus, in the next paragraph the presence and role of high-risk HPVs in human CRCs will be

Papillomaviruses were first identified in rabbits in 1933, and they were found to be involved in transmissible growth of benign papillomas [54]. HPVs were first identified in 1956, and they were associated with a variety of benign growths in humans [55]. However, it was later observed that HPVs, a highly prevalent sexually transmitted infection, have potentially serious health consequences in males and females. HPV infections have received considerable attention in recent years. So far, more than 150 HPV types have been isolated and characterized. While the involvement of HPV in causing benign warts was already known, the first evidence of the association between human cancer and certain HPV types was proposed more than

The common mode of transmission and acquisition of HPV is by horizontal transmission consequent to sexual activity. Occasionally, HPV may be transmitted through modes other

associated with a significant degree of inter-observer variability [36, 37] .

other organs including the bone and the brain [38].

172 Human Papillomavirus - Research in a Global Perspective

**3. Human papillomaviruses (HPVs)**

thirty years ago by zur Hausen and his colleagues [56].

reviewed.

HPVs are small, double-stranded DNA viruses that generally infect cutaneous and mucosal epithelial tissues of the anogenital tract. The HPV DNA genome encodes approximately eight open reading frames (ORFs) [52, 62]. It is divided into three functional parts: the early (E) region, the late (L) region, and a long control region (LCR). The E region is important for replication, cellular transformation, and for the control of viral transcription, whereas the L region encodes the structural proteins (L1-L2) that take part in assembly [12]. The LCR is necessary for viral DNA replication and transcription. The seven proteins of the E region are E1, E2, E3, E4, E5, E6, and E7. E1 is necessary for viral DNA replication, while E2 has a role in viral gene transcription and replication. The function of E3 is still not understood. On the other hand, E4 protein interacts with the keratin cytoskeleton and intermediate filaments. Moreover, it facilitates virus assembly and release. The E5 protein interacts with the receptors of growth factors and stimulates cellular proliferation and inhibits apoptosis. E6 induces DNA synthesis, prevents cell differentiation, and interacts with tumor suppressor proteins and repair factors. In fact, E7 induces cell proliferation and interacts with negative regulators of cell cycle and tumor suppressor proteins. E5, E6, and E7 proteins act as oncogenes which are associated with carcinogenesis [12, 20, 63–66] (please see below).

As we mentioned above, over 150 different viral types have been identified, and about onethird of these infect epithelial cells in the genital tract [67]. HPVs are classified as either high risk or low risk. Infections with low-risk types are generally self-limiting and do not lead to malignancy. However, infections with high-risk HPVs (type 16, 18, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68, 73, 82, and 83) are associated with the development of cervical cancers since more than 96% of these cancers are positive for high-risk HPVs [7, 9, 68–70].

It is well known that high-risk HPV early proteins, including E5, E6, and E7 oncoproteins, increase cellular alteration and probably lead to HPV induced carcinogenesis [20, 71–73]. More specifically, the E5 oncoprotein interacts with EGF-R1 signaling pathways (MAP Kinase and P13K-Akt) and proapoptotic proteins [74–76]; and therefore, it can play an important role in cell transformation and tumor formation. On the other hand, E6 and E7 of the high-risk HPV types, such as HPV16, are thought to work together in lesions caused by this virus, since, the two proteins are expressed from bicistronic mRNA [77] and initiated from the viral early promoter (p97). These proteins have functions that stimulate cell cycle progression and both can associate with regulators of the cell cycle [70, 72, 78].

Several studies have shown that the viral E6 protein complements the role of E7 and is thought to prevent the induction of apoptosis in response to unscheduled S-phase entry mediated by E7 [70, 79]. The E6 protein is also involved in the inactivation of p53-mediated growth suppression and/or apoptosis and can also associate with other proapoptotic proteins includ‐ ing Bak [80] and Bax [81]. In addition, E6 stimulates cell proliferation independently from E7 through its C-terminal PDZ-ligand domain [70, 82]. E6-PDZ binding is sufficient to mediate suprabasal cell proliferation [83, 84] and may contribute to the development of metastatic tumours by disrupting normal cell adhesion. On the other hand, the E7 viral is involved with members of the pocket protein family such as pRb, which is well documented. E7 binding to pRb displaces E2F, irrespective of the presence of external growth factors and leads to the expression of proteins necessary for DNA replication [70, 71, 78, 85].

To address the role of E6/E7 genes in high-risk HPV-associated carcinogenesis *in vivo*, transgenic mice have been developed expressing E6/E7 of HPV type 16 individually and together under the human K14 promoter [86, 87]. These transgenic mice developed skin tumors, in general, and cervical cancer with chronic estrogen administration [87, 88]. On the other hand, and to examine the oncogenic properties of E5 *in vivo,* K14-E5 transgenic mice were generated in which the expression of E5 was directed to the basal layer of the stratified squamous epithelia. These mice exhibited the epidermal hyperplasia, aberrant differentiation of the epithelium, and were susceptible to spontaneous skin tumors [89]. Recently, it was reported that K14-E6/E7 transgenic mice have high susceptibility to colorectal cancers and precancerous lesions after dimethylbenz[a]anthracene-treatment, which is a chemical carci‐ nogen that is known to induce squamous cell carcinomas in other sites [90]. These studies show clearly that high-risk HPVs play an important role in cancer initiation and/or progression of several anatomical sites, which could include colorectal, through their E5, E6, and E7 onco‐ proteins.

## **4. High-risk HPVs in colorectal cancers**

High-risk HPVs have been established as etiological agents of invasive cervical cancer, as more than 96% of these cancers are positive for high-risk HPVs which are the most common viral sexually transmitted infection worldwide [7–9]. Infection with high-risk HPVs is important for the development of premalignant lesions and/or progression of the disease [10, 11]. Additionally, it was revealed that high-risk HPVs have carcinogenic effects at several other anatomical regions in women and men such as HN as well as colorectal [12–15]. These studies showed that high-risk HPVs are present in roughly 30 and 70% of HN and colorectal cancers, respectively, especially in their invasive form [14, 15]. Therefore, several recent studies including one from our group pointed-out that high-risk HPVs are present in human CRCs, specifically types 16, 18, 31, 33, and 35 [7, 12, 15, 16, 52]. Moreover, six recent meta-analysis studies confirmed the presence of high-risk HPVs in human CRCs [70, 91–95]; however, the prevalence of high-risk HPVs varied from one geographic location to another [7, 52]. Mean‐ while, it was stated that high-risk HPVs are present especially in the invasive form of these malignancies worldwide [15].

Nevertheless, it is important to mention that high-risk HPV infection alone is not sufficient to induce neoplastic transformation of human normal epithelial cells; the infected cells must undergo additional genetic changes and/or coinfection with another oncovirus to reach full transformation and consequently tumor formation. Based on this fact, we have developed a new model to study the cooperation effect between high-risk HPVs and other oncogenes in human carcinogenesis using human normal epithelial (HNE) cells. In this model, we established that E6/E7 oncoproteins of high-risk type 16 cooperate with the ErbB-2 receptor to induce cellular transformation of HNE cells; this was accompanied by a delocalization of βcatenin from the undercoat membrane to the nucleus in HNE cells. Furthermore, we reported that cyclin D1 is the target of E6/E7/ErbB-2 cooperation via the conversion of β-catenin's role from a cell–cell adhesion molecule to a transcriptional regulator [96]. In parallel, we revealed that D-type cyclins (D1, D2, and D3) are essential for cell transformation induced by E6/E7/ ErbB-2 cooperation in human HNE and mouse normal embryonic fibroblast (NEF) cells [96, 97]. Finally, we were able to show that the cooperation effect of E6/E7 with ErbB-2, in human normal epithelial and cancer cells, occurs via β-catenin tyrosine phosphorylation through pp60 (c-Src) kinase activation [98, 99]. Thus, the cooperation between E6/E7 oncoproteins of highrisk HPVs and other oncogenes could occur in colorectal carcinogenesis.

suprabasal cell proliferation [83, 84] and may contribute to the development of metastatic tumours by disrupting normal cell adhesion. On the other hand, the E7 viral is involved with members of the pocket protein family such as pRb, which is well documented. E7 binding to pRb displaces E2F, irrespective of the presence of external growth factors and leads to the

To address the role of E6/E7 genes in high-risk HPV-associated carcinogenesis *in vivo*, transgenic mice have been developed expressing E6/E7 of HPV type 16 individually and together under the human K14 promoter [86, 87]. These transgenic mice developed skin tumors, in general, and cervical cancer with chronic estrogen administration [87, 88]. On the other hand, and to examine the oncogenic properties of E5 *in vivo,* K14-E5 transgenic mice were generated in which the expression of E5 was directed to the basal layer of the stratified squamous epithelia. These mice exhibited the epidermal hyperplasia, aberrant differentiation of the epithelium, and were susceptible to spontaneous skin tumors [89]. Recently, it was reported that K14-E6/E7 transgenic mice have high susceptibility to colorectal cancers and precancerous lesions after dimethylbenz[a]anthracene-treatment, which is a chemical carci‐ nogen that is known to induce squamous cell carcinomas in other sites [90]. These studies show clearly that high-risk HPVs play an important role in cancer initiation and/or progression of several anatomical sites, which could include colorectal, through their E5, E6, and E7 onco‐

High-risk HPVs have been established as etiological agents of invasive cervical cancer, as more than 96% of these cancers are positive for high-risk HPVs which are the most common viral sexually transmitted infection worldwide [7–9]. Infection with high-risk HPVs is important for the development of premalignant lesions and/or progression of the disease [10, 11]. Additionally, it was revealed that high-risk HPVs have carcinogenic effects at several other anatomical regions in women and men such as HN as well as colorectal [12–15]. These studies showed that high-risk HPVs are present in roughly 30 and 70% of HN and colorectal cancers, respectively, especially in their invasive form [14, 15]. Therefore, several recent studies including one from our group pointed-out that high-risk HPVs are present in human CRCs, specifically types 16, 18, 31, 33, and 35 [7, 12, 15, 16, 52]. Moreover, six recent meta-analysis studies confirmed the presence of high-risk HPVs in human CRCs [70, 91–95]; however, the prevalence of high-risk HPVs varied from one geographic location to another [7, 52]. Mean‐ while, it was stated that high-risk HPVs are present especially in the invasive form of these

Nevertheless, it is important to mention that high-risk HPV infection alone is not sufficient to induce neoplastic transformation of human normal epithelial cells; the infected cells must undergo additional genetic changes and/or coinfection with another oncovirus to reach full transformation and consequently tumor formation. Based on this fact, we have developed a new model to study the cooperation effect between high-risk HPVs and other oncogenes in

expression of proteins necessary for DNA replication [70, 71, 78, 85].

174 Human Papillomavirus - Research in a Global Perspective

**4. High-risk HPVs in colorectal cancers**

malignancies worldwide [15].

proteins.

On the other hand, and to determine the role of high-risk HPVs infection in human cancer cells, we examined the effect of E6/E7 of HPV type 16 in two noninvasive human breast cancer cell lines. We reported that E6/E7 of HPV type 16 induce cell invasive and metastatic abilities of the two cell lines *in vitro* and *in vivo*, respectively, in comparison with their wild-type cells [100]. This is accompanied by an overexpression of Id-1, a family member of helix-loop-helix transcription factors which regulates cell invasion and metastasis of human cancer cells [101, 102]. We also demonstrated that E6/E7 oncoproteins upregulate Id-1 promoter activity in human cancer cells. These data suggest that high-risk HPVs could play an important role in the progression of human carcinomas via Id-1 deregulation. Thus, we believe that E6/E7 oncoproteins of high-risk HPVs could play a similar role in the progression of human CRCs.

In order to investigate the role of high-risk HPV infection in human colorectal carcinogenesis, we examined the effect of E6/E7 of HPV type 16 in two human primary normal colorectal "mesenchymal" cell lines, NCM1 and NCM5, which were established in our laboratory [20].

**Figure 1.** E6/E7 oncoproteins of high-risk HPV type 16 induce cellular transformation in human primary normal color‐ ectal "mesenchymal" cell lines, NCE1, and NCE5 cells [103]. We note that NCE1 and NCE5 cells are unable to grow in soft agar. In contrast, NCE1 and NCE5 cells expressing E6/E7 oncoproteins form colonies in soft agar assay, which is an important characteristic of cancer cells.

We found that the expression of E6/E7 oncoproteins stimulate cell proliferation and induce cellular transformation (**Figure 1**) and migration of NCM1 and NCM5 cell lines. Moreover, our data revealed that E6/E7 of HPV type 16 provoke the upregulation of D-type cyclins and Cyclin E as well as Id-1 in these cell lines [103]. It is important to highlight that there are no other studies regarding the role of E6/E7 oncoproteins of high-risk HPVs in human colorectal cancers. Meanwhile, the function of E5 oncoprotein, in these malignancies, has not been investigated yet.

Additionally, we have recently investigated the incidence of high-risk HPVs in human CRCs in the Syrian population in a cohort of 78 cancer samples using PCR and tissue microarray analyses. We reported, for the first time, that high-risk HPVs are present in 42 samples (53.84%), which represent the majority of invasive colorectal cases; more significantly, our data pointedout that the most frequent high-risk HPV types in the Syrian population are 16, 33, 18, 35, and 31, respectively. Furthermore, the expression of E6 oncoprotein of high-risk HPVs was found to be correlated with Fascin, Id-1, and P-cadherin expression/overexpression in the majority of cancer tissue samples, which are major regulators of cell invasion and metastasis [17–19, 52]. Our data imply that high-risk HPVs are present in human CRCs, and their presence is associated with invasive and metastatic phenotype [16, 52, 104]. Collectively, these data suggest that high-risk HPVs are present in CRCs and therefore could play an important role in the initiation and progression of these cancers. Thus, we believe that high-risk HPVs can be associated with a subset of colorectal cancers. However, future large-scale multicenter case– control studies with data on risk factors such as lifestyle and sexual behavior are needed; meanwhile, molecular and cellular studies are necessary to determine the role of E5 and E6/E7 oncoproteins in human colorectal cancer and normal cells since it was proposed that E5 can cooperate with E6/E7 oncoproteins to enhance cancer progression of other human malignancies via the EMT event [20, 52]. Thus, we believe that E5 and E6/E7 of high-risk HPVs can cooperate with other oncogenes and/or risk factors such as smoking or alcohol to initiate colorectal cancer; in addition, E5 could cooperate with E6/E7 to enhance cancer progression of this malignancy via the EMT event (**Figure 2**).

**Figure 2.** E5 and E6/E7 of high-risk HPVs cooperation and colorectal carcinogenesis. We believe that E5 and E6/E7 of high-risk HPVs can cooperate with other oncogene overexpressions that are linked to lifestyle or/and environmental factors to induce cellular transformation and consequently tumor formation. On the other hand, E5 and E6/E7 together can enhance cancer progression of colorectal cancer via the initiation of the epithelial-mesenchymal transition (EMT) event .

Finally, we think it is important to talk about the prevention strategy of HPV infections and their related cancers, which is essentially based on HPV vaccines. These vaccines are made of virus-like particles (VLPs) that contain inactive L1 HPV proteins—proteins from and specific to each type of HPV viruses [105, 106]. Thus, the quadrivalent vaccine Gardasil (Merck and Co) was developed and approved by the FDA in 2006 for protection against low-risk HPV types 6 and 11, which cause genital warts—and rarely, nongenital warts [107] and high-risk HPV types 16 and 18 [108]. The quadrivalent vaccine will not protect against anogenital disease other than HPV types 6, 11, 16, and 18 [109, 110]. In 2010, the FDA approved the quadrivalent vaccine for the prevention of CRC [106]. The efficacy of prevention of rectal intraepithelial neoplasia in some group of patients is 77.5% [111]. In 2009, a bivalent vaccine (Cervarix; GlaxoSmithKline) was approved for the prevention of HPV infections from high-risk types 16 and 18 [112]. On December 10, 2014, the FDA approved a 9-valent HPV vaccine (Gardasil-9; Merck and Co) that was approved to be given in three intramuscular doses to males 9–15 years of age and females 9–26 years of age [106, 113]. The 9-valent HPV vaccine targets high-risk HPV type 16 (responsible for 50% of cervical carcinogenesis) [114], high-risk HPV type 18 (detected in 20% of cervical cancers) [115], and types 31, 33, 45, 52, and 58, which are responsible for 25% of cervical cancers. Immunizations against low-risk HPV types 6 and 11, which cause genital warts, are also included in the 9-valent vaccine [106, 116]. Approval of the 9-valent vaccine was based on a randomized control study with 14,000 females 16–26 years of age; it noted efficacy of 97% [106]. Therefore, this vaccine will have an important role in preventing HPV infections and their related cancers including colorectal malignancies and their metasta‐ sis.

## **5. Conclusion and perspectives**

We found that the expression of E6/E7 oncoproteins stimulate cell proliferation and induce cellular transformation (**Figure 1**) and migration of NCM1 and NCM5 cell lines. Moreover, our data revealed that E6/E7 of HPV type 16 provoke the upregulation of D-type cyclins and Cyclin E as well as Id-1 in these cell lines [103]. It is important to highlight that there are no other studies regarding the role of E6/E7 oncoproteins of high-risk HPVs in human colorectal cancers. Meanwhile, the function of E5 oncoprotein, in these malignancies, has not been

Additionally, we have recently investigated the incidence of high-risk HPVs in human CRCs in the Syrian population in a cohort of 78 cancer samples using PCR and tissue microarray analyses. We reported, for the first time, that high-risk HPVs are present in 42 samples (53.84%), which represent the majority of invasive colorectal cases; more significantly, our data pointedout that the most frequent high-risk HPV types in the Syrian population are 16, 33, 18, 35, and 31, respectively. Furthermore, the expression of E6 oncoprotein of high-risk HPVs was found to be correlated with Fascin, Id-1, and P-cadherin expression/overexpression in the majority of cancer tissue samples, which are major regulators of cell invasion and metastasis [17–19, 52]. Our data imply that high-risk HPVs are present in human CRCs, and their presence is associated with invasive and metastatic phenotype [16, 52, 104]. Collectively, these data suggest that high-risk HPVs are present in CRCs and therefore could play an important role in the initiation and progression of these cancers. Thus, we believe that high-risk HPVs can be associated with a subset of colorectal cancers. However, future large-scale multicenter case– control studies with data on risk factors such as lifestyle and sexual behavior are needed; meanwhile, molecular and cellular studies are necessary to determine the role of E5 and E6/E7 oncoproteins in human colorectal cancer and normal cells since it was proposed that E5 can cooperate with E6/E7 oncoproteins to enhance cancer progression of other human malignancies via the EMT event [20, 52]. Thus, we believe that E5 and E6/E7 of high-risk HPVs can cooperate with other oncogenes and/or risk factors such as smoking or alcohol to initiate colorectal cancer; in addition, E5 could cooperate with E6/E7 to enhance cancer progression of

**Figure 2.** E5 and E6/E7 of high-risk HPVs cooperation and colorectal carcinogenesis. We believe that E5 and E6/E7 of high-risk HPVs can cooperate with other oncogene overexpressions that are linked to lifestyle or/and environmental factors to induce cellular transformation and consequently tumor formation. On the other hand, E5 and E6/E7 together can enhance cancer progression of colorectal cancer via the initiation of the epithelial-mesenchymal transition (EMT)

investigated yet.

176 Human Papillomavirus - Research in a Global Perspective

event .

this malignancy via the EMT event (**Figure 2**).

This chapter presented substantial evidence that high-risk HPVs are present in human CRCs, thereby these viruses, through their E5 and E6/E7 oncoproteins, could play an important role in the initiation and progression of these malignances (Figure 2) [20, 100]. However, we believe that further studies are required to determine the function of E5 and E6/E7 oncogenes in human colorectal normal and cancer cells. Thus, developing new *in vitro* and *in vivo* models, such as cell lines and animal models, are necessary to identify the exact role of these oncoproteins and their potential cooperation in human colorectal carcinogenesis. Such studies can lead to the discovery of new targets to manage these malignances and other human carcinomas related to high-risk HPVs.

Alternatively, and with regards to colorectal malignancies as well as other human carcinomas prevention, we assume that the elimination of a number of known risk factors especially unprotected sexual activity, physical inactivity, smoking, alcohol, high consumption of red meat, and oncovirus infections such as high-risk HPVs could diminish the development of these malignancies and their metastases [20, 23, 26]. Additionally, prevention methodologies of high-risk HPVs using presently available vaccines could greatly reduce high-risk HPVsassociated cancers, including colorectal, and their progression to invasive form, which is responsible for the majority of cancer-related deaths.

## **Acknowledgements**

We are grateful to Mrs. A. Kassab for her critical reading of the chapter. The research works from Dr. Al Moustafa's laboratory is supported by the College of Medicine of Qatar University.

## **Author details**

Ala-Eddin Al Moustafa1,2,3\*, Noor Al-Antary1 and Amber Yasmeen4

\*Address all correspondence to: aalmoustafa@qu.edu.qa; ala-eddin.almoustafa@mcgill.ca

1 College of Medicine & Biomedical Research Centre, Qatar University, Doha, Qatar

2 Oncology Department, McGill University, Montreal, Quebec, Canada

3 Syrian Research Cancer Centre of the Syrian Society against Cancer, Aleppo, Syria

4 Segal Cancer Centre, Lady Davis Institute for Medical Research, JGH, McGill University, Montreal, Quebec, Canada

### **References**


[7] Al Moustafa AE, Al-Awadhi R, Missaoui N, Adam I, Durusoy R, et al. Human papil‐ lomaviruses-related cancers. Presence and prevention strategies in the Middle east and north African regions. Human Vaccines & Immunotherapeutics. 2014a;10:1812—21.

**Acknowledgements**

178 Human Papillomavirus - Research in a Global Perspective

**Author details**

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Ala-Eddin Al Moustafa1,2,3\*, Noor Al-Antary1

We are grateful to Mrs. A. Kassab for her critical reading of the chapter. The research works from Dr. Al Moustafa's laboratory is supported by the College of Medicine of Qatar University.

\*Address all correspondence to: aalmoustafa@qu.edu.qa; ala-eddin.almoustafa@mcgill.ca

1 College of Medicine & Biomedical Research Centre, Qatar University, Doha, Qatar

3 Syrian Research Cancer Centre of the Syrian Society against Cancer, Aleppo, Syria

4 Segal Cancer Centre, Lady Davis Institute for Medical Research, JGH, McGill University,

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**Section 4**

## **HPV - Vaccines**

## **The Involvement of Epigenetic Mechanisms in HPV‐ Induced Cervical Cancer**

Adriana Plesa, Iulia V. Iancu, Anca Botezatu, Irina Huica, Mihai Stoian and Gabriela Anton

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62833

### **Abstract**

High‐risk human papillomavirus (HPV) genotypes infection associates with cervical dysplasia and carcinogenesis. hr‐HPV transforming potential is based on E6 and E7 viral oncoproteins actions on cellular proteins. A persistent infection with hr‐HPV leads to progression from precursor lesions to invasive cervical cancer inducing changes in host genome and epigenome. Pathogenesis and development of cancer associated with both genetic and epigenetic defects alter transcriptional program. An important role for malignant transformation in HPV‐induced cervical cancer is played by epigenetic changes that occur in both viral and host genome. Furthermore, there are observations demon‐ strating that oncogenic viruses, once they integrated into host genome, become susceptible to epigenetic alterations made by host machinery. Epigenetic regulation of viral gene expression is an important factor in HPV‐associated disease. Gene expression control is complex and involves epigenetic changes: DNA methylation, histone modification, and non‐coding RNAs activity. Persistent infection with hr‐HPV can cause viral DNA integration into host genome attracting defense mechanisms such as methylation machinery. In this chapter, we aim to review HPV infection role in chromatin modification/ remodeling and the impact of HPV infection on non‐coding RNAs in cervix oncogene‐ sis. The reversible nature of epigenetic alterations provides new opportunities in the development of therapeutic agents targeting epigenetic modification in oncogenesis.

**Keywords:** HPV, epigenetic regulation, DNA methylation, histone modification, ncRNAs

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Cervical cancer accounts for almost 12% of all cancers in women, representing the second most frequent gynecological malignancy in the world, human papillomavirus (HPV) being consid‐ ered as etiologic agent of this malignancy [1, 2]. HPVs exhibit tropism for skin or mucosal epithelium where they cause warts, benign lesions that usually regress. HPV prevalence is a combination of incidental and persistent infections that have accumulated over time, due to lack of clearance. Infection with a high‐risk HPV (hr‐HPV) type is considered necessary for the development of cervical cancer, but by itself, it is not sufficient to cause cancer [3, 4].

The persistent infection with hr‐HPVs that have tropism for mucosal epithelia has been defined as the cause of more than 98% of cervical carcinomas as well as a high proportion of other cancers of the anogenital region (vulvar, vaginal, and penial) and oropharyngeal region [5]. It is known that persistent infection with hr‐HPV genotypes is necessary but not sufficient for the development of high‐grade cervical lesions and progression to malignancy. Persistent infection is characterized by continuous detection of the virus or its intermittent detection, due to latency, although the mechanism of latency has not yet been established but it is clear that the differences between active and latent cervical infection are qualitative and/or quantitative. The high prevalence of HPV infection in precancerous and cancerous cervical lesions confirms its oncogenic potential, different genotypes seem to be responsible for invasive cancer development. Approximately, 40 HPV genotypes were found to be associated with anogenital infections and are generally classified according to their oncogenic potential into low‐, high‐, and intermediate‐risk types. High‐risk or oncogenic types such as HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered so due to their presence in high‐grade squamous intraepithelial lesions (HSIL) or cervical cancer [6]. hr‐HPV genotypes 16 and 18 are causing more than 70% cervical cancer cases and most of the anogenital cancer as well as oropharyngeal tumors in men and women.

The molecular mechanism of cellular transformation induction involves epigenetic abnormal‐ ities along with genetic alterations. HPV disrupts normal cell‐cycle control, promoting uncontrolled cell division, and the accumulation of genetic damage. The transforming properties of hr‐HPV E6 and E7 oncoproteins are in interaction with many host cell proteins resulting in the maintenance and the reentering into cell cycle, permitting the virus to replicate, as it is dependent on the host cell DNA replication machinery.

Both E6 and E7 oncoproteins are able to interfere with key cellular processes (cell cycle, senescence, apoptosis and telomere shortening, differentiation). Furthermore, because of the frequent integration of the hr‐HPV genome into a host cell chromosome, those two proteins are the only viral proteins known to be consistently expressed in HPV‐associated cancers [7, 8]. Persistent infection with hr‐HPV genotypes determines progression from precursor lesions to invasive cervical cancer by inducing changes in the host genome and epigenome. Tran‐ scriptional modification program through genetic and epigenetic alterations leads to cancer development. Gene expression control is complex and involves epigenetic changes.

hr‐E6 protein is one of the most studied HPV proteins due to its many functions and is found to be interacting with many host cell proteins. Although E6 protein leads to p53 protein loss, an important element of cell transformation [9], many studies have identified a number of additional cellular targets that may play an important role. HPV E6 interferes with different apoptosis pathways by additional interactions with key mediators (TNFR‐1, FADD, CASP8, BAK, DFF40, GADD34/PP1, and TIP60) [10–15]. E6 protein is found to interact with proteins involved in cell cycle, cell-cell contact and polarity (MUPPI, E6BP, MAGI 1/3, DLG, PAT, paxillin, interacts with many proteins directly involved in DNA repair such as BRCA1, XRCC1, and MGMT [16–24] and targets other cell proteins involved in chromosomal and DNA stability for instance NFX1, hTERT, MCM7 [25, 26]. The E6 protein appears to have role in immune evasion as it interacts with tyk‐2 and IRF‐3 proteins both of which are involved in interferon signaling [27, 28].

**1. Introduction**

192 Human Papillomavirus - Research in a Global Perspective

tumors in men and women.

Cervical cancer accounts for almost 12% of all cancers in women, representing the second most frequent gynecological malignancy in the world, human papillomavirus (HPV) being consid‐ ered as etiologic agent of this malignancy [1, 2]. HPVs exhibit tropism for skin or mucosal epithelium where they cause warts, benign lesions that usually regress. HPV prevalence is a combination of incidental and persistent infections that have accumulated over time, due to lack of clearance. Infection with a high‐risk HPV (hr‐HPV) type is considered necessary for the

The persistent infection with hr‐HPVs that have tropism for mucosal epithelia has been defined as the cause of more than 98% of cervical carcinomas as well as a high proportion of other cancers of the anogenital region (vulvar, vaginal, and penial) and oropharyngeal region [5]. It is known that persistent infection with hr‐HPV genotypes is necessary but not sufficient for the development of high‐grade cervical lesions and progression to malignancy. Persistent infection is characterized by continuous detection of the virus or its intermittent detection, due to latency, although the mechanism of latency has not yet been established but it is clear that the differences between active and latent cervical infection are qualitative and/or quantitative. The high prevalence of HPV infection in precancerous and cancerous cervical lesions confirms its oncogenic potential, different genotypes seem to be responsible for invasive cancer development. Approximately, 40 HPV genotypes were found to be associated with anogenital infections and are generally classified according to their oncogenic potential into low‐, high‐, and intermediate‐risk types. High‐risk or oncogenic types such as HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered so due to their presence in high‐grade squamous intraepithelial lesions (HSIL) or cervical cancer [6]. hr‐HPV genotypes 16 and 18 are causing more than 70% cervical cancer cases and most of the anogenital cancer as well as oropharyngeal

The molecular mechanism of cellular transformation induction involves epigenetic abnormal‐ ities along with genetic alterations. HPV disrupts normal cell‐cycle control, promoting uncontrolled cell division, and the accumulation of genetic damage. The transforming properties of hr‐HPV E6 and E7 oncoproteins are in interaction with many host cell proteins resulting in the maintenance and the reentering into cell cycle, permitting the virus to replicate,

Both E6 and E7 oncoproteins are able to interfere with key cellular processes (cell cycle, senescence, apoptosis and telomere shortening, differentiation). Furthermore, because of the frequent integration of the hr‐HPV genome into a host cell chromosome, those two proteins are the only viral proteins known to be consistently expressed in HPV‐associated cancers [7, 8]. Persistent infection with hr‐HPV genotypes determines progression from precursor lesions to invasive cervical cancer by inducing changes in the host genome and epigenome. Tran‐ scriptional modification program through genetic and epigenetic alterations leads to cancer

hr‐E6 protein is one of the most studied HPV proteins due to its many functions and is found to be interacting with many host cell proteins. Although E6 protein leads to p53 protein loss,

development. Gene expression control is complex and involves epigenetic changes.

as it is dependent on the host cell DNA replication machinery.

development of cervical cancer, but by itself, it is not sufficient to cause cancer [3, 4].

E7 protein key activity is to overcome tumor suppressor block controlled by the pRb family proteins (RB, p107, p130) through disruption of pRb–E2F complexes thereby initiating the E2F mediated transcription [29]. E7‐pRB complex leads to functional inactivation and disruption of cell‐cycle progression in S phase. Another E7 hr‐HPV function is as cell‐cycle regulator, in doing so, the oncoprotein binds to p21/p27 and subsequent inactivates the CDK inhibitors [29, 30] and also to cyclins A, E in order to regulate cell cycle through pRb, p107 binding [31, 32]. On the other hand, E7 is known to be involved in transcription modulation by targeting host cell proteins AP1, TBP, MPP2, E2F6, and Skip [33–37]. In addition, E7 hr‐HPV protein binds to histone deacetylases (HDACs) in a pRb‐independent manner, which promotes cell growth. The E7 protein can also associate, directly or indirectly, with histone acetyl transferases (HATs) (p300, pCAF, and SRC1) and abrogates SRC1 associated HAT activity [38].

Following persistent infections with hr‐HPVs, E6, and E7 oncoproteins acts on the DNA and cause epigenetic changes. The cooperation between genetic and epigenetic alterations leads to the malignant phenotype and cancer progression. In contradiction to genetic alterations, the epigenetic changes are reversible, making them therapeutic targets in various conditions, and do not affect DNA sequence of the genes, but determine the gene expression regulation acting on the genome. It is well supported that cancers are epigenetically deregulated. Disruption of epigenetic processes determines altered gene function leading to imprinting disorders, developmental abnormalities and cancer. Epigenetic regulation of viral gene expression is an important factor in HPV associated diseases, due to processes that arise independently of changes in the underlying DNA sequence. Gene expression control is complex and involves epigenetic changes such as DNA methylation [39], histone modifications, and chromatin‐ remodeling proteins [40] and DNA silencing by non‐coding RNAs (ncRNAs) [41].

Taking into account that molecular mechanism induction of cellular transformation involves epigenetic abnormalities along with genetic alterations, in this chapter, we aim to review: (1) DNA methylation and cervical cancer; (2) the role of HPV infection in chromatin modification/ remodeling; (3) the impact of HPV infection on ncRNA in cervix oncogenesis; (4) epigenetic changes involved in viral gene expression; (5) potential epigenetic biomarkers in cervical cancer.

## **2. DNA methylation and cervical cancer**

The most studied epigenetic mechanism is DNA methylation. DNA methylation is a general term for processes of DNA bases (adenine, cytosine, and guanine) change by addition of a methyl group. Methylation of DNA bases can be achieved either under physiological condi‐ tions after a specific endogenous enzyme reaction by transferring the methyl group from a donor (biological methylation), or non‐physiological conditions through the action of chemical compounds: alkylating agents. DNA methylation plays an important role in various cellular processes including gene expression, silencing of transposable elements, as well as in the defense mechanism against viral infection.

In several types of cancer, many genes have been reported to be hypermethylated. DNA hypermethylation results in blocking of affected gene transcription, causing silencing them. In cancer, hypermethylation is considered one of the most important mechanisms for tumor suppressor gene silencing, responsible for the control of the normal cellular differentiation and/or inhibition of cell growth. The main chemical DNA modification is methylation of cytosine, commonly found in areas with CpG dinucleotides islands. Almost 60% of promoters of genes encoding proteins in the human genome contain CpG islands, and the majority are methylated in varying degrees, depending on the tissue [42].

Cytosine methylation is a stable inherited and reversible hallmark and is generally associated with transcriptional repression. The methylation inhibits transcription factors that bind to recognized DNA sequences by the recruitment of methyl cytosine binding protein (MECP and MBD) with corepressor molecules. The way that 5‐methylcytosine (5‐mC) repress transcription at the promoter level is by the recruitment of methyl binding proteins (MeCP2, MBD1, MBD2, MBD3, MBD4), which subsequently interacts with another protein to repress DNA transcrip‐ tion as well as HDAC and other chromatin remodeling enzymes [43].

DNA methylation is controlled by DNA methyltransferase (DNMT), which catalyses the transfer of the methyl group from S‐adenosyl methionine donor (SAM). Three active catalytic DNA methyltransferases were identified as follows: DNMT1, DNMT3A, and DNMT3B.

First tumor suppressor gene identified as hypermethylated was pRB, and then was followed by multiple publications describing similar phenomena for a variety of tumor suppressor genes such as p16, MLH1, VHL, and E‐cadherin [44, 45]. It remains controversial whether tumor suppressor gene hypermethylation is a cause or a consequence of silencing them. DNA methylation is reversible and various chemical compounds are known that can reactivate gene expression [46].

On the other hand, DNA hypermethylation may be a secondary process, due to changes in chromatin role in maintaining the status repression of gene expression. More evidence of this hypothesis, resulting from experiments showing that when DNA methyltransferase expres‐ sion was blocked *in vitro*, histone H3K9 methylation determined silencing of p16 gene in the absence of promoter DNA methylation [47, 48]. It was shown in cervical carcinoma that tumor suppressor genes are silent or abnormal diminished expressed due promoter hypermethyla‐ tion (**Table 1**).


**2. DNA methylation and cervical cancer**

194 Human Papillomavirus - Research in a Global Perspective

defense mechanism against viral infection.

expression [46].

tion (**Table 1**).

methylated in varying degrees, depending on the tissue [42].

tion as well as HDAC and other chromatin remodeling enzymes [43].

The most studied epigenetic mechanism is DNA methylation. DNA methylation is a general term for processes of DNA bases (adenine, cytosine, and guanine) change by addition of a methyl group. Methylation of DNA bases can be achieved either under physiological condi‐ tions after a specific endogenous enzyme reaction by transferring the methyl group from a donor (biological methylation), or non‐physiological conditions through the action of chemical compounds: alkylating agents. DNA methylation plays an important role in various cellular processes including gene expression, silencing of transposable elements, as well as in the

In several types of cancer, many genes have been reported to be hypermethylated. DNA hypermethylation results in blocking of affected gene transcription, causing silencing them. In cancer, hypermethylation is considered one of the most important mechanisms for tumor suppressor gene silencing, responsible for the control of the normal cellular differentiation and/or inhibition of cell growth. The main chemical DNA modification is methylation of cytosine, commonly found in areas with CpG dinucleotides islands. Almost 60% of promoters of genes encoding proteins in the human genome contain CpG islands, and the majority are

Cytosine methylation is a stable inherited and reversible hallmark and is generally associated with transcriptional repression. The methylation inhibits transcription factors that bind to recognized DNA sequences by the recruitment of methyl cytosine binding protein (MECP and MBD) with corepressor molecules. The way that 5‐methylcytosine (5‐mC) repress transcription at the promoter level is by the recruitment of methyl binding proteins (MeCP2, MBD1, MBD2, MBD3, MBD4), which subsequently interacts with another protein to repress DNA transcrip‐

DNA methylation is controlled by DNA methyltransferase (DNMT), which catalyses the transfer of the methyl group from S‐adenosyl methionine donor (SAM). Three active catalytic DNA methyltransferases were identified as follows: DNMT1, DNMT3A, and DNMT3B.

First tumor suppressor gene identified as hypermethylated was pRB, and then was followed by multiple publications describing similar phenomena for a variety of tumor suppressor genes such as p16, MLH1, VHL, and E‐cadherin [44, 45]. It remains controversial whether tumor suppressor gene hypermethylation is a cause or a consequence of silencing them. DNA methylation is reversible and various chemical compounds are known that can reactivate gene

On the other hand, DNA hypermethylation may be a secondary process, due to changes in chromatin role in maintaining the status repression of gene expression. More evidence of this hypothesis, resulting from experiments showing that when DNA methyltransferase expres‐ sion was blocked *in vitro*, histone H3K9 methylation determined silencing of p16 gene in the absence of promoter DNA methylation [47, 48]. It was shown in cervical carcinoma that tumor suppressor genes are silent or abnormal diminished expressed due promoter hypermethyla‐

**Table 1.** Hypermethylated tumor suppressor genes in invasive[R1] cervical cancer (Adapted with permission from Dueñas‐González et al. [49]).

On the other hand, microRNA genes undergo methylation‐mediated transcriptional repres‐ sion in cervical cancer miR‐149, miR‐375, miR‐432, miR‐1286, miR‐641, miR‐1290, miR‐1287, and miR‐95 [75–77].

CADM1, MAL, PAX1, and ADCYAP1 genes promoter hypermethylation were found to be involved in HPV‐mediated transformation and may be significantly associated with the development of cervical cancer [78].

It was shown that hTERT, mir124‐2, and PRDM14 were the first genes that became methylated during experimental immortalization. Following immortalization, ROBO3 methylation and CYGB was methylated, followed by CADM1, FAM19A4, MAL, PHACTR3, and SFRP2 [79].

## **3. The role of HPV infection in chromatin modification/remodeling**

Nucleosomes are the basic repetitive units of chromatin and are intended to pack huge eukaryotic genome in the nucleus (mammalian cells contain approximately 2 m linear DNA packed into a nucleus‐sized 10 mm diameter). Nucleosomes are further compacted to form chromosomes. These structures confer DNA compaction, but also create a base for the gene expression regulation. Nucleosome core particle is approximately 147 base pairs wrapped around a histone octamer made up of two copies of the histones H2A, H2B, H3, and H4. Histone H1 (linker histone) and its isoforms are involved in chromatin compaction and underlying nucleosomes condensation. Compaction of chromatin in the cell nucleus is necessary but is not fully understood. In nucleosomes compacting DNA linker—10 nm has an important role. A chain of nucleosomes can be arranged in 30 nm chromatin fibers whose formation is dependent of histone H1. A 30‐nm chromatin fiber is arranged as a loop around a central protein scaffold to generate the active form of the transcription‐euchromatin. Compacting the fibers can lead to transcriptionally inactive form—heterochromatin [80].

### **3.1. Histone modifications**

Covalent modifications of histones (epigenetic changes) are regulatory elements important in many biological processes. They affect chromatin interactions by structural changes in the histones or by modifying the electrostatic interactions and non‐histone proteins recruit [81]. Histones can undergo a variety of post‐translational modifications at the N‐terminus, which is represented by acetylation, methylation, phosphorylation, sumoylation, ADP–ribosylation, and ubiquitination. They can alter the DNA histone interaction, with a major impact on chromatin structure. Some covalent modifications of histones are involved in transcription and are associated with DNA repair process. On the other hand, the phosphorylation of histone H2AX appears to be unique modification in DNA repair. Histone modifications may influence both among themselves and in interaction with methylated DNA, and the presence of numerous changes in the combined space‐time context creates a program of genome expres‐ sion profile specific to each cell to keep identity.

### **3.2. Histone acetylation**

Histone acetylation is a modification of the lysine aminoacid that neutralizes the positive charges occurring at specific targets of nucleosome core. It has been speculated that histone acetylation can alter DNA interaction, helping to create more open chromatin architecture. Acetylation of lysine residues is catalyzed by HATs by transfer of an acetyl group from acetyl‐ coenzyme A to ε nitrogen of the lysine aminoacid. With few exceptions, these changes tend to create a relaxed form of chromatin, open for transcription, while deacetylation performed by HDACs is associated with transcriptional repression. Many transcriptional activators have been identified possessing intrinsic activity acetyl transferase: Gcn5/PCAF, CBP/p300, and SRC‐1. Similar to these co‐activators that exhibit HAT activity, there are co‐repressors with HDAC activity, such as mSin3a, NcoR/SMRT and NURD/Mi‐2 [82]. Rpd3 complex is an exception, being a complex with HDAC activity, associated with the active form of RNA polymerase II. Through this association is Rpd3 complex transcriptional repressor of initiation [83].

### **3.3. Histone methylation**

Methylation of H3 and H4 histone at lysine and arginine residues arises in mono‐, di‐, or tri‐ methylated form and is conducted using a specific histone methyltransferase (HMT) that acts at the level of these residues. HMTs can catalyze the addition of up to three methyl groups on the ε nitrogen of lysine [84]. Co‐activators, such as arginine methyltransferase (CARM1) and protein arginine methyltransferase (PRMT1), are essential for histone H3 and H4 arginine methylation [85]. Most of HMTs contain catalytic domains, named SET conserved domains named after *D. melanogaster* Su (var)3‐9 Enhancer of Z‐(E(z)) and trithorax (TRX), although there are some exceptions of HMTs without SET domain [85–88]. H3K4 and H3K36 methyla‐ tion is carried out by several methyltransferases. This redundancy makes the study of histone methylation more complex. Mammalian H3K79 methyltransferases include Suv39h1,2, G9a, and ESET [89]. EZH2 catalyses methylation of histone H3K27 and PR‐Set2 (also known SET 8) with Suv4‐20h1,2 catalyzes histone H4K20 methylation [90]. Methylation of H3K9, H3K27, and H4K20 is generally linked to the formation of heterochromatin in the presence of a transcrip‐ tional repressor HP1, while methylation of H3K4 and H3K36 is associated with transcription‐ ally active regions [91]. Methylation of histones lysine is reversible, being made by two enzymes families: aminooxidases (LSD1) and hydroxylases (JmjC family members), which may also demethylate trimethylated lysine [92, 93]. It was identified a series of proteins that bind to post‐translationally modified histones. For example, methylated lysine residues can bind to protein with conserved regions, like plant‐homeodomain (PHD) and chromodomain (CHD), whereas acetylated lysine residues bind to proteins with bromodomain [94]. These recruitment and recognition events can serve as a regulatory mechanism for other mechanisms that lead to other modifications of the histones. Two main complexes were identified, accom‐ panying epigenetic changes, and containing members of trithorax (TrxG) and Polycomb (PCG) group. Some components of complex PCG and TrxG exhibit histone–methyltransferase activity, while other members interpret histones modifications playing a central role in gene regulation, coordinating such DNA availability for development and to establish cell faith. This are accomplished by pausing the state of balance between silent transcriptionally heterochromatin (PCG) and competent transcriptionally euchromatin (TrxG) [95].

### **3.4. Histone phosphorylation**

Histone H1 (linker histone) and its isoforms are involved in chromatin compaction and underlying nucleosomes condensation. Compaction of chromatin in the cell nucleus is necessary but is not fully understood. In nucleosomes compacting DNA linker—10 nm has an important role. A chain of nucleosomes can be arranged in 30 nm chromatin fibers whose formation is dependent of histone H1. A 30‐nm chromatin fiber is arranged as a loop around a central protein scaffold to generate the active form of the transcription‐euchromatin. Compacting the fibers can lead to transcriptionally inactive form—heterochromatin [80].

Covalent modifications of histones (epigenetic changes) are regulatory elements important in many biological processes. They affect chromatin interactions by structural changes in the histones or by modifying the electrostatic interactions and non‐histone proteins recruit [81]. Histones can undergo a variety of post‐translational modifications at the N‐terminus, which is represented by acetylation, methylation, phosphorylation, sumoylation, ADP–ribosylation, and ubiquitination. They can alter the DNA histone interaction, with a major impact on chromatin structure. Some covalent modifications of histones are involved in transcription and are associated with DNA repair process. On the other hand, the phosphorylation of histone H2AX appears to be unique modification in DNA repair. Histone modifications may influence both among themselves and in interaction with methylated DNA, and the presence of numerous changes in the combined space‐time context creates a program of genome expres‐

Histone acetylation is a modification of the lysine aminoacid that neutralizes the positive charges occurring at specific targets of nucleosome core. It has been speculated that histone acetylation can alter DNA interaction, helping to create more open chromatin architecture. Acetylation of lysine residues is catalyzed by HATs by transfer of an acetyl group from acetyl‐ coenzyme A to ε nitrogen of the lysine aminoacid. With few exceptions, these changes tend to create a relaxed form of chromatin, open for transcription, while deacetylation performed by HDACs is associated with transcriptional repression. Many transcriptional activators have been identified possessing intrinsic activity acetyl transferase: Gcn5/PCAF, CBP/p300, and SRC‐1. Similar to these co‐activators that exhibit HAT activity, there are co‐repressors with HDAC activity, such as mSin3a, NcoR/SMRT and NURD/Mi‐2 [82]. Rpd3 complex is an exception, being a complex with HDAC activity, associated with the active form of RNA polymerase II. Through this association is Rpd3 complex transcriptional repressor of initiation

Methylation of H3 and H4 histone at lysine and arginine residues arises in mono‐, di‐, or tri‐ methylated form and is conducted using a specific histone methyltransferase (HMT) that acts at the level of these residues. HMTs can catalyze the addition of up to three methyl groups on the ε nitrogen of lysine [84]. Co‐activators, such as arginine methyltransferase (CARM1) and

**3.1. Histone modifications**

196 Human Papillomavirus - Research in a Global Perspective

**3.2. Histone acetylation**

**3.3. Histone methylation**

[83].

sion profile specific to each cell to keep identity.

Histones are phosphorylated at specific sites (serine residues) during cell division [96]. Phosphorylation process requires certain kinases. All four histone suffer phosphorylation; their biological meanings depend on the context. For example, histone H4S1 is evolutionary conserved role in chromatin compaction during the late stages of gametogenesis [97]. Phos‐ phorylation of histone H2A (human—Ser14 in yeast—Ser10) is correlated with meiotic chromosome condensation, but disappears during meiotic division [98]. Chromatin conden‐ sation in apoptosis has been linked to the phosphorylation of histone H2B, in both humans and yeast. Histone phosphorylation seems to have a role in transcription. It was shown that phosphorylation of histone H3 determines the competence of transcriptional response for JUN and FOS genes immediately. These changes occur due to activation of Ras‐MAP kinase pathway by growth factors.

### **3.5. Histone ubiquitination**

Ubiquitin is a polypeptide that is attached covalently to other proteins as a result of a steps series involving activation and conjugation enzymes of ubiquitin E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme) and ubiquitin ligase (E3) [99]. Polyubiquitination or more ubiquitin molecules addition to a protein is a classic signal for degradation via the proteasome. Histone H2A was first protein identified to serve as ubiquitin substrate [100]. Histone ubiquitination may be reversible using deubiquitinases. Like histone acetylation, ubiquitination is important in regulating gene expression. Highly ubiquitinated histones H2A and H2B have been associated with transcriptionally active sequences. Removing the ubiquitin residues on histone H2A leads to transcriptional repression [101].

### **3.6. ADP‐ribosylation**

ADP‐ribosylation is a post‐translational modification of proteins, including histones, which involves the addition of one or more residues of ADP and ribose. Mono or poly ADP-ribosy‐ lation is mediated by MARTs (Mono-ADP-ribosyltransferases) or PARPs (poly-ADP-ribose polymerases) enzymes [102]. ADP‐ribosylation of histones is carried out in a single‐site H2BE2ar1 [103]. Recently it was demonstrated the role of PARP‐1 in transcriptional activity, but only if that DNA repair process was induced [104].

### **3.7. Crosstalk between DNA methylation and histone modifications**

Several studies have shown that the relationship between DNA methylation and histone modifications is mediated by a group of proteins whose function is the binding to methyl groups from DNA, including proteins which bind to CpG methylated islands (MeCP2), proteins with binding domain to CpG methylated islands (methyl‐CpG binding domain protein 1, MBD1), and Kaiso protein‐also known as ZBTB 33 (Zinc finger and BTB domain containing protein 33). These proteins are localized to the methylated promoters and recruit a protein complex containing HDACs and HMTs [105–107]. These studies suggest that DNA methylation may induce structural changes to the chromatin by altering the histone modifi‐ cations. It is also known that DNA methylation inhibits methylation of histone H3K4me [108, 109]. In embryonic stem cells gene, Oct3/4 is inactivated after the fate is determined to a particular cell type. The silencing process is realized by recruiting a co‐repressor complex consisting of G9a methyltransferase and with HDAC enzyme activity. DNMT3A and DNMT3B DNA methyltransferases are subsequently recruited, catalysing the *de novo* DNA methylation at the gene promoter level [110]. Interaction between G9a protein and DNA methyltransferases (DNMT3A and DNMT3B) depends on the ankyrin motif of G9a protein [111]. In exchange, the SET domain responsible for methyl transferase activity of the G9a protein does not interact with DNA methyltransferases [112, 113]. These data suggest that DNA methylation at the promoter level depends on recruiting especially G9a protein and less of its methyltransferase activity. Interaction between histone H3K9 methylation and DNA methylation represents a model in which these two changes determine a strong silencing loop or bidirectional interference.

Recently, it was established with the aid ChIP and bioinformatics a link between methylation‐ mediated by PCG on histone H3K27 and *de novo* DNA methylation in cancers, which claims that the signal required by PRC2 during development predisposing certain genes to *de novo* methylation later [113–115]. In tumor cells were observed interactions between DNA methyl‐ ation and histone H3K9 methylated, thereby contributing to a stable silencing mechanism. PCG and EZH2 proteins are members of Polycomb repressor complex 2 (PRC2) which has methyltransferase activity with substrate specificity for histone H3K27. Histone H3K27me3 serves as specific binding signal to a chromodomain of another Polycomb repressor complex (PRC1). PRC1 blocks transcriptional factors recruitment, therefore the presence of PRC1 stops transcription initiation.

Biochemical studies have also shown that DNA methyltransferase binds EZH2 to certain conditions [115–117]. Histone H3K9 and H3K27 methylation presence does not always lead to the *de novo* DNA methylation. A subset of target genes for complex PCG can be methylated in cancer. Additional factors are required for DNA methylation in genes showing changes in the histones. Recently, it has been shown that the histone H3K27 trimethylation is PCG mediated and is a mechanism that determines tumor suppressor gene silencing in cancer, which is independent of promoter methylation [118, 119]. This lack of dependence between DNA methylation and histone modifications of these studies demonstrated conflicting results of previous studies. It should be noted that most of the genes presenting at their level histone H3K27me3 in prostate cancer do not show islands CpG motifs in the promoter, instead gene targeted by PCG complexes show generally the CpG promoters islands in embryonic stem cells ES [120]. This indicates that the histone H3K27 methylation processes mediated by the PCG complex in ES, normal and tumor cells are different because the tumor cells by removing the functional path of histone H3K27me3 usurps silencing mechanisms. Therefore, it was established the existence of 3 directions involved in silencing machinery associated H3K27 methylated histone mediated by PCG complex. The first relates to *de novo* repressed genes by methylation of histone H3K27, PCG mediated, and targets certain gene in particular which do not present CpG islands at promoter level. The second direction supports that during onco‐ genesis an early gene subset became methylated and CpG islands of the promoters are initially marked by PCG complex. This includes also those genes which undergo epigenetic reprog‐ ramming and are silent initially by the PCG and then suffer DNA methylation process like an alternated silencing mechanism. This epigenetic silencing switch through DNA methylation reduces epigenetic plasticity, blocking key regulators and contributes to tumourigenesis [121]. Third mechanism supports the fact that DNA methylation and histone H3K27me3 co‐ exist at the same promoter and methylation H3K27me3 histone by PCG silencing machinery is dominant. The silencing machinery may contribute to oncogenesis process in various forms, which can constitute in a repressor mechanism from flexible to plastic up to stable inactivation maintained by DNA methylation.

### **3.8. Chromatin and cancer**

enzyme), E2 (ubiquitin conjugating enzyme) and ubiquitin ligase (E3) [99]. Polyubiquitination or more ubiquitin molecules addition to a protein is a classic signal for degradation via the proteasome. Histone H2A was first protein identified to serve as ubiquitin substrate [100]. Histone ubiquitination may be reversible using deubiquitinases. Like histone acetylation, ubiquitination is important in regulating gene expression. Highly ubiquitinated histones H2A and H2B have been associated with transcriptionally active sequences. Removing the ubiquitin

ADP‐ribosylation is a post‐translational modification of proteins, including histones, which involves the addition of one or more residues of ADP and ribose. Mono or poly ADP-ribosy‐ lation is mediated by MARTs (Mono-ADP-ribosyltransferases) or PARPs (poly-ADP-ribose polymerases) enzymes [102]. ADP‐ribosylation of histones is carried out in a single‐site H2BE2ar1 [103]. Recently it was demonstrated the role of PARP‐1 in transcriptional activity,

Several studies have shown that the relationship between DNA methylation and histone modifications is mediated by a group of proteins whose function is the binding to methyl groups from DNA, including proteins which bind to CpG methylated islands (MeCP2), proteins with binding domain to CpG methylated islands (methyl‐CpG binding domain protein 1, MBD1), and Kaiso protein‐also known as ZBTB 33 (Zinc finger and BTB domain containing protein 33). These proteins are localized to the methylated promoters and recruit a protein complex containing HDACs and HMTs [105–107]. These studies suggest that DNA methylation may induce structural changes to the chromatin by altering the histone modifi‐ cations. It is also known that DNA methylation inhibits methylation of histone H3K4me [108, 109]. In embryonic stem cells gene, Oct3/4 is inactivated after the fate is determined to a particular cell type. The silencing process is realized by recruiting a co‐repressor complex consisting of G9a methyltransferase and with HDAC enzyme activity. DNMT3A and DNMT3B DNA methyltransferases are subsequently recruited, catalysing the *de novo* DNA methylation at the gene promoter level [110]. Interaction between G9a protein and DNA methyltransferases (DNMT3A and DNMT3B) depends on the ankyrin motif of G9a protein [111]. In exchange, the SET domain responsible for methyl transferase activity of the G9a protein does not interact with DNA methyltransferases [112, 113]. These data suggest that DNA methylation at the promoter level depends on recruiting especially G9a protein and less of its methyltransferase activity. Interaction between histone H3K9 methylation and DNA methylation represents a model in which these two changes determine a strong silencing loop

Recently, it was established with the aid ChIP and bioinformatics a link between methylation‐ mediated by PCG on histone H3K27 and *de novo* DNA methylation in cancers, which claims that the signal required by PRC2 during development predisposing certain genes to *de novo* methylation later [113–115]. In tumor cells were observed interactions between DNA methyl‐

residues on histone H2A leads to transcriptional repression [101].

but only if that DNA repair process was induced [104].

**3.7. Crosstalk between DNA methylation and histone modifications**

**3.6. ADP‐ribosylation**

198 Human Papillomavirus - Research in a Global Perspective

or bidirectional interference.

The involvement of DNA methylation process and chromatin changes in oncogenesis is indisputable, but separation of the genetic from epigenetic events is artificial. New evidence has shown that primary genetic defects (mutations in the genes coding for the receptors of growth factors, adhesion molecules, the gene that affects the DNA methylation and histone modifications as DNMT, HAT, or HDAC) lead to altered DNA methylation and changes in chromatin pattern. Both the endogenous and exogenous carcinogens do not cause genetic mutations but first epigenetic alterations, which highlights that epigenetic alteration is a step in oncogenesis.

All classical genetic alterations as mutations in tumor suppressor genes and in oncogenes can affect gene transcription (e.g., mutations in Ras gene, HER2 gene amplification). It is not surprising that the control of gene transcription machinery can be directly involved in oncogenesis. Although the complex nature of transcriptional regulation is uncertain, balance disruption of enzymatic activity responsible for maintaining acetylated histones status is expected to occur in cancer. p300/CBP histone acethyltransferase gene exhibits mutations in various cancer type (lung tumors, esophageal, ovarian, and gastric) [122–125]. Chromosomal translocations that targeted CBP/p300 gene locus affects transcription by their merger the translocated fragment with genes located in the chromatid area where they were joined (event met in hematological cancers such as acute myeloid leukemia) [126, 127].

Limited data regarding the global profile of histone modifications in oncogenesis can be found, but as a highlight is the overall loss of H4K16 monoacetylation and H4K20 trimethylation [128]. It has also been found that an important role in tumourigenesis is represented by changes of histone from promoters of tumor suppressor genes that determine their silencing. Such modifications are the loss of histone H3K9 acetylation and di/trimethylation of H3K4, H3K9 dimethylation, or trimethylation of H3K27 [129]. Several studies have reported a high level of EZH2 expression, which promotes tumor growth in both *in vitro* and *in vivo*, as identified in a number of human cancers such as melanoma, leukemia, prostate, and breast cancer [130, 131]. It has been shown that EZH2 could be a potential biomarker, and its expression was correlated with aberrant H3K27 trimethylation and silencing of tumor‐suppressor genes [119, 132].

Another frequent mechanism in cancer is the inactivation H3K27 demethylase‐UTX/KDM6A (lysine *(K)‐specific demethylase 6A*). KDM6A gene mutations have been reported in many types of tumors: multiple myeloma, esophageal squamous cell carcinoma, and renal cell carcinoma [133].

### **3.9. Alteration of histones changes in cervical cancer**

Histone modifications and alterations have recently begun to be studied in the cervical cancer. Analysis of histone modifications in the progression of cervical lesions is relatively at the beginning, there are few studies which indicate an association between alterations of histones and cervical cancer development. There are some data supporting that chromatin pattern in cervical samples may help in cervical neoplasia diagnosis, particularly for glandular lesions. The molecular basis of chromatin modifications is not fully determined [134]. E6 and E7 viral oncogenes expression is essential but not sufficient for neoplastic transformation, many studies highlight the important role of epigenetic changes in cervical carcinogenesis. Recently, it has been shown that E6 and E7 oncoproteins interacts with histone‐modulating enzyme, which regulates transcription via the host cell chromatin [84]. A recent report showed that in tumourigenesis, tumor cells lose monoacethylated and trimethylated histone H4 (acetylated Lys16 and trimethylated Lys20) form, that being associated with hypomethylation of repetitive DNA sequences [135]. Huang et al. [136] showed that the expression levels of HDACs were found to be increased in cervical dysplasia and invasive carcinoma.

mutations but first epigenetic alterations, which highlights that epigenetic alteration is a step

All classical genetic alterations as mutations in tumor suppressor genes and in oncogenes can affect gene transcription (e.g., mutations in Ras gene, HER2 gene amplification). It is not surprising that the control of gene transcription machinery can be directly involved in oncogenesis. Although the complex nature of transcriptional regulation is uncertain, balance disruption of enzymatic activity responsible for maintaining acetylated histones status is expected to occur in cancer. p300/CBP histone acethyltransferase gene exhibits mutations in various cancer type (lung tumors, esophageal, ovarian, and gastric) [122–125]. Chromosomal translocations that targeted CBP/p300 gene locus affects transcription by their merger the translocated fragment with genes located in the chromatid area where they were joined (event

Limited data regarding the global profile of histone modifications in oncogenesis can be found, but as a highlight is the overall loss of H4K16 monoacetylation and H4K20 trimethylation [128]. It has also been found that an important role in tumourigenesis is represented by changes of histone from promoters of tumor suppressor genes that determine their silencing. Such modifications are the loss of histone H3K9 acetylation and di/trimethylation of H3K4, H3K9 dimethylation, or trimethylation of H3K27 [129]. Several studies have reported a high level of EZH2 expression, which promotes tumor growth in both *in vitro* and *in vivo*, as identified in a number of human cancers such as melanoma, leukemia, prostate, and breast cancer [130, 131]. It has been shown that EZH2 could be a potential biomarker, and its expression was correlated with aberrant H3K27 trimethylation and silencing of tumor‐suppressor genes [119,

Another frequent mechanism in cancer is the inactivation H3K27 demethylase‐UTX/KDM6A (lysine *(K)‐specific demethylase 6A*). KDM6A gene mutations have been reported in many types of tumors: multiple myeloma, esophageal squamous cell carcinoma, and renal cell carcinoma

Histone modifications and alterations have recently begun to be studied in the cervical cancer. Analysis of histone modifications in the progression of cervical lesions is relatively at the beginning, there are few studies which indicate an association between alterations of histones and cervical cancer development. There are some data supporting that chromatin pattern in cervical samples may help in cervical neoplasia diagnosis, particularly for glandular lesions. The molecular basis of chromatin modifications is not fully determined [134]. E6 and E7 viral oncogenes expression is essential but not sufficient for neoplastic transformation, many studies highlight the important role of epigenetic changes in cervical carcinogenesis. Recently, it has been shown that E6 and E7 oncoproteins interacts with histone‐modulating enzyme, which regulates transcription via the host cell chromatin [84]. A recent report showed that in tumourigenesis, tumor cells lose monoacethylated and trimethylated histone H4 (acetylated Lys16 and trimethylated Lys20) form, that being associated with hypomethylation of repetitive

met in hematological cancers such as acute myeloid leukemia) [126, 127].

**3.9. Alteration of histones changes in cervical cancer**

in oncogenesis.

200 Human Papillomavirus - Research in a Global Perspective

132].

[133].

It was reported that MGMT a DNA repair protein silencing seems to be associated with a reduction in acetylated histones [137]. Moreover, the activation of Wnt signaling pathway may be realized by a transcriptionally repressed Wnt antagonist DICKKOPF‐1 (DKK‐1), by histone deacetylation in HPV‐infected cervical cells [138]. HDAC function is necessary for HIF‐1 (hypoxia inducible factor‐1) activity, and it was found that E7HPV protein can block the interaction of HDACs with HIF‐1α, activating HIF‐1‐dependent transcription for a range of pro‐angiogenic factors [139, 140]. Silencing of proliferation repressor protein osteo‐protegerin (OPG) and retinoic acid receptor β2 (RAR‐β2) was found to occur through histone modification as well as DNA methylation [141, 142].

It has been shown that phosphorylated and acetylated forms of histone H3 in cervical swabs are associated with progression from CIN I to CIN II and CIN III [143]. The balance between HDACs and HATs activity has a key role in regulating gene transcription [144]. This balance must be maintained in normal cells, to prevent an uncontrolled proliferation and cell death. E6 and E7 HPV target numerous cellular proteins to disrupt cell growth and proliferation, including HDACs and HATs. E7 hr‐HPV protein binds to HDACs, this interaction being performed by Mi2β, a member of the nucleosomes remodeling complex and acetylation of histones (NuRD), which possess the ability to modify chromatin structure by both the deacetylation of histones and by the repositioning ATP‐dependent nucleosomes [145]. The interaction the E7‐HDACs is independent of binding to Rb protein and E7 gene mutations abolish its ability to target the HDACs and to transform mouse fibroblasts [84]. E6 hr‐HPV protein shares with other DNA tumorigenic viruses' ability to target CBP/p300. The interaction involves C‐terminus zinc finger of E6 protein and 1808–1826 residues of CBP; as a result, the p53 transcriptional activity is reduced, independently of p53 protein removal through the proteasome degradation pathway [146]. E7, E6 protein binds to the transcriptional co‐activator p300/CBP, being a crucial step in cellular transformation [147].

Histone methylation is acknowledged to be a dynamically process controlled by two types of enzymes that work together to maintain global histone methylation patterns: HMTs and histone lysine demethylases (KDMs) [42, 95]. Histone methylation can occur at different lysine residues. The interaction between HMTs and KDMs locally adjusts the degree of methylation which results in the activation or repression of gene expression, depending on the specific target lysine residue [95]. Thus, the degree of methylation and the position of methylated lysine have different consequences: overall methylation of H3K9 (histone 3 lysine at position 9), H3K27 (histone 3 lysine at position 27), and H4K20 (histone 4 lysine at position 20) is linked to the heterochromatin formation in the presence of a transcriptional repressor associated with HP1, while the methylation of H3K4 (histone 3 lysine in position 4) and H3K36 (histone 3 lysine in position 4) is associated with transcriptionally active regions [148–152]. Generally, methy‐ lated H3K4, H3K36, and H3K79 are considered activating marks, whereas methylation of H3K9, H3K27, and H4K20 are often associated with gene silencing [150–154].

E6, E7 oncoproteins can associate with enzymes that modulate histone acetylation, and thus, regulate the transcriptional capacity of host cell chromatin [151, 152, 155, 156]. Especially, KDMs expression was found to be deregulated and associated with cancer aggressiveness. KDMs were further proposed as potential tumor biomarkers and could play distinct role in cancer progression acting either as putative oncogene or tumor suppressor based on different transcriptional role (gene activation/repression) [157, 158].

McLaughlin‐Drubin *et al*. [159] sustain that E7 HPV16 can induce epigenetic and transcrip‐ tional alterations by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27 (H3K27)‐specific demethylases.

KDM5C demethylase role in the pathogenesis of HPV‐induced has been described in the literature. KDM5C is recruited by the E2 viral protein for E6 and E7 oncogenes transcriptional repression through the LCR region of HPV. The results obtained indicate KDM5C as a good marker for severe lesions and SCC [160].

Another recent study showed that KDM4C, KDM5C, KDM6A, and KDM6B genes expression significantly increase in high‐grade lesions (CIN 2+) and SCC presenting a positive correlation with HPV infection. A significantly increased of KDM4C expression levels in SCC samples compared with precancerous lesions propose it as a suitable tumor marker. KDM4C/GASC1/ JMJD2C/ is a histone demethylase that is mainly regarded as oncogene due to its role in demethylating heterochromatic H3K9me3/2 [161]. Another good marker for high‐risk lesions and SCC seems to be KDM5C whose expression levels were found increased in CIN2+ lesions and significantly increased in SCC cases [160, 161].

p16 gene expression in normal cells is generally low due to gene silencing by H3K27 trime‐ thylation and PRC complex action. It was observed that the E7 oncogene expression may reduce residues H3K27 required for repression of PRC1 complex, leading to transcriptional activation of histone H3K27, histone demethylases KDM6A, and KDM6B through an unknown mechanism. In response to the stimulation of the RAS/RAF transcriptional activation of KDM6B occurs possible via AP1, leading to the removal of H3K27me3 (histone 3 lysine at position 27 trimethylated) residues and increasing expression of p16INK4a [162]

The literature data suggest an important role as biomarker for p16INK4a tumor‐suppressor gene in HPV‐induced lesions and cervical cancers. The mechanism of induction of p16 expression by the E7 viral oncogene is believed to be achieved by the activation of E2F transcription factor [163]. Later it was observed that from the p16 promoter are missing response elements to E2F and E7 HPV16 mutated variants that are defective in binding/ degradation of pRb and E2F transcription are not activated; p16 expression can be induced by the wild‐type and variants [159]. p16INK4 expression is induced by demethylation of H3K27 residues KDM6B mediated, that underpins the induction of senescence by oncogenes (*Oncogene induced senescence—*OIS), an intrinsic cellular innate tumor suppressor mechanism triggered by oncogenes such as RAS [164]. E7 oncogene causes degradation of pRB, the main mediator of halting cell growth and senescence induced by p16, repealing the mechanism of induction of senescence by oncogenes. The mechanism of inactivation of pRB by E7 can be explained by the necessity to avoid eliminating E7 HPV positive cells targeted by OIS. Such high levels of p16 observed in this study correlated with an increased expression of KDM6B histone demethylase due to E7 oncogene activity on H3K27 modulators.

## **4. The impact of HPV infection on ncRNA in cervix oncogenesis**

KDMs expression was found to be deregulated and associated with cancer aggressiveness. KDMs were further proposed as potential tumor biomarkers and could play distinct role in cancer progression acting either as putative oncogene or tumor suppressor based on different

McLaughlin‐Drubin *et al*. [159] sustain that E7 HPV16 can induce epigenetic and transcrip‐ tional alterations by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27

KDM5C demethylase role in the pathogenesis of HPV‐induced has been described in the literature. KDM5C is recruited by the E2 viral protein for E6 and E7 oncogenes transcriptional repression through the LCR region of HPV. The results obtained indicate KDM5C as a good

Another recent study showed that KDM4C, KDM5C, KDM6A, and KDM6B genes expression significantly increase in high‐grade lesions (CIN 2+) and SCC presenting a positive correlation with HPV infection. A significantly increased of KDM4C expression levels in SCC samples compared with precancerous lesions propose it as a suitable tumor marker. KDM4C/GASC1/ JMJD2C/ is a histone demethylase that is mainly regarded as oncogene due to its role in demethylating heterochromatic H3K9me3/2 [161]. Another good marker for high‐risk lesions and SCC seems to be KDM5C whose expression levels were found increased in CIN2+ lesions

p16 gene expression in normal cells is generally low due to gene silencing by H3K27 trime‐ thylation and PRC complex action. It was observed that the E7 oncogene expression may reduce residues H3K27 required for repression of PRC1 complex, leading to transcriptional activation of histone H3K27, histone demethylases KDM6A, and KDM6B through an unknown mechanism. In response to the stimulation of the RAS/RAF transcriptional activation of KDM6B occurs possible via AP1, leading to the removal of H3K27me3 (histone 3 lysine at

The literature data suggest an important role as biomarker for p16INK4a tumor‐suppressor gene in HPV‐induced lesions and cervical cancers. The mechanism of induction of p16 expression by the E7 viral oncogene is believed to be achieved by the activation of E2F transcription factor [163]. Later it was observed that from the p16 promoter are missing response elements to E2F and E7 HPV16 mutated variants that are defective in binding/ degradation of pRb and E2F transcription are not activated; p16 expression can be induced by the wild‐type and variants [159]. p16INK4 expression is induced by demethylation of H3K27 residues KDM6B mediated, that underpins the induction of senescence by oncogenes (*Oncogene induced senescence—*OIS), an intrinsic cellular innate tumor suppressor mechanism triggered by oncogenes such as RAS [164]. E7 oncogene causes degradation of pRB, the main mediator of halting cell growth and senescence induced by p16, repealing the mechanism of induction of senescence by oncogenes. The mechanism of inactivation of pRB by E7 can be explained by the necessity to avoid eliminating E7 HPV positive cells targeted by OIS. Such high levels of p16 observed in this study correlated with an increased expression of KDM6B

position 27 trimethylated) residues and increasing expression of p16INK4a [162]

histone demethylase due to E7 oncogene activity on H3K27 modulators.

transcriptional role (gene activation/repression) [157, 158].

(H3K27)‐specific demethylases.

marker for severe lesions and SCC [160].

202 Human Papillomavirus - Research in a Global Perspective

and significantly increased in SCC cases [160, 161].

In the latest years, thanks to a growing number of studies focusing on high‐throughput next generation sequencing (NGS), large‐scale genome, and genome‐wide transcriptome methods, a new world of RNA molecules: ncRNAs have emerged [165].

More recently, through deep sequencing data obtained by transcriptome projects such as ENCODE (Encyclopedia of DNA Elements Consortium), it has been revealed that around 90% of genomic DNA in eukaryotes is transcribed with just 1–2% of the transcript encoding for proteins, the vast majority being transcribed as ncRNAs [166].

Some of the ncRNAs molecules appear to be important players in genome functioning acting as "regulatory RNAs". Experimentally data gathered so far sustain the ncRNAs involvement in many biological processes; they seem to have important roles in genes transcriptional and posttranscriptional regulation, RNA splicing, translation and turnover, also in epigenetic modifications [167, 168].

Furthermore, given the regulatory role that these non‐coding molecules possess in normal biological processes, it has been presumed that they might play a significant role also in different types of pathologies. There are accumulating evidence highlighting a major role for these molecules in various diseases where they appear to have aberrant expression and contributes to disease development and progression.

Several studies showed ncRNAs involvement in diseases such as neurodegenerative, cardio‐ vascular, immune diseases and in neoplastic transformation [169]. Regulatory ncRNAs could be classified according to their length in three categories [170]:


miRNAs molecules are approximately 18–24 nucleotides in length, ncRNAs that regulate genes expression in eukaryotic organisms. These RNA molecules are known to be a part of RISC complex (RNA‐induced silencing complex) and are involved in gene silencing by pairing with complementary sequences at 3' UTR (untranslated regions) or coding region of a target messenger RNAs (mRNAs) that leads to mRNA degradation and blocking protein synthesis [171–173].

Through this interaction miRNAs molecules play an important role in specific cellular processes including cellular development, proliferation, differentiation, apoptosis, and thereby controlling the expression level of hundreds important genes involved in these processes [174, 175].

Numerous studies have reported miRNAs aberrant expression profiles in different types of cancer. Until recently, the most extensively studied ncRNAs in oncogenesis, miRNAs appear to have a dual nature in neoplastic transformation acting either as tumor suppressors and/or as oncogenes depending on the cellular context [176].

miRNAs known to have oncogenic functions also called "oncomiRs" have frequently been demonstrated to control processes such as cell differentiation, apoptosis, and tumor develop‐ ment through tumor suppressor genes inhibition. Several examples of well‐known oncomiRs linked to malignant transformation are miR‐15, miR‐16 found upregulated in many types of leukemia's and lymphomas [177, 178]; miR‐155 overexpressed in chronic lymphocytic leukemia (CLL), B‐cell lymphoma, anaplastic large cell lymphoma (ALCL), Hodgkin's or Burkitt's lymphoma and in breast tumors [179–182]; miRNA‐17‐92 cluster (oncomiR‐1) deregulated in multiple types of cancer: lung (particularly in small‐cell lung cancer and aggressive forms), pancreatic, hepatocellular, colorectal, breast, ovarian, and hematopoietic cancers [183, 184]; meanwhile, miR‐106a has an oncogenic role in pancreatic, colon cancer, and T‐cell lymphoma [185–187]; another promising oncomiR is miR‐21 found overexpressed in various cancers including breast cancer, lung cancer, colorectal cancer, hepatocellular carci‐ noma, glioblastoma [188–196].

On the other hand, in cancer, it was shown that some miRNAs are consistently downregulated and act as tumor suppressor with example such as: miR‐15a and miR‐16 cluster that is often deleted or downregulated in tumor cells [197–200] or miR‐34 family members that were identified as potential tumor suppressor in many cancers [201–203], also miR‐124 was found significantly downregulated in several types of human cancers [203–206]; miR‐122 demon‐ strated to regulate intrahepatic metastasis in hepatocellular carcinoma and thus acting as a tumor suppressor for this pathology [207] and mir‐203 was shown to suppress cell proliferation and migration in various types of cancer [208–210].

A malignancy where miRNAs role has been extensively investigated is cervical cancer. There are many reports that emphasize a substantial role for these non‐coding molecules in cervical oncogenesis.

An interesting research direction in miRNAs field is their relationships with viral infections. Various studies support the fact that some cellular miRNAs expression can be regulated by virus infection and these observations are not surprising given the host defense mechanisms against pathogen agents such as bacteria and viruses.

Researchers also have identified several cellular miRNAs whose levels can be modulated by HPV infection, respectively, by viral E6 or E7 oncoprotein of high‐risk genotypes [211].

Studies based on miRNAs expression profiles revealed a differentially pattern of expression in cervical tumors tissue compared with normal tissue, still due to different detection methods and experimental systems use in some cases the observations are contradictory (**Table 2**).

Recently based on the observation that some viruses could express their own set of miRNAs, there is an ongoing effort to identifying these miRNAs and to establish their role during viral infection. Reports revealed that miRNAs encoded by viruses target host genes involved cell The Involvement of Epigenetic Mechanisms in HPV‐Induced Cervical Cancer http://dx.doi.org/10.5772/62833 205


**Table 2.** Examples of miRNAs aberrant expressed in cervical cancer.

Numerous studies have reported miRNAs aberrant expression profiles in different types of cancer. Until recently, the most extensively studied ncRNAs in oncogenesis, miRNAs appear to have a dual nature in neoplastic transformation acting either as tumor suppressors and/or

miRNAs known to have oncogenic functions also called "oncomiRs" have frequently been demonstrated to control processes such as cell differentiation, apoptosis, and tumor develop‐ ment through tumor suppressor genes inhibition. Several examples of well‐known oncomiRs linked to malignant transformation are miR‐15, miR‐16 found upregulated in many types of leukemia's and lymphomas [177, 178]; miR‐155 overexpressed in chronic lymphocytic leukemia (CLL), B‐cell lymphoma, anaplastic large cell lymphoma (ALCL), Hodgkin's or Burkitt's lymphoma and in breast tumors [179–182]; miRNA‐17‐92 cluster (oncomiR‐1) deregulated in multiple types of cancer: lung (particularly in small‐cell lung cancer and aggressive forms), pancreatic, hepatocellular, colorectal, breast, ovarian, and hematopoietic cancers [183, 184]; meanwhile, miR‐106a has an oncogenic role in pancreatic, colon cancer, and T‐cell lymphoma [185–187]; another promising oncomiR is miR‐21 found overexpressed in various cancers including breast cancer, lung cancer, colorectal cancer, hepatocellular carci‐

On the other hand, in cancer, it was shown that some miRNAs are consistently downregulated and act as tumor suppressor with example such as: miR‐15a and miR‐16 cluster that is often deleted or downregulated in tumor cells [197–200] or miR‐34 family members that were identified as potential tumor suppressor in many cancers [201–203], also miR‐124 was found significantly downregulated in several types of human cancers [203–206]; miR‐122 demon‐ strated to regulate intrahepatic metastasis in hepatocellular carcinoma and thus acting as a tumor suppressor for this pathology [207] and mir‐203 was shown to suppress cell proliferation

A malignancy where miRNAs role has been extensively investigated is cervical cancer. There are many reports that emphasize a substantial role for these non‐coding molecules in cervical

An interesting research direction in miRNAs field is their relationships with viral infections. Various studies support the fact that some cellular miRNAs expression can be regulated by virus infection and these observations are not surprising given the host defense mechanisms

Researchers also have identified several cellular miRNAs whose levels can be modulated by HPV infection, respectively, by viral E6 or E7 oncoprotein of high‐risk genotypes [211].

Studies based on miRNAs expression profiles revealed a differentially pattern of expression in cervical tumors tissue compared with normal tissue, still due to different detection methods and experimental systems use in some cases the observations are contradictory (**Table 2**).

Recently based on the observation that some viruses could express their own set of miRNAs, there is an ongoing effort to identifying these miRNAs and to establish their role during viral infection. Reports revealed that miRNAs encoded by viruses target host genes involved cell

as oncogenes depending on the cellular context [176].

204 Human Papillomavirus - Research in a Global Perspective

and migration in various types of cancer [208–210].

against pathogen agents such as bacteria and viruses.

noma, glioblastoma [188–196].

oncogenesis.

proliferation, apoptosis, host immunity regulation, in order to maintain their survival and to escape from immune system response.

Over 200 miRNAs encoded by several virus families have been identified to date, many of them being found for herpes viruses and Epstein–Barr virus (EBV) [237]. For instance, it was found that EBV encodes more than 40 miRNAs that presents different expression levels during viral infection and some are involved in maintaining viral latency [238, 239]. From our knowledge to date, there are no reports on the existence of HPV‐encoded microRNAs.

Although researcher's attention in latest years was mainly focus on short ncRNAs molecules and their functions in normal/pathological conditions, at present great efforts are put into investigating the major part of the non‐coding transcriptome namely lncRNAs transcripts.

It has been revealed that certain lncRNAs can control gene expression through a range of different mechanism including transcriptional, splicing, and post‐transcriptional regulation or at epigenetic levels by chromatin remodeling and histone modification regulation [240–242].

Even though few lncRNAs have been well characterized, from the knowledge accumulated so far it is clear that they represent significant gene regulators and play critical roles in many cellular and development processes. Therefore, taken into account, the wide functions that lncRNAs hold, it is not surprising that their alterations are associated with an extensive range of disease.

Several studies have reported lncRNAs involvement in cardiovascular diseases, neurological disorders, immune disease, and also in cancer, data indicates a differential lncRNAs expression in many types of malignancy including, breast cancer, colon cancer, prostate cancer, hepato‐ cellular carcinoma, pancreatic cancer, lymphomas [243].

Currently, the expression profile of various ncRNAs has become an important feature of oncogenesis process. There are numerous publications indicating an association between lncRNAs expression and malignant transformation and the number is still rising. Despite the keen interest shown by these molecules for many of them, the functional role in normal/ pathological condition is still unclear, additional studies are needed. Recently with the help of the latest NGS techniques, new information is brought to light for better understanding lncRNAs role, mechanisms of action, and also the potential use of them in various cancer therapies.

Among the best well‐characterized lncRNAs are XIST (*X inactive specific transcript*) a 17‐kb‐ long transcript known for its role in dosage compensation involving X chromosome inactiva‐ tion and H19 transcript 2.5‐kb‐long that plays an important role in imprinting [244, 245].

Experimentally data sustain a potential oncogenic role for H19, an aberrant expression have been identified in a variety of cancers: breast, ovarian, hepatocellular, gastric, lung, colon, esophagus [246–251]. It has been shown that H19 oncogenic role is also due to the fact that the transcript acts as a precursor for miARN‐675 leading to pRB gene expression decrease [252].

Another lncRNA having oncogenic potential is MALAT1 (metastasis‐associated lung adeno‐ carcinoma transcript 1) who it was suggested in many studies that can promote cell prolifer‐ ation apoptosis, invasion, and metastasis. Significantly high levels of MALAT1 expression were detected in lung cancer, prostate cancer, colorectal cancer, hepatocellular carcinomas, gynecologic (endometrial, cervical) cancer, osteosarcoma [253–262].

There are identified lncRNAs that appear to have tumor suppressor function in carcinogenesis. For GAS5 (growth arrest specific 5) lncRNA, it was found to play an important role in apoptosis induction; studies have reported a significantly reduced GAS5 levels of expression in breast cancer and prostate cancer [263, 264]. Another report shows that GAS5 low level of expression is associated with poor prognosis in hepatocellular carcinoma [265].

MEG3 (maternally expressed 3) is lncRNAs that presents a reduced expression in several types of cancer. Several experimental evidences demonstrate that MEG3 interacts with p53 tumor suppressor gene and regulates p53 target gene expression, therefore, inhibits tumor cell proliferation and cancer progression. Aberrant levels of MEG3 expression have been identified in glioblastoma, ovarian, colon, cervical, lung cancer [266–271].

In literature, there are a relatively small number of experimental data showing an association regarding lncRNAs involvement in cervical carcinogenesis, but due to high interest shown toward these molecules the number is growing fast. The data collected so far on lncRNAs involvement in cervical cancer are presented in **Table 3**.


**Table 3.** List of lncRNAs expressed in cervical cancer [272–274].

cellular and development processes. Therefore, taken into account, the wide functions that lncRNAs hold, it is not surprising that their alterations are associated with an extensive range

Several studies have reported lncRNAs involvement in cardiovascular diseases, neurological disorders, immune disease, and also in cancer, data indicates a differential lncRNAs expression in many types of malignancy including, breast cancer, colon cancer, prostate cancer, hepato‐

Currently, the expression profile of various ncRNAs has become an important feature of oncogenesis process. There are numerous publications indicating an association between lncRNAs expression and malignant transformation and the number is still rising. Despite the keen interest shown by these molecules for many of them, the functional role in normal/ pathological condition is still unclear, additional studies are needed. Recently with the help of the latest NGS techniques, new information is brought to light for better understanding lncRNAs role, mechanisms of action, and also the potential use of them in various cancer

Among the best well‐characterized lncRNAs are XIST (*X inactive specific transcript*) a 17‐kb‐ long transcript known for its role in dosage compensation involving X chromosome inactiva‐ tion and H19 transcript 2.5‐kb‐long that plays an important role in imprinting [244, 245].

Experimentally data sustain a potential oncogenic role for H19, an aberrant expression have been identified in a variety of cancers: breast, ovarian, hepatocellular, gastric, lung, colon, esophagus [246–251]. It has been shown that H19 oncogenic role is also due to the fact that the transcript acts as a precursor for miARN‐675 leading to pRB gene expression decrease [252].

Another lncRNA having oncogenic potential is MALAT1 (metastasis‐associated lung adeno‐ carcinoma transcript 1) who it was suggested in many studies that can promote cell prolifer‐ ation apoptosis, invasion, and metastasis. Significantly high levels of MALAT1 expression were detected in lung cancer, prostate cancer, colorectal cancer, hepatocellular carcinomas,

There are identified lncRNAs that appear to have tumor suppressor function in carcinogenesis. For GAS5 (growth arrest specific 5) lncRNA, it was found to play an important role in apoptosis induction; studies have reported a significantly reduced GAS5 levels of expression in breast cancer and prostate cancer [263, 264]. Another report shows that GAS5 low level of expression

MEG3 (maternally expressed 3) is lncRNAs that presents a reduced expression in several types of cancer. Several experimental evidences demonstrate that MEG3 interacts with p53 tumor suppressor gene and regulates p53 target gene expression, therefore, inhibits tumor cell proliferation and cancer progression. Aberrant levels of MEG3 expression have been identified

In literature, there are a relatively small number of experimental data showing an association regarding lncRNAs involvement in cervical carcinogenesis, but due to high interest shown

gynecologic (endometrial, cervical) cancer, osteosarcoma [253–262].

is associated with poor prognosis in hepatocellular carcinoma [265].

in glioblastoma, ovarian, colon, cervical, lung cancer [266–271].

cellular carcinoma, pancreatic cancer, lymphomas [243].

206 Human Papillomavirus - Research in a Global Perspective

of disease.

therapies.

## **5. Epigenetic changes involved in viral gene expression**

Methylation status of integrated HPV depends of viral life cycle as well as of neoplastic transformation, this making HPV methylome a potential tool in cancer diagnostic. HPV genome methylation status depends on the viral life cycle and is associated with neoplastic progression.

According to Johanssen and Lambert study, viral genome is subjected to *de nov*o methylation by host DNMTs. Methylation of the viral genome may be a part of a mechanism involved in innate response to pathogens by which the host attempts to suppress viral gene expression. The authors note that in HeLa cells, HPV18 genome chromatin histone modification status correlates with the occupancy of host transcriptional machinery specifically within the LCR [292]. E7 and E6 oncoproteins of hr‐HPVs appear to modulate host epigenetic machinery through their interplay with both DNA methylation enzymes as well as chromatin remodeling enzymes [159].

Mirabello et al. [293] reported in 2013 that they found 3‐region in L1 strongly methylated in cancers and only in a small percentage in CIN I and CIN II lesions. In addition, the authors shown that methylation at certain CpG sites can indicate an evolution toward CIN II+ years before it happens.

Evaluation of cervical samples from HPV positive women, presenting precancerous lesions or invasive ones, showing that the hypomethylation degree in LCR and E6 gene region increase with the increasing of lesion severity. These data convinced the authors to conclude that neoplastic transformation could be suppressed by hypermethylation, while hypomethylation accompanies or leads to progression toward cancer [294].

Using laser microdissection on different layers from samples with HPV‐infected lesions, Vinokurova and von Knebel Doeberitz [295] found dynamic changes in HPV16 LCR methyl‐ ation in the context of the viral life cycle. A decrease in methylation in the transcriptional enhancer region within the LCR was observed in terminally differentiated epithelial compart‐ ment and meanwhile an increase in methylation within the region of the LCR containing the early promoter was noted [295].

Another study highlighted heterogeneity of methylation status among patients, even in samples from the same patient. Methylation frequency was found to be approximately 30% in L1 region, less than in CpG islands around enhancer and promoter of HPV16. In most of the HPV genome sites, hypermethylation is associated better with carcinoma than with dysplastic lesions [296].

On the other hand, a study regarding methylation status in HPV18 immortalized cell lines (HeLa and C4‐1) and in samples from patients, determined a clonally heterogeneity of methylation status in different regions of viral genome. The clinical samples showed partial or total methylation in HPV enhancer region, while in asymptomatic patient's, samples were fully unmethylated. Viral promoter was reported to be methylated in tumor samples and in cervical smears [297].

These studies indicate that methylation status of viral oncogenes in cervical lesions could be the result of transcriptional activity level and not an event that leads toward neoplastic progression. Further studies regarding the influence of DNA methylation on viral life cycle focused on E2 (early gene involved in viral transcription and replication) gene methylation. *In vitro* studies revealed that HPV16 URR (upstream regulatory region) methylation inhibit E2 protein capacity to bind DNA [298]. By looking at methylation status of E2BS (E2 binding sites) in immortalized epithelial cells from a HPV16 positive patient, Kim *et al*. found this region to be selectively hypomethylated in highly differentiated cell population, while heavily methy‐ lated in basal‐like differentiated cells. The conclusion was that methylation status of E2BS may vary during the viral life cycle, this giving an insight on E2 modulation function during progression of infection [298]. E2BS is more frequently found in a hypermethylated state in cervical lesions with extrachromosomal state of viral genome, while upon integration in the host genome, it was found to be hypomethylated, except the cases in which viral genome integrates as a concatemer, when only a small proportion are found hypomethylated and most of them hypermethylated [292].

All this experimentally observations conclude that HPV genome methylation status could hold a prognostic and progression value for cervical lesions.

## **6. Potential epigenetic biomarkers in cervical cancer**

The authors note that in HeLa cells, HPV18 genome chromatin histone modification status correlates with the occupancy of host transcriptional machinery specifically within the LCR [292]. E7 and E6 oncoproteins of hr‐HPVs appear to modulate host epigenetic machinery through their interplay with both DNA methylation enzymes as well as chromatin remodeling

Mirabello et al. [293] reported in 2013 that they found 3‐region in L1 strongly methylated in cancers and only in a small percentage in CIN I and CIN II lesions. In addition, the authors shown that methylation at certain CpG sites can indicate an evolution toward CIN II+ years

Evaluation of cervical samples from HPV positive women, presenting precancerous lesions or invasive ones, showing that the hypomethylation degree in LCR and E6 gene region increase with the increasing of lesion severity. These data convinced the authors to conclude that neoplastic transformation could be suppressed by hypermethylation, while hypomethylation

Using laser microdissection on different layers from samples with HPV‐infected lesions, Vinokurova and von Knebel Doeberitz [295] found dynamic changes in HPV16 LCR methyl‐ ation in the context of the viral life cycle. A decrease in methylation in the transcriptional enhancer region within the LCR was observed in terminally differentiated epithelial compart‐ ment and meanwhile an increase in methylation within the region of the LCR containing the

Another study highlighted heterogeneity of methylation status among patients, even in samples from the same patient. Methylation frequency was found to be approximately 30% in L1 region, less than in CpG islands around enhancer and promoter of HPV16. In most of the HPV genome sites, hypermethylation is associated better with carcinoma than with dysplastic

On the other hand, a study regarding methylation status in HPV18 immortalized cell lines (HeLa and C4‐1) and in samples from patients, determined a clonally heterogeneity of methylation status in different regions of viral genome. The clinical samples showed partial or total methylation in HPV enhancer region, while in asymptomatic patient's, samples were fully unmethylated. Viral promoter was reported to be methylated in tumor samples and in

These studies indicate that methylation status of viral oncogenes in cervical lesions could be the result of transcriptional activity level and not an event that leads toward neoplastic progression. Further studies regarding the influence of DNA methylation on viral life cycle focused on E2 (early gene involved in viral transcription and replication) gene methylation. *In vitro* studies revealed that HPV16 URR (upstream regulatory region) methylation inhibit E2 protein capacity to bind DNA [298]. By looking at methylation status of E2BS (E2 binding sites) in immortalized epithelial cells from a HPV16 positive patient, Kim *et al*. found this region to be selectively hypomethylated in highly differentiated cell population, while heavily methy‐ lated in basal‐like differentiated cells. The conclusion was that methylation status of E2BS may vary during the viral life cycle, this giving an insight on E2 modulation function during

accompanies or leads to progression toward cancer [294].

enzymes [159].

208 Human Papillomavirus - Research in a Global Perspective

before it happens.

early promoter was noted [295].

lesions [296].

cervical smears [297].

Cancer epigenome is currently in the researchers spotlight due to the fact that all the epigenetic changes that accompany cervical carcinogenesis can be exploited as biomarkers. Thus, once deciphered, the epigenetic peculiarities of cervical cancer might be used in the development of new alternatives for screening or for the assessment of prognostic [299]. On the other hand, the reversible nature of epigenetic alterations makes them attractive targets for new therapeu‐ tic approaches. Some of these discoveries have been proposed as investigation methods or resulted in new treatment approaches and commercial tests [300]. By far, the most studied epigenetic changes are the methylation patterns, especially the methylation markers of the host. Abnormal methylation of promoters of tumor suppressor genes is common in different type of cancers with the prospect of becoming a biomarker in oncology [301]. As the methyl‐ ation profile of these genes increases with the severity of cervical lesions, their status might be used as potential biomarker for early detection of cervical cancer disease [302]. For a better stratification of cervical cancer and precursor lesions, different specific methylation panels have been suggested [303]. Using DMH (differential methylation hybridization) technique and qPCR, Lai et al. [304] found in scrapings isolated from CIN3 lesions, a higher frequency of methylation for SOX1, NKX6‐1, PAX1, WT1, and LMX1A genes. Siegel et al. [305] demon‐ strated that aberrant methylation levels of DAPK1, RARB, WIF1, and SLIT2 might increase specificity to identify cervical cancer compared to viral testing alone. Also, the methylation patterns of GGTLA4 (183 bp) and ZNF516 (241 bp) genes were proposed in a patent as biomarkers for diagnosis of premalignant cervical lesions [306], while aberrant methylation of PAX1, PTPRR, SOX1, and ZNF582 promoters were suggested as markers for AC screening [307]. The studies that associate the methylation profile with cervical lesion severity have resulted in a commercial test (GynTect) [308]. GynTect assay is based on methylation‐specific PCR (MS‐PCR) and, if positive, detects specific methylated DNA sites in cervical smears. Manufacturer recommend the test for cervical cancer screening, allowing the triage of women over 30 years who tested positive for HPV. Moreover, GynTect may be performed using residual material from the HPV test. So, this methylation assays might be use as a secondary marker after HPV DNA testing in order to guide the subsequent clinical approach (referral to colposcopy or initiating a certain therapy) [309].

Other authors correlated changes in host DNA methylation with the development of drug resistance. Chen et al. [310] identified both genome‐wide and within individual loci changes in an oxaliplatin‐resistant cervical cancer cell line derived from SiHa cell line. The methylation of *Casp8AP2* gene resulted in increased drug resistance in different cells.

Masuda et al. [311] reported that aberrant methylation of Werner (WRN) gene that encode for a DNA helicase, increased the sensitivity to CPT‐11 (an inhibitor of DNA topoisomerase I). Iida et al. [312] reported aberrant hypermethylation of CHFR (checkpoint with forkhead and ring finger) in adenocarcinoma and HeLa cell line (immortalized with HPV18) and correlated this profile with lower sensitivity to anticancer therapy when compared to SSC, proposing this pattern in adenocarcinoma as a potential biomarker for sensitivity to paclitaxel. Therefore, the identification of methylation patterns associated with drug‐resistance might become a valuable tool in cervical treatment with demethylation agents that can revert this epigenetic change.

Regarding the methylation of viral DNA, data are still under debate. While some authors have suggested that it is a defense mechanism of the host cell, others considered it is a way by which the virus contributes to persistent infection. [313]. Other researchers considered that neoplastic transformation may be suppressed by HPV CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression [314]. Several authors proposed HPV16 L1 ORF methylation as a predictive marker for CIN3+ [315] and elevated levels of CpG 6367 L1HPV16 methylation as marker to predict future CIN2+ in women older than 28 years [293]. Also, Mirabello et al. [316] correlated elevated levels of CpG methylation in the L1, L2, E2/E4 with CIN3 or worse and data were confirmed by other papers [317]. Moreover, Went‐ zensen et al. [317] found differential methylation patterns in CIN3 patients with multiple infections thus suggesting a possible way to identify the causal type of HPV.

Cervical carcinogenesis is accompanied also by altered expression of methyltransferases. For therapeutic purpose, Hamamoto et al. [318] had synthesized double‐stranded molecules that inhibit the expression of SUV39H2 (suppressor of variegation 3–9 homolog 2) gene. This gene encodes a HMT that methylate the H3K9 lysine residue and its hyperexpression correlates with carcinogenesis. The silencing of CHFR through its promoter hypermethylation leads also to the activation of DNA methyltransferases (including DNMT1). Different patterns of demethylation obtained by silencing DNMT1 in experimental model (HeLa and SiHa cell lines) indicate the inhibition of DNMT1 as a target for the treatment of cervical cancer with HPV18 infection [312]. These results showed that infection with different HPV genotypes differently interfere with epigenetic mechanisms.

Molecular investigations of cervical tumors and cell lines immortalized with HPV have shown that, from all ncRNA molecules, miRNAs profile is significantly changed when compared to normal tissue, even in early stages of carcinogenesis [309]. Zheng et al. [319] provided data that viral E6 and E7 oncoproteins deregulate the expression of several miRNAs via the E6‐p53 and E7‐pRb pathways. In turn, miRNAs may influence the expression of HPV genes by targeting viral RNA transcripts, these recommended miRNAs as new biomarkers in cervical screening. The panel of four circulating miRNAs (miR‐16‐2\*, miR‐195, miR‐2861, miR‐49) [320] are suggested as predictive biomarkers for the prognosis of cervical cancer patients, upregulate expression of serum miR‐205 [321] and serum pattern of miR‐29a and miR‐200a may indicate tumor histological grade and progression stage [322]. Li et al. [323] found lower levels of miR‐ 218 levels in patients with high‐risk HPV comparing with control or those with low‐risk or intermediate‐risk HPV. In Chinese population, Zhou et al. [324] reported a good correlation between a miR‐218 polymorphism and its target laminin 5B3 in cervical cancer invasiveness. Epigenetic changes through methylation of miRNAs might correlate with cervical disease. A panel of three miRs (miR‐149, miR‐203, and miR‐375) was found hypermethylated in HPV‐ positive cell lines [76] and miR‐203 and miR‐375 hypermethylation correlated with uterine precancerous lesions [325].

miRNAs might be also used for cervical cancer therapy. On animal model, Liu et al. [227] who noticed an inverse correlation between the expression of miR‐143 and Bcl2 suggested the possibility of a therapeutic approach by targeting this pathway. Also, miRNAs might modulate the sensitivity to chemotherapy. For example, miR‐375 might be a therapeutic target in paclitaxel‐resistance of cervical cancer cells [326], while miR‐155 and miR‐281 increase sensitivity to cisplatin [325]. Therefore, miRNA deregulation may become a target of the investigations for evaluating the effectiveness of treatments in cervical cancer [327].

## **7. Conclusions**

Masuda et al. [311] reported that aberrant methylation of Werner (WRN) gene that encode for a DNA helicase, increased the sensitivity to CPT‐11 (an inhibitor of DNA topoisomerase I). Iida et al. [312] reported aberrant hypermethylation of CHFR (checkpoint with forkhead and ring finger) in adenocarcinoma and HeLa cell line (immortalized with HPV18) and correlated this profile with lower sensitivity to anticancer therapy when compared to SSC, proposing this pattern in adenocarcinoma as a potential biomarker for sensitivity to paclitaxel. Therefore, the identification of methylation patterns associated with drug‐resistance might become a valuable tool in cervical treatment with demethylation agents that can revert this epigenetic

Regarding the methylation of viral DNA, data are still under debate. While some authors have suggested that it is a defense mechanism of the host cell, others considered it is a way by which the virus contributes to persistent infection. [313]. Other researchers considered that neoplastic transformation may be suppressed by HPV CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression [314]. Several authors proposed HPV16 L1 ORF methylation as a predictive marker for CIN3+ [315] and elevated levels of CpG 6367 L1HPV16 methylation as marker to predict future CIN2+ in women older than 28 years [293]. Also, Mirabello et al. [316] correlated elevated levels of CpG methylation in the L1, L2, E2/E4 with CIN3 or worse and data were confirmed by other papers [317]. Moreover, Went‐ zensen et al. [317] found differential methylation patterns in CIN3 patients with multiple

Cervical carcinogenesis is accompanied also by altered expression of methyltransferases. For therapeutic purpose, Hamamoto et al. [318] had synthesized double‐stranded molecules that inhibit the expression of SUV39H2 (suppressor of variegation 3–9 homolog 2) gene. This gene encodes a HMT that methylate the H3K9 lysine residue and its hyperexpression correlates with carcinogenesis. The silencing of CHFR through its promoter hypermethylation leads also to the activation of DNA methyltransferases (including DNMT1). Different patterns of demethylation obtained by silencing DNMT1 in experimental model (HeLa and SiHa cell lines) indicate the inhibition of DNMT1 as a target for the treatment of cervical cancer with HPV18 infection [312]. These results showed that infection with different HPV genotypes differently

Molecular investigations of cervical tumors and cell lines immortalized with HPV have shown that, from all ncRNA molecules, miRNAs profile is significantly changed when compared to normal tissue, even in early stages of carcinogenesis [309]. Zheng et al. [319] provided data that viral E6 and E7 oncoproteins deregulate the expression of several miRNAs via the E6‐p53 and E7‐pRb pathways. In turn, miRNAs may influence the expression of HPV genes by targeting viral RNA transcripts, these recommended miRNAs as new biomarkers in cervical screening. The panel of four circulating miRNAs (miR‐16‐2\*, miR‐195, miR‐2861, miR‐49) [320] are suggested as predictive biomarkers for the prognosis of cervical cancer patients, upregulate expression of serum miR‐205 [321] and serum pattern of miR‐29a and miR‐200a may indicate tumor histological grade and progression stage [322]. Li et al. [323] found lower levels of miR‐ 218 levels in patients with high‐risk HPV comparing with control or those with low‐risk or intermediate‐risk HPV. In Chinese population, Zhou et al. [324] reported a good correlation

infections thus suggesting a possible way to identify the causal type of HPV.

interfere with epigenetic mechanisms.

210 Human Papillomavirus - Research in a Global Perspective

change.

All these data underline the importance of epigenetic modification in tumor development and cervical cancer risk assessment. Epigenetic alterations could be used as biomarkers for the prognosis and evolution of the disease and for therapy response prediction. New techniques in epigenetic investigations may yield better detection systems in order to identify new and sensitive biomarkers that might contribute to improved screening assays, new therapeutic approaches, and prediction biomarkers. The reversible nature of epigenetic alterations provides new opportunities in the development of therapeutic agents targeting epigenetic modification in oncogenesis.

### **Conflict of interest**

The authors declare no conflict of interest.

## **Acknowledgements**

### **POSCCE SMIS 14049**

This work is supported by The Executive Agency for Higher Education, Research, Develop‐ ment and Innovation Funding (UEFISCDI) under grant: PNII‐RU‐TE‐2014‐4‐2502 (Onco‐ NuRD).

## **Author details**

Adriana Plesa\* , Iulia V. Iancu, Anca Botezatu, Irina Huica, Mihai Stoian and Gabriela Anton

\*Address all correspondence to: adrianaplesa@yahoo.com

"Stefan S. Nicolau" Institute of Virology, Bucharest, Romania

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0248‐3


**HPV Infections, Related Diseases and Cancers - Prevention and Control - Human Papillomavirus and Cancer - Immunological Consequences of MHC Class 1 Down - Regulation**

## **Pathogenesis of Human Papillomavirus – Immunological Responses to HPV Infection**

G. Hossein Ashrafi and Nadia Aziz Salman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63965

### **Abstract**

Papillomavirus is an oncogenic virus which infects mucosal and cutaneous epithelia where it induces benign hyperproliferative lesions. Few studies have been conducted on the causative factors associated with the development of cancer. Infections by highrisk human papillomaviruses (HPVs) have been implicated as causative agents in a variety of cancers such as anogenital, and head and neck cancers. HPVs appear to have evolved mechanisms resulting in escape from host immune surveillance and delay of resolution of infection. The HPV E5 oncoprotein is one of the possible effectors that allows the virus to escape from host immune system through the downregulation of surface classical major histocompatibility complex class I (MHC I) and not the nonclassical MHC I. Lack of classical MHC I in infected cells expressing E5 would allow evasion of cytotoxic T lymphocytes (CTLs) killing and thus establishment and persistence of viral infection.

In this chapter we discuss the process of immunomodulation by HPV and review our recent discoveries on the association of HPV with cancers and its implication in medicine.

**Keywords:** cancer, HR-HPV, E5, MHC I, CTL

## **1. Introduction**

The global cancer burden is markedly increasing and is thus a leading cause of death, second only to cardiovascular disease [1–3]. Based on the most recent available data collected by the international agency for cancer, an estimated 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012, compared with 12.7 million and 7.6 million, respec‐ tively, in 2008 [4].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The human body is dependent on regulated cell division and elimination which forms homeostasis within the body. Certain genes play vital roles in regulating the cell cycle. These genes are grouped into three categories, tumour suppressor genes (TSGs), proto-oncogenes and DNA repair genes. Any alteration in these groups of genes downregulates the cell control mechanisms and ultimately causes cancer cell development and malignant transformation [5]. Genetic changes in the progression of cancer typically affect two different types of genes, oncogenes (e.g., *Ras*) and TSGs (e.g., P53 and pRB). Both of the oncogenes and TSGs encode many kinds of proteins that play a key role in cancer induction. These genes control cell growth and proliferation, and mutations in these genes can contribute to the development of cancer.

Despite of the evolving medical significance of cancers, few studies have been conducted to identify the possible risk factors that implicated in the initiation of cancer. However, there are well-established risk factors that have been identified for its association with the development of cancer such as clinical, genetic and epidemiological factors. In addition, biological agents have been implicated in various cancers including viruses, bacteria and parasites.

Viruses have been the most studied in their relation to tumour formation. They are thought to be associated with 15–20% of all human cancers worldwide of which about 80% are cancers of the cervix and liver [6, 7]. Viruses have evolved multiple strategies to transform host cell. One common route is to alter the expression of cellular genes by integration of the viral genome into the cellular DNA. Viruses also help cause malignancy by introducing an oncogene into a cell to disrupt the regulation of cell division [8–10].

Infectious agents have been implicated, either as direct carcinogens or as promoters. Viral infections, in particular, human papillomaviruses (HPVs) are recognised as carcinogenic agents in humans and are responsible for a significant share of the global cancer burden [7, 11, 12].

HPVs are small double-stranded DNA oncogenic viruses of approximately 8 kbp. HPVs are ubiquitous in the human population, and occasionally infection leads to cervical cancer. Although the body is working to get the infection under control, HPVs infect and disturb cutaneous and mucosal epithelial cells of the anogenital tract, hands or feet which can lead to a variety of diseases with a range of severities depending on the types of HPV infection.

To date, over 100 different types of HPV have been identified, and about one-third of these infect epithelial cells in the genital tract. The type HPV family is divided into two categories: low-risk HPVs and high-risk HPVs (HR-HPVs). The low-risk HPV types such as HPV 6 and HPV 11 commonly cause benign genital warts (condylomas). These lesions can regress even without treatment [13], due to cell-mediated immune responses [14]. While the high-risk types of HPV which include HPV 16, 18, 31, 35 and 45 are associated with the development of anogenital cancers and found in up to 99% of all cervical carcinomas [15, 16]. These HR-HPV types cause benign genital epithelial hyperproliferative lesions, such as low-grade premalig‐ nant cervical intraepithelial neoplasia (CIN I). In most premalignant cases, lesions can regress even without treatment. However, in a limited number of cases, the lesions persist or progress to invasive cancer due to the lack or ineffective immunological responses.

It has been reported that the low-grade CIN I would progress to high-grade lesion (CIN III) and eventually invasive cervical cancer [17]. Consequently, the associations of the HPV infection in the development of cancer are an area of ongoing interest.

## **2. The host immune evasion by HPV**

The human body is dependent on regulated cell division and elimination which forms homeostasis within the body. Certain genes play vital roles in regulating the cell cycle. These genes are grouped into three categories, tumour suppressor genes (TSGs), proto-oncogenes and DNA repair genes. Any alteration in these groups of genes downregulates the cell control mechanisms and ultimately causes cancer cell development and malignant transformation [5]. Genetic changes in the progression of cancer typically affect two different types of genes, oncogenes (e.g., *Ras*) and TSGs (e.g., P53 and pRB). Both of the oncogenes and TSGs encode many kinds of proteins that play a key role in cancer induction. These genes control cell growth and proliferation, and mutations in these genes can contribute to the development of cancer.

Despite of the evolving medical significance of cancers, few studies have been conducted to identify the possible risk factors that implicated in the initiation of cancer. However, there are well-established risk factors that have been identified for its association with the development of cancer such as clinical, genetic and epidemiological factors. In addition, biological agents

Viruses have been the most studied in their relation to tumour formation. They are thought to be associated with 15–20% of all human cancers worldwide of which about 80% are cancers of the cervix and liver [6, 7]. Viruses have evolved multiple strategies to transform host cell. One common route is to alter the expression of cellular genes by integration of the viral genome into the cellular DNA. Viruses also help cause malignancy by introducing an oncogene into a

Infectious agents have been implicated, either as direct carcinogens or as promoters. Viral infections, in particular, human papillomaviruses (HPVs) are recognised as carcinogenic agents in humans and are responsible for a significant share of the global cancer burden [7, 11,

HPVs are small double-stranded DNA oncogenic viruses of approximately 8 kbp. HPVs are ubiquitous in the human population, and occasionally infection leads to cervical cancer. Although the body is working to get the infection under control, HPVs infect and disturb cutaneous and mucosal epithelial cells of the anogenital tract, hands or feet which can lead to a variety of diseases with a range of severities depending on the types of HPV infection.

To date, over 100 different types of HPV have been identified, and about one-third of these infect epithelial cells in the genital tract. The type HPV family is divided into two categories: low-risk HPVs and high-risk HPVs (HR-HPVs). The low-risk HPV types such as HPV 6 and HPV 11 commonly cause benign genital warts (condylomas). These lesions can regress even without treatment [13], due to cell-mediated immune responses [14]. While the high-risk types of HPV which include HPV 16, 18, 31, 35 and 45 are associated with the development of anogenital cancers and found in up to 99% of all cervical carcinomas [15, 16]. These HR-HPV types cause benign genital epithelial hyperproliferative lesions, such as low-grade premalig‐ nant cervical intraepithelial neoplasia (CIN I). In most premalignant cases, lesions can regress even without treatment. However, in a limited number of cases, the lesions persist or progress

to invasive cancer due to the lack or ineffective immunological responses.

have been implicated in various cancers including viruses, bacteria and parasites.

cell to disrupt the regulation of cell division [8–10].

244 Human Papillomavirus - Research in a Global Perspective

12].

The papillomaviruses are small double-stranded DNA viruses which belong to the papillo‐ maviridae family [6]. HPVs infect cutaneous or mucosal epithelial cells initiating benign or cancerous lesions that depend on the HPV types. The lifecycle, oncogenic characteristics and molecular-based evidence of HR-HPVs are suggestive of a causal role for cancer. HPV infections are normally cleared by the immune system; however, the persistence of HPV could trigger a progression to malignant lesion in the presence of other risk factors. For instance, it is well documented that the persistent infection of the cervix with HR-HPV types 16 and 18 is an initiating event of the cervical cancer [18, 19]. Therefore, the establishment, persistence of HR-HPV infection and evasion of the host immune system are necessary for premalignant lesions to initiate and progress towards squamous carcinoma [20–22].

The host immune response mechanism plays a significant role in controlling and limiting HPV infection. The suppression of HPV-induced lesion depends on the host inflammatory reaction and penetration of lymphocytes to the infected tissue [14]. Thus, the prevalence of HPVinduced lesions is higher in immunosuppressed individuals such as transplant recipients or human immunodeficiency virus (HIV)-infected patients [23, 24].

Lack of HPV clearance and persistence of viral infection for many months are necessary before the onset of an immune response. The reasons of this fact are still unknown. And perhaps one of the most important ongoing questions in the field of papillomavirus research is the latency of the host immune response in eliminating the virus in immunocompetent hosts as well as immunosuppressed hosts.

The nonlytic feature of HPV is one of the explanations for evading the recognition of HPV infection. HPV does not lyse the infected cell or cause viremia, and this will reduce the exposure of viral antigen to cell-mediated immunity and consequent lack of inflammation. HPV life cycle characterised by the physical evasion from the immune cells' recognition through HPV restoration and protection within infected cells' nuclei [25, 26]. Additionally, HPV has the ability to downregulate major histocompatibility complex class I (MHC I) and disrupt the interferon (IFN) pathway. HPV facilitates this mechanism using early oncoproteins, E5, E6 and E7, which have the ability to interfere and actively participate to the downregulation of host immune system. The role of E6 and E7 is to inhibit the production of IFN in natural killer (NK) cells or the expression of transporter associated with antigen processing (TAP) [27, 28].

E5 oncoprotein downregulates surface MHC class I by retaining it in the Golgi apparatus (**Figures 1** and **2**) [20, 29]. Downregulation of MHC class 1 has been observed with E5 from different PVs, including HPV-85 and HPV-16 (**Figure 3**), indicating that key functions of E5 are conserved between the PV species and HPV types [21].

**Figure 1.** The expression of MHC I in control and transformed PalF cells using Define FACS as "Fluorescence-activated cell sorting" (**A and B**), control cells (**C and D**), 4-E5 transfected cells (**E and F**) and 1-E5 transfected cells (**G and H**) were incubated with anti MHC 1 antibody. Surface MHC I (**A, C, E and G**) and total MHC I (**B, D, F and H**) [**29**].

**Figure 2. Visualisation of the GA and MHC I**. HaCaT cells carrying empty vectors or cells expressing HPV-16 E5 were costained with anti-HLA class I antibody (mAb W6/32) and anti-golgi GM130 antibody (mAb 4A3) and analysed using Leica TCS SP2 fluorescence confocal microscopy. Representative cells are shown. (**A**) Control HaCaT cells carry‐ ing either pcDNA or pL2 empty vector. (**B**) HeCaT cells expressing HPV-16 E5 in either pcDNA (pc-16E5) or pL2 (pL2-16E5). N = nucleus [**20**].

**Figure 3.** Total and surface expression of MHC-I using FACS analysis in normal HaCaT cells transfected with pcDNA (empty vectors), pcHPV16E5, HPV83-E5a (antisense orientation) and HPV83-E5s (sense orientation) [**21**].

**Figure 1.** The expression of MHC I in control and transformed PalF cells using Define FACS as "Fluorescence-activated cell sorting" (**A and B**), control cells (**C and D**), 4-E5 transfected cells (**E and F**) and 1-E5 transfected cells (**G and H**) were incubated with anti MHC 1 antibody. Surface MHC I (**A, C, E and G**) and total MHC I (**B, D, F and H**) [**29**].

**Figure 2. Visualisation of the GA and MHC I**. HaCaT cells carrying empty vectors or cells expressing HPV-16 E5 were costained with anti-HLA class I antibody (mAb W6/32) and anti-golgi GM130 antibody (mAb 4A3) and analysed using Leica TCS SP2 fluorescence confocal microscopy. Representative cells are shown. (**A**) Control HaCaT cells carry‐ ing either pcDNA or pL2 empty vector. (**B**) HeCaT cells expressing HPV-16 E5 in either pcDNA (pc-16E5) or pL2

(pL2-16E5). N = nucleus [**20**].

246 Human Papillomavirus - Research in a Global Perspective

E5 is the smallest HPV oncogenic protein that is located in the membranes of the endoplasmic reticulum (ER) and Golgi apparatus of the transformed cells. This hydrophobic protein is a structure of 83 amino acids in HR-HPV type 16, is expressed before the onset of viral replica‐ tion. E5 contribute to cell transformation through interaction with several cellular proteins, including the epidermal growth factor receptor (EGF-R), the human receptor for colony stimulating factors (CSF-1). E5 may also reduce the processing and presentation of viral antigen through the interaction with 16 kDa subunit of the vacuolar H+ -ATPase acidification of endosome [30].

The human MHC class I molecules are known as human leukocyte antigen (HLA) system, which have a critical function in the recognition of virally infected cells. The proteasomes and other cytoplasmic proteases are playing a role in viral protein degradation into short chain peptides of 8–10 amino acids long. HLA molecules and intracellular viral antigenic peptides complex are transported through the Golgi apparatus to the cell surface of infected cells where it is presented and recognised by the cytotoxic CD8+ T cells. The activated cytotoxic T lym‐ phocytes (CTLs) are able to destroy the infected cell through the mechanism of apoptosis that mediated either by granulate exocytosis (perforin and granzymes) or by Fas-Fas ligand interaction [26].

High frequency of virus-specific CTLs is a characteristic of persistent viral infection such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), however low frequency of HPVspecific CTL has been detected in CIN III and cervical cancer patients [31, 32].

CD4+ T helper lymphocytes (Ths) are able to identify foreign antigens that presented on MHC class II molecules (antigen-MHC II complex). MHC class II molecules are mostly expressed on professional antigen presenting cells (APCs) such as dendritic cells, macrophages and B cells, but they can also be expressed on the epithelial cells (target for HPV) by IFN treatment.

T helper cells are capable of secreting cytokines that allow the proliferation and maintenance of cytotoxic lymphocytes. Additionally, they play a role in activation of B cells for antibody production, as well as dendritic cells for antigen presentation. Th cells potentially have an important role in cell-mediated immunity against HPV infection. The reactivity of HPVrestricted Th cells was found in patients with a persistent papillomavirus infection [33, 34].

While E6 and E7 are presented throughout the course of the HPV infection, their functions are necessary for the maintenance of a transformed status. Expression of E5 takes place in the early stage of papillomavirus infection and in the deep layers of the infected epithelium. Ashrafi et al. [29] were the first to report that E5 protein of HPV downregulates the classical HLA (A and B) but not the nonclassical HLA-E. This potentially allows the cell to escape CTL and also NK cells' killing [20].

The downregulation of MHC class I by E5 oncoprotein allows the infected cell to evade cellmediated immune response and this potentially enables other HPV oncoproteins in the establishment and persistence of virus infection. Interestingly, it has been reported that the oncogenic E5 also inhibits the Fas receptor [35] and HLA II [36]. This strongly indicates that E5 might have additional major role in negatively regulating the immune response.

The downregulation of MHC I is an imperative mechanism to evade CTL-mediated immune clearance, however, the lack of surface MHC I will activate NK cells to attack and destroy the infected cells. Human NK cells express surface receptors (NKRs) that interact with HLA class I molecules, including killer cell immunoglobulin-like receptors (KIRs) that mainly recognise classical HLA-C and also C type lectin receptors which identify nonclassical HLA-E molecule. In the absence of classical HLA-C and non-classical HLA-E, NK-mediated cell lysis would be inhibited due to the recognition of MHC class I molecules by their inhibitory receptors. Consequently, certain viruses, including HIV and CMV, have the ability to escape both CTL and NK cells' killing. HIV-negative proteins (Nef) and CMV US3/UL40 proteins have evolved to selectively downregulate HLA (A and B), the main presenters of peptides to CTLs, but not HLA-C or nonclassical HLA-E [37, 38].

## **3. HR-HPVs and cancers**

HR-HPV types are considered as the most important aetiological factors for many types of cancers such as cervical cancer. The risk of cervical cancer has increased in parallel with the incidence of certain genotypes of HR-HPV. Therefore, the presence of these genotypes indicates a significant risk factor for the initiation and progression of almost 90% of cervical cancer cases [18].

Despite the medical significance of HR-HPV infection, there is still a lack of information on the incidence of cancers that are caused by different HR-HPV genotypes in different popula‐ tions. An investigation on the incidence and distribution of HR-HPV genotypes in cervical cancer patients confirmed the presence of additional high-risk types (HPV-45 and HPV-39) other than common types (HPV-16 and HPV-18) [19].

Being a sexually contagious virus, HPV virus has the ability to spread through sexual and skinto-skin contact. HR-HPVs have also been found to be the causative agents for almost up to half of vaginal, penile, anal and oral cancers. These findings suggest that HR-HPV virions might be spread from the original infected site to other organs and lead to cancer development in various organs [18].

To date, studies on the role of HPV in breast carcinogenesis have generated considerable controversies and it is still not clear whether HPV infection is implicated in breast cancer pathogenesis [39]. HPV infection is a sexually transmissible disease, and most breast cancers originate from mammary duct epithelia. Therefore, the relationship between HPV and breast cancer is imperative for many reasons. The exposure of the mammary ducts to the external environment increases the risk of HPV infection. Our unpublished data have shown the presence of HR-HPV types of viral DNA other than 16 and 18 in freshly collected human breast cancer tissue and this provides a solid basis to advance research in a crucial health problem affecting women.

These initial findings support the association of HPV and breast cancer and highlight possible causative agents of breast cancer. Therefore, further research is required to investigate whether HR-HPV infection plays a role in the pathogenesis of breast cancer. The information gained will pave the way to better awareness of breast cancer risk factors other than those recognised to date.

## **4. Conclusions**

CD4+ T helper lymphocytes (Ths) are able to identify foreign antigens that presented on MHC class II molecules (antigen-MHC II complex). MHC class II molecules are mostly expressed on professional antigen presenting cells (APCs) such as dendritic cells, macrophages and B cells, but they can also be expressed on the epithelial cells (target for HPV) by IFN treatment.

T helper cells are capable of secreting cytokines that allow the proliferation and maintenance of cytotoxic lymphocytes. Additionally, they play a role in activation of B cells for antibody production, as well as dendritic cells for antigen presentation. Th cells potentially have an important role in cell-mediated immunity against HPV infection. The reactivity of HPVrestricted Th cells was found in patients with a persistent papillomavirus infection [33, 34]. While E6 and E7 are presented throughout the course of the HPV infection, their functions are necessary for the maintenance of a transformed status. Expression of E5 takes place in the early stage of papillomavirus infection and in the deep layers of the infected epithelium. Ashrafi et al. [29] were the first to report that E5 protein of HPV downregulates the classical HLA (A and B) but not the nonclassical HLA-E. This potentially allows the cell to escape CTL and also NK

The downregulation of MHC class I by E5 oncoprotein allows the infected cell to evade cellmediated immune response and this potentially enables other HPV oncoproteins in the establishment and persistence of virus infection. Interestingly, it has been reported that the oncogenic E5 also inhibits the Fas receptor [35] and HLA II [36]. This strongly indicates that

The downregulation of MHC I is an imperative mechanism to evade CTL-mediated immune clearance, however, the lack of surface MHC I will activate NK cells to attack and destroy the infected cells. Human NK cells express surface receptors (NKRs) that interact with HLA class I molecules, including killer cell immunoglobulin-like receptors (KIRs) that mainly recognise classical HLA-C and also C type lectin receptors which identify nonclassical HLA-E molecule. In the absence of classical HLA-C and non-classical HLA-E, NK-mediated cell lysis would be inhibited due to the recognition of MHC class I molecules by their inhibitory receptors. Consequently, certain viruses, including HIV and CMV, have the ability to escape both CTL and NK cells' killing. HIV-negative proteins (Nef) and CMV US3/UL40 proteins have evolved to selectively downregulate HLA (A and B), the main presenters of peptides to CTLs, but not

HR-HPV types are considered as the most important aetiological factors for many types of cancers such as cervical cancer. The risk of cervical cancer has increased in parallel with the incidence of certain genotypes of HR-HPV. Therefore, the presence of these genotypes indicates a significant risk factor for the initiation and progression of almost 90% of cervical

Despite the medical significance of HR-HPV infection, there is still a lack of information on the incidence of cancers that are caused by different HR-HPV genotypes in different popula‐

E5 might have additional major role in negatively regulating the immune response.

cells' killing [20].

HLA-C or nonclassical HLA-E [37, 38].

248 Human Papillomavirus - Research in a Global Perspective

**3. HR-HPVs and cancers**

cancer cases [18].

Identification of HPV pathogenesis offers means of therapeutic intervention targeted against HPV oncoproteins (E5, E6 and E7) which will facilitate early lesion eradication. This will also provide results central to our understanding of HPV pathogenesis and help elucidate early events in HPV infection that may determine persistence and disease development.

Moreover, our findings on the presence of high-risk HPV-39 and HPV-45 types in cervical cancer other than types 16 and 18 [19] and the presence of different types of HR-HPV in breast cancer will postulate a need for further assessment of the influence of current prophylactic vaccination programs that is protective against the two most common oncogenic papilloma‐ viruses, HPV-16 and HPV-18, but not against other high-risk mucosal HPVs detected in our studies.

Viral carcinogenesis and cancer prevention are rapidly developing sectors of this field, and the future translation of this chapter lead to a faster resolution of HPV infection, and with obvious advantages for all HPV-affected patients, and in particular for individuals affected by early HPV-related cancer diseases.

## **Author details**

G. Hossein Ashrafi1\* and Nadia Aziz Salman2

\*Address all correspondence to: h.ashrafi@kingston.ac.uk

1 Cancer Study Group, SEC Faculty, Kingston University London, Kingston Upon Thames, UK

2 School of Life Science, Pharmacy and Chemistry, SEC Faculty, Kingston University Lon‐ don, Kingston Upon Thames, UK

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## **Human Papillomavirus in Head and Neck Cancer**

Makbule Tambas, Musa Altun and Deniz Tural

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63014

### **Abstract**

Throughout the last three decades, there has been a notable shift in the epidemiology of head and neck cancer (HNC) worldwide. A rapidly spreading subtype of HNCs is caused by human papillomavirus (HPV) infection. HPV-related cancers are now considered to constitute 30–65% of all HNC cases and 50–80% of oropharyngeal cancers. HPVpositive oropharyngeal cancers have a unique demographic profile and tumor biology characteristics. HPV-associated patients predominantly consist of younger men with better performance status and fewer comorbid diseases. They have better dentition, higher numbers of oral sex partners, and use less amount of tobacco or alcohol, higher amount of marijuana compared with HPV-negative patients. In addition, patients with HPVpositive tumors have a 60–80% reduced mortality rates, a finding that was confirmed by multiple trials and led to several ongoing deintensification studies. This chapter describes epidemiologic features of HPV-positive HNC, risk factors for HPV infection and HPVassociated oropharyngeal cancer, HPV detection methods, mechanisms of carcinogene‐ sis and improved treatment response, and the impact of HPV status on clinical outcome as well as deintensification approaches and potential of vaccination.

**Keywords:** head and neck cancer, human papillomavirus, epidemiology, carcinogene‐ sis, treatment

### **1. Introduction**

Head and neck cancer (HNC) involves a wide field of tumors that originate from the skull base to the clavicles including the orbits, paranasal sinuses, nasopharynx, oropharynx, hypophar‐ ynx, oral cavity, and larynx. Worldwide, the incidence of HNC accounts for more than half a million each year, 5% of all cancer cases, with being the fifth most common cancer in the world [1, 2]. The distribution of HNC varies around world, such that it is 3–4% of all cancer diag‐ nosed in North America and the Europe, whereas HNC consists of 30% of all cancer cases in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

men in India [3]. Yet, around 300,000 people die from HNC that can be seen in the global picture of the disease [4].

The well-known risk factors for HNC comprise tobacco, alcohol, poor oral health, human papillomavirus (HPV) infection (for oropharyngeal cancer), and Epstein-Barr virus (EBV) infection (for nasopharyngeal carcinoma) [5, 6]. Traditionally, HNC was related to tobacco use and alcohol exposure, and the synergistic increased risk with the combination of them [7]. HNCs are most common among 50–60 year-old individuals who are heavy smokers and alcohol users with lower socioeconomic status [8]. Additionally, the squamous cell carcinoma constitutes more than 90% of histological subtype of HNC [1].

## **2. Epidemiology**

### **2.1. Changing trends in epidemiology of HPV-associated HNCs**

Throughout the last three decades, there has been a notable shift in the epidemiology of HNC worldwide. The decrease in tobacco consumption has resulted in an entire reduction in the incidence of HNC during the past 30 years [9]. Since smoking maintains as the primary risk factor for the oral cavity, larynx, and hypopharynx, this declining trend is marked for these sites. However, up to 25% of all HNCs recently diagnosed are not related to tobacco use. A rapidly spreading subtype of HNCs is caused by HPV infection [10].

First suggested in 1983 by Syrjanen, HPV was reported as an initiative factor of HNC owing to its oncogenic potential, the parallel clinical characteristics in oral and genital damages, epithelia similarities, and HPV affinity for epithelial cells [11]. The evidence for a significant correlation was insufficient, until recently. In the 2000s, several reports, mainly from Sweden, established that a remarkable ratio of tonsillar cancers included HPV DNA. The increases by 2.8- and 2.9-fold were detected in the incidence of tonsillar cancer and ratio (23–68%) of HPVpositive tonsillar cancer between 1970 and 2002, in Sweden, in 2006 [12]. Immediately after this, in 2007, an arising HPV epidemic correlating with oropharyngeal SCC was reported in the United States [13]. The striking increases up to sevenfold in both HPV-positive tonsillar and base of tongue were demonstrated during the periods of 1970–2007 and 1998–2006 in 2009 and 2010 from Sweden, respectively [14, 15]. Following Dalianis group definition of "epidemic of viral-induced carcinoma," due to observation that most of the tonsillar carcinomas were HPV-associated in 2009 [14], a retrospective analysis of clinical trial material detected that HPV-related oropharyngeal cancer rate was 64% in the USA [16]. Similar retrospective analyses on oropharyngeal tumor samples revealed an increase in HPV incidence from 23, 28 to 57% from 1970s, 1980s to 1990s, respectively. Also, a constant trend in the increase was detected for recent term (68, 77, 93% incidence rates during 2000–2002, 2003–2005, 2006–2007, respectively [14]. Similarly, HPV association rate in oropharyngeal cases was increased from 16% to 73%, from 1984–1989 to 2000–2004 in the United States [17]. Furthermore, during the same periods, reports delivering similar striking rises in both oropharyngeal and HPV-positive oropharyngeal cancers have accumulated from several Western countries [3, 12–15, 17–23].

The oropharynx is distinctively sensitive to HPV, and as high as 70% of oropharyngeal cancers in the USA are HPV-related oropharyngeal squamous cell carcinomas [24]. The past 30-year Surveillance, Epidemiology, and End Results (SEER) population-based data were analyzed by Chaturvedi et al, and they evaluated the incidence trends of HPV-related and -unrelated HNC. They demonstrated a significant decline (1.85% annual reduction) in the incidence of HNC in HPV-unrelated fields (hypopharynx, oral cavity, larynx) and a remarkable rise (0.8% annual increase) in the incidence of HNC in HPV-associated oropharyngeal regions during the same period [9]. In addition, the incidence of hypopharyngeal, oral cavity, and laryngeal carcinomas notably reduced due to smoking and alcohol consumption declining in many countries [25]. On the contrary, the oropharyngeal carcinoma incidence has been reported to be increased in many countries including the UK [26], Canada [27], Australia [28], Norway [29], Denmark [30], and the Netherlands [20] over the last two decades, besides Sweden [12] and the USA [9]. HPVrelated cancers are now considered to constitute 30–65% of all HNC cases and 50–80% of oropharyngeal cancers [9, 16]. HPV-positive HNC, which has distinctive epidemiologic and prognostic features [5–14] has become an expanding public health issue, is expected to be the main factor for HNC development in the forthcoming period [17].

Currently, the highest HPV-positive HNC incidence rates have been reported in Sweden and the USA. The genital HPV infection prevalence, sexual habits, and smoking and alcohol use may affect the population-specific HPV-positive HNC incidences. The focus of HPV-related malignancies has been shifted from anogenital cancers developing predominantly in women to oropharyngeal cancers developing mainly in men due to the fact that men were reported to have >70% HPV-positive oropharyngeal cancer cases [9, 12, 15, 16, 20–22]. By 2020, the number of HPV-mediated oropharyngeal patients per year is reflected to outnumber HPV-related cervical cancer cases in the USA [17]. The HPV-mediated oropharyngeal cancer has become an epidemic of our era [31].

### **2.2. HPV positivity in non-oropharyngeal sites of HNC**

men in India [3]. Yet, around 300,000 people die from HNC that can be seen in the global picture

The well-known risk factors for HNC comprise tobacco, alcohol, poor oral health, human papillomavirus (HPV) infection (for oropharyngeal cancer), and Epstein-Barr virus (EBV) infection (for nasopharyngeal carcinoma) [5, 6]. Traditionally, HNC was related to tobacco use and alcohol exposure, and the synergistic increased risk with the combination of them [7]. HNCs are most common among 50–60 year-old individuals who are heavy smokers and alcohol users with lower socioeconomic status [8]. Additionally, the squamous cell carcinoma

Throughout the last three decades, there has been a notable shift in the epidemiology of HNC worldwide. The decrease in tobacco consumption has resulted in an entire reduction in the incidence of HNC during the past 30 years [9]. Since smoking maintains as the primary risk factor for the oral cavity, larynx, and hypopharynx, this declining trend is marked for these sites. However, up to 25% of all HNCs recently diagnosed are not related to tobacco use. A

First suggested in 1983 by Syrjanen, HPV was reported as an initiative factor of HNC owing to its oncogenic potential, the parallel clinical characteristics in oral and genital damages, epithelia similarities, and HPV affinity for epithelial cells [11]. The evidence for a significant correlation was insufficient, until recently. In the 2000s, several reports, mainly from Sweden, established that a remarkable ratio of tonsillar cancers included HPV DNA. The increases by 2.8- and 2.9-fold were detected in the incidence of tonsillar cancer and ratio (23–68%) of HPVpositive tonsillar cancer between 1970 and 2002, in Sweden, in 2006 [12]. Immediately after this, in 2007, an arising HPV epidemic correlating with oropharyngeal SCC was reported in the United States [13]. The striking increases up to sevenfold in both HPV-positive tonsillar and base of tongue were demonstrated during the periods of 1970–2007 and 1998–2006 in 2009 and 2010 from Sweden, respectively [14, 15]. Following Dalianis group definition of "epidemic of viral-induced carcinoma," due to observation that most of the tonsillar carcinomas were HPV-associated in 2009 [14], a retrospective analysis of clinical trial material detected that HPV-related oropharyngeal cancer rate was 64% in the USA [16]. Similar retrospective analyses on oropharyngeal tumor samples revealed an increase in HPV incidence from 23, 28 to 57% from 1970s, 1980s to 1990s, respectively. Also, a constant trend in the increase was detected for recent term (68, 77, 93% incidence rates during 2000–2002, 2003–2005, 2006–2007, respectively [14]. Similarly, HPV association rate in oropharyngeal cases was increased from 16% to 73%, from 1984–1989 to 2000–2004 in the United States [17]. Furthermore, during the same periods, reports delivering similar striking rises in both oropharyngeal and HPV-positive oropharyngeal cancers have accumulated from several Western countries [3, 12–15, 17–23].

constitutes more than 90% of histological subtype of HNC [1].

**2.1. Changing trends in epidemiology of HPV-associated HNCs**

rapidly spreading subtype of HNCs is caused by HPV infection [10].

of the disease [4].

256 Human Papillomavirus - Research in a Global Perspective

**2. Epidemiology**

HPV detection rates were currently reported to be between 12.6% and 90.9% in oropharyngeal carcinoma [32]. One-quarter to one-half of unknown primaries originates from oropharyngeal region [33, 34] and the HPV presence in cervical lymph node metastases is a strong marker of oropharyngeal subsite for unknown primary regions [33, 35]. Although the association between oropharyngeal carcinoma (predominantly soft palate, the tonsils, base of tongue) and HPV is well known, the role of HPV in other head and neck subsites etiology is not well established. The presence of HPV DNA was not demonstrated in laryngeal, nasopharyngeal, hypopharyngeal, and oral cavity cancers in older studies, whereas recently published studies using more advanced methodologies and analyzed HPV gene expression products (e.g., p16) reported that HPV existed in non-oropharyngeal sites in small proportions which explains the increased incidence of oropharyngeal carcinoma compared with other HNCs [1, 36–38].

The prevalence of HPV-mediated HNCs was found as 25.9% in a systematic review of 60 published studies using PCR-based methods while it was higher in oropharyngeal carcinoma (35.6%) than both oral (23.5%) and laryngeal (24.0%) carcinoma. HPV-16 and the second most common high-risk type HPV-18 were detected in 86.7% and only 2.8% of the HPV-positive oropharyngeal carcinoma [39]. In addition, in a meta-analysis of 39 publications with 3649 HNC patients which investigated HPV in European population, the prevalence of HPV was found to be 40.0%, while it was most dominant in tonsillar cancer (66.4%) and lowest in pharyngeal (15.3%) and tongue (25.7%) cancers [36].

Unlike oropharyngeal cancers, HPV etiologic role in other HNC sites remains unclear. Concerning other head and neck subsites, HPV may play a role in the supraglottic larynx cancer [40], which might constitute the high-risk HPV infection rate established in laryngeal cancer, since its marginal field is adjacent with the oropharynx [28, 29, 41, 42]. Overall, contemporary studies using gold standard methods (type-specific HPV E6/E7 mRNA) from the United States detected the presence of HPV in less than 5% of non-oropharyngeal HNCs [17, 43]. Nevertheless, the estimate for the HPV infection-mediated non-oropharyngeal HNCs varies widely in the literature. While the estimated prevalence of HPV is 24% in the larynx, 31% in the nasopharynx, oral cavity 6–20%, and 21% in the sinonasal tract [44, 45], the studies mainly from the USA based on *in situ* hybridization (ISH) assays and E6/E7 mRNA detection suggest that the HPV association was found in 3% of oral cavity, 7% of larynx, and 0% of hypopharynx cancers [43]. Many factors including the border of anatomic classification of head and neck subsites and HPV detection methods contribute to this instability. The identification of certain markers of HPV-induced carcinogenesis and the correct rule out of the oropharynx as tumor origin site are substantial forthcoming issues in HPV-related non-oropharyngeal HNC estimation [43]. In addition, another topic which remains to be clarified is whether HPV tumor status in non-oropharyngeal sites may be implicated as a strong independent prognostic indicator such as in oropharyngeal cancers [16, 46–48].

### **2.3. The characteristics of HPV-positive HNC cancer patients**

The demographic and risk features of HPV-positive and HPV-negative HNC patients differ remarkably. First, HPV(+) patients are younger than HPV(−) ones [49]. The HPV-related cancers develop in those aged 40–55 [50] who are 4–10 years younger than HPV-negative patients [16, 51]. This age difference might explain the increase in oropharyngeal cancer incidence in younger individuals in developed countries [17, 52]. Given that HPV-associated patients are younger at diagnosis, they have better performance status [16, 47, 50] with fewer comorbid diseases [16, 51, 53].

Second, although HNC is generally more common in men than women, a notably larger rate of HPV-positive oropharyngeal cancer is diagnosed in men than HPV-negative ones [17]. This is convenient with the data that oral HPV16 infection is five times more common in men compared with women in the USA [54].

Third, HPV-HNC has been found to develop more frequently in white than black patients. HPV-positivity rate is 29–34% in whites compared to only 0–4% in blacks [55, 56], while HPVpositive versus -negative rumors occurring in whites are 92–97% and 75–78%, respectively [16, 46, 55]. Thus, in contrast to blacks, the incidence of oropharyngeal cancer has increased in whites which is probably caused by higher HPV-HNC rates in whites compared with blacks in the USA [57]. Furthermore, the socioeconomic status differs in HPV-positive patients from HPV-negative ones. HPV-positive patients have higher income and are more educated with higher rates of being married [51, 54].

oropharyngeal carcinoma [39]. In addition, in a meta-analysis of 39 publications with 3649 HNC patients which investigated HPV in European population, the prevalence of HPV was found to be 40.0%, while it was most dominant in tonsillar cancer (66.4%) and lowest in

Unlike oropharyngeal cancers, HPV etiologic role in other HNC sites remains unclear. Concerning other head and neck subsites, HPV may play a role in the supraglottic larynx cancer [40], which might constitute the high-risk HPV infection rate established in laryngeal cancer, since its marginal field is adjacent with the oropharynx [28, 29, 41, 42]. Overall, contemporary studies using gold standard methods (type-specific HPV E6/E7 mRNA) from the United States detected the presence of HPV in less than 5% of non-oropharyngeal HNCs [17, 43]. Nevertheless, the estimate for the HPV infection-mediated non-oropharyngeal HNCs varies widely in the literature. While the estimated prevalence of HPV is 24% in the larynx, 31% in the nasopharynx, oral cavity 6–20%, and 21% in the sinonasal tract [44, 45], the studies mainly from the USA based on *in situ* hybridization (ISH) assays and E6/E7 mRNA detection suggest that the HPV association was found in 3% of oral cavity, 7% of larynx, and 0% of hypopharynx cancers [43]. Many factors including the border of anatomic classification of head and neck subsites and HPV detection methods contribute to this instability. The identification of certain markers of HPV-induced carcinogenesis and the correct rule out of the oropharynx as tumor origin site are substantial forthcoming issues in HPV-related non-oropharyngeal HNC estimation [43]. In addition, another topic which remains to be clarified is whether HPV tumor status in non-oropharyngeal sites may be implicated as a strong independent prognostic

The demographic and risk features of HPV-positive and HPV-negative HNC patients differ remarkably. First, HPV(+) patients are younger than HPV(−) ones [49]. The HPV-related cancers develop in those aged 40–55 [50] who are 4–10 years younger than HPV-negative patients [16, 51]. This age difference might explain the increase in oropharyngeal cancer incidence in younger individuals in developed countries [17, 52]. Given that HPV-associated patients are younger at diagnosis, they have better performance status [16, 47, 50] with fewer

Second, although HNC is generally more common in men than women, a notably larger rate of HPV-positive oropharyngeal cancer is diagnosed in men than HPV-negative ones [17]. This is convenient with the data that oral HPV16 infection is five times more common in men

Third, HPV-HNC has been found to develop more frequently in white than black patients. HPV-positivity rate is 29–34% in whites compared to only 0–4% in blacks [55, 56], while HPVpositive versus -negative rumors occurring in whites are 92–97% and 75–78%, respectively [16, 46, 55]. Thus, in contrast to blacks, the incidence of oropharyngeal cancer has increased in whites which is probably caused by higher HPV-HNC rates in whites compared with blacks in the USA [57]. Furthermore, the socioeconomic status differs in HPV-positive patients from

pharyngeal (15.3%) and tongue (25.7%) cancers [36].

258 Human Papillomavirus - Research in a Global Perspective

indicator such as in oropharyngeal cancers [16, 46–48].

comorbid diseases [16, 51, 53].

compared with women in the USA [54].

**2.3. The characteristics of HPV-positive HNC cancer patients**

The HPV-positive HNC patients have better dentition, higher numbers of oral sex partners, and use less amount of tobacco or alcohol, higher amount of marijuana compared with HPVnegative patients. In addition, these risk factors have a powerful dose effect, revealing the distinguishing risk profile for HPV-positive patients [17]. Furthermore, these patients without environmental risk factors have persistent infection with high-risk HPVs [17].

## **3. Risk factors for HPV infection and HPV(+) oropharyngeal cancers**

HPV-16 which is detected in about 90% of the HPV(+) oropharyngeal cancers is the most common among several high-risk HPV types. Currently, HPV-16 is the only HPV type which is accepted as cancer inducing in the HNC [58, 59]. In addition, there are other high-risk HPV types with a less significant function and different manner than HPV-16 [37]. Among these, HPV-58, HPV-35, HPV-33, and HPV- 45 were detected in 10–15% of HPV(+) oropharyngeal cancer [38, 60, 61]. At present, HPV 16 constitutes 15- to 230-fold increased risk for orophar‐ yngeal cancer [50, 62]. HPV has been confirmed to induce and promote the development of oropharyngeal cancer [63].

With the identification of HPV as a powerful and independent risk factor for HNC, data are accumulating concerning the oral HPV infection epidemiology. The prevalence of oral HPV infection has been estimated as 7% in population-based studies in the United States and significantly correlated with male gender, older age, current smoking, and various sexual habits (e.g., the number of oral sexual partners during lifetime) [54]. HPV infection which may be contaminated by any type of sexual contact has become the most frequent disease that transmits sexually in the world [64]. The estimated number of individuals who are currently infected and expected to be infected each year is 20 million and 6.2 million in the United States alone [65]. Even though the orally infected rate among the population aged between 14 and 69 (10% of men, 4% of women) is 7%, the cancer-causing HPV subtypes consist of only 1% of these infections [54].

HPV-infected persons are unaware of the infection, since there are no correlated signs or symptoms. No effective treatment has been developed for active HPV infection, currently. Fortunately, the virus will be cleared in most of the infected persons within 2 years. It remains unknown how to identify those in whom the infection will become chronic and progress to HPV-HNC in time. Since the most of carcinogenesis occurs deep in the crypts of the tonsils where simple "Pap smear equivalents" are inaccessible in contrast to anus or cervix, an efficient screening test for early detection of HPV-related oropharyngeal cancer does not exist, yet [31].

Marijuana smoking has been shown to be an independent risk factor for HPV-mediated HNC, while the duration, intensity, and total years of marijuana use increase the risk. Marijuana use is suggested to cause oropharyngeal cancer development since the cannabinoids bind to the CB2 receptor of immune modulatory cells found in the tonsillar tissue which results in reduced immune response, lower resistance to viral infections, and antitumor functions [66].

It has been shown that HPV is less frequent in ex-, current-smokers, and tobacco chewers than nonsmokers and nonchewers [67]. Interestingly, in contrast to HPV(−) HNC, HPV(+) HNC decreases with increasing lifetime tobacco consumption [50]. On the one hand, rates of past and current tobacco use in HPV-oropharyngeal squamous cell carcinoma (OSCC) are reported to be 65% as opposed to 74% in HPV-negative OSCCs [6]. On the other hand, oral HPV infection presence is separately correlated with current smoking, but not lifetime smoking history [54]. Similarly, heavy alcohol consumption is less common in HPV(+) HNC patients compared with HPV-negative HNC ones [50]. Although heavy alcohol use (>21 drinks per week) is related to increased incidence of both HPV(+) and HPV(−) HNC, it is not correlated with oral HPV infection with the adjustment for sexual habits [54]. The complex role of alcohol remains to be identified.

Other significant factors in the increasing incidence of HPV infection and HPV(+) HNC are the changes in sexual behaviors, early sex debut, and number of oral-vaginal partners [68]. In addition, oral-oral contact and HPV transmission at birth may also lead to oral HPV infection [69, 70]. HPV transmits mainly through sexual contact directly with vaginal or anal intercourse, oral sex, or any mucosal contact. Also, oral HPV infection is more correlated with a genital HPV infection status, compared with oral sex activity in young males, suggesting autoinocu‐ lation as a potential HPV transmission route [71]. The first sexual encounter at younger ages, increasing number of sexual partners as well as more oral sex are reported by Americans [72, 73]. Additionally, the lifetime prevalence of oral sex was reported to be increased from 50% in 1970 to 90% in 2006 in a French study [73]. Furthermore, HPV exposure increases the HNC development risk, and HPV-16 seropositivity leads to cancer development 9 years earlier [74].

## **4. Diagnosis and histopathology**

### **4.1. Clinical and pathologic presentation**

An asymptomatic neck mass is the presentation of the disease in up to 90% of HPV(+) oro‐ pharyngeal cancer patients [75]. The neck nodes are generally in cystic structure which results in nondiagnostic aspiration materials. The diagnosis may be delayed due to no suspicious history because of being a nonsmoker, insufficient examination, and non-diagnostic aspirates of cystic lymph nodes. Ultrasound-guided fine-needle aspirates taken from cystic neck nodes may increase the chance of early diagnosis [76]. The utility of HPV detection in theses aspirates using p16 immunohistochemistry (IHC) and ISH has been shown [77]. In cases of unknown primary, a palatine and/or lingual tonsillectomy is superior to random biopsies.

HPV(+) oropharyngeal carcinoma exhibits a nonkeratinizing, basaloid, well-differentiated histology with diffuse nuclear and cytoplasmic p16 staining [66]. The HPV-positive tumor pathologic properties differ from HPV-negative ones showing lobular growth, having infiltrating lymphocytes, but not surface dysplasia or keratinization [66]. In addition, HPV- positive tumors present frequently with smaller primary tumors but advanced nodal meta‐ stasis [46, 50]. Despite being not pathognomonic, major histopathological features of HPVmediated tumors are the presence of classic koilocytes, perinuclear cytoplasmic halos, nuclear dysplasia with the addition of presence of dyskeratosis, atypical immature metaplasia, macrocytes, and binucleation as minor properties [78].

### **4.2. HPV detection tests/methods**

CB2 receptor of immune modulatory cells found in the tonsillar tissue which results in reduced

It has been shown that HPV is less frequent in ex-, current-smokers, and tobacco chewers than nonsmokers and nonchewers [67]. Interestingly, in contrast to HPV(−) HNC, HPV(+) HNC decreases with increasing lifetime tobacco consumption [50]. On the one hand, rates of past and current tobacco use in HPV-oropharyngeal squamous cell carcinoma (OSCC) are reported to be 65% as opposed to 74% in HPV-negative OSCCs [6]. On the other hand, oral HPV infection presence is separately correlated with current smoking, but not lifetime smoking history [54]. Similarly, heavy alcohol consumption is less common in HPV(+) HNC patients compared with HPV-negative HNC ones [50]. Although heavy alcohol use (>21 drinks per week) is related to increased incidence of both HPV(+) and HPV(−) HNC, it is not correlated with oral HPV infection with the adjustment for sexual habits [54]. The complex role of alcohol remains to be

Other significant factors in the increasing incidence of HPV infection and HPV(+) HNC are the changes in sexual behaviors, early sex debut, and number of oral-vaginal partners [68]. In addition, oral-oral contact and HPV transmission at birth may also lead to oral HPV infection [69, 70]. HPV transmits mainly through sexual contact directly with vaginal or anal intercourse, oral sex, or any mucosal contact. Also, oral HPV infection is more correlated with a genital HPV infection status, compared with oral sex activity in young males, suggesting autoinocu‐ lation as a potential HPV transmission route [71]. The first sexual encounter at younger ages, increasing number of sexual partners as well as more oral sex are reported by Americans [72, 73]. Additionally, the lifetime prevalence of oral sex was reported to be increased from 50% in 1970 to 90% in 2006 in a French study [73]. Furthermore, HPV exposure increases the HNC development risk, and HPV-16 seropositivity leads to cancer development 9 years earlier [74].

An asymptomatic neck mass is the presentation of the disease in up to 90% of HPV(+) oro‐ pharyngeal cancer patients [75]. The neck nodes are generally in cystic structure which results in nondiagnostic aspiration materials. The diagnosis may be delayed due to no suspicious history because of being a nonsmoker, insufficient examination, and non-diagnostic aspirates of cystic lymph nodes. Ultrasound-guided fine-needle aspirates taken from cystic neck nodes may increase the chance of early diagnosis [76]. The utility of HPV detection in theses aspirates using p16 immunohistochemistry (IHC) and ISH has been shown [77]. In cases of unknown

HPV(+) oropharyngeal carcinoma exhibits a nonkeratinizing, basaloid, well-differentiated histology with diffuse nuclear and cytoplasmic p16 staining [66]. The HPV-positive tumor pathologic properties differ from HPV-negative ones showing lobular growth, having infiltrating lymphocytes, but not surface dysplasia or keratinization [66]. In addition, HPV-

primary, a palatine and/or lingual tonsillectomy is superior to random biopsies.

immune response, lower resistance to viral infections, and antitumor functions [66].

identified.

**4. Diagnosis and histopathology**

260 Human Papillomavirus - Research in a Global Perspective

**4.1. Clinical and pathologic presentation**

The detection of cyclin-dependent kinase inhibitor 2A (p16Ink4A or p16) by IHC is recognized as a standard test for HPV positivity in a tumor in many clinics and during clinical trial enrollment. The HPV-16 E7 protein downregulates Rb and frees E2F, its regulatory partner which upregulates p16. IHC detection of p16 is a fast, cheap, and easily available method and is the standard method for HPV status assessment in clinics [79].With a false-negative rate of 4%, p16 IHC is a reliable marker in HPV infection, given that strong nuclear and cytoplasmic staining is extremely predictive for HPV(+) HNC [80]. By contrast, in cases with intermediate p16 expression levels, ISH or reverse transcription-polymerase chain reaction (RT-PCR) is required [80]. The inability of PCR-based assays to distinguish integrated DNA from episomal viral DNA decreases the test specificity remarkably in contrast to ISH in which sensitivity is lower compared to PCR [81]. Furthermore, the RNA ISH E6/E7 microRNA probes enable the direct scanning of viral transcripts which means accurate HPV detection [82]. It may be used as a HPV confirmation test in p16-positive samples, due to the fact that p16 is also overex‐ pressed in non-virally induced situations. Given the different survival rates of the p16(+)/ HPV(−) subgroups, considering personalized approaches, these patients should be evaluated as a distinct subcategory that is why using both ISH and p16 is recommended for HPV assessment [83]. In a recent study, which used both three methods, the sensitivity of p16 was detected as 100%, whereas its specificity was 74% in oral cancer and 93% in oropharyngeal cancer [84]. Cancer Care Ontario recommends routine HPV test in oropharyngeal cancer, in metastatic cervical nodes from an unknown head and neck primary and the use of p16 IHC as an initial test for its high sensitivity in patients with HNC [31].

Antibodies against HPV in the serum can be used to measure the cumulative exposure to HPV infection [85]. HPV16 E6 or E7 antibodies were detected in 65% of HPV(+) oropharyngeal cancer patients which indicates that they are useful tools as HPV markers in the unavailability of appropriate cytologic or histologic materials [67]. The detection of HPV antibodies in saliva instead of serum may lead to common false negative results, since antibodies in oral sampling are fewer compared with those in serum [85].

Currently, several methods including The Roche linear array HPV genotyping test, the Hybrid Capture 2, and a PCR bead-based multiplex method present for HPV typing [86–88]. Never‐ theless, since E6 and E7 mRNA by RT-PCR demonstrate functional HPV expression, it is widely accepted as the gold standard [89]. The correlation between the steps of HPV infection to cell, detection targets, and methods is shown in **Table 1**.


**Table 1.** Correlation between the steps of HPV infection to cell, detection targets and methods.

## **5. Carcinogenesis**

Currently, HPVs consist of over 81 different subtypes of HPVs classified on the basis of their L1 protein, separated based on cutaneous and mucosal site tropism, and divided in low-, and high-risk viruses according to correlation with cancers [90]. The high-risk HPV types may be classified as follows: highest risk types (HPV 16, 18, 31, 45), other high-risk types (HPV 33, 35, 39, 51, 52, 56, 58, 59), and probably high-risk types (HPV 26, 53, 66, 68, 73, 82). Oncogenic HPV types 16, 18, 31, 33, and 35 are related to HPV(+) HNC, while HPV-16 is most frequently found in oropharyngeal cancer [91, 92]. Majority of the cases of HPV(+) HNC arise from the oro‐ pharyngeal region due to epithelial injury predisposition of its location and lack of protective keratin layer that leads to easy virus exposure of the basal cells [93].

HPVs are circular, non-enveloped, epitheliotrophic, double-stranded DNA viruses belonging to family *Papovaviridae* with an approximately 8000 base pair-sized viral genome carrying early open-reading frame (ORF), late ORF, and noncoding control region between these two regions with histones within a 52–55-nm virion [94]. HPV genome encodes two structural capsid proteins (L1 and L2), two regulatory proteins (E1, E2), three oncoproteins (E5, E6, E7) [94]. The early region encodes the regulatory proteins, E1-E2, E4-E7, which are responsible for gene regulation, replication, and pathogenesis, while the late region encodes the two structural proteins, L1, L2, which form the viral capsid and have no known role in carcinogenesis but are substantial immune response targets to HPV infection. The E4 protein is thought to ease viral particle release into the surroundings and be responsible for G2 arrest in HPV-infected cells [95, 96].

The best-known relation between high-risk HPVs and cancer is established for the uterine cervix. However, HPV is also correlated with vaginal, vulvar, anal, and penile cancers, and especially since 2007, it has also been demonstrated to be a risk factor for oropharyngeal cancer [15, 95, 96]. HPV-16 and HPV-18 remain the main mediating factors in most HPV-related cancers. For instance, they are associated with 70% of cases with cervical cancer [94]. Approx‐ imately 50% of penile cancers include HPV DNA, predominantly HPV-16 [93], while HPV-16 accounts for more than 90% of HPV(+) oropharyngeal cancers [74].

**Step HPV infection Detection target Suitable methods**

**Table 1.** Correlation between the steps of HPV infection to cell, detection targets and methods.

keratin layer that leads to easy virus exposure of the basal cells [93].

DNA detection PCR

RNA detection PCR

Currently, HPVs consist of over 81 different subtypes of HPVs classified on the basis of their L1 protein, separated based on cutaneous and mucosal site tropism, and divided in low-, and high-risk viruses according to correlation with cancers [90]. The high-risk HPV types may be classified as follows: highest risk types (HPV 16, 18, 31, 45), other high-risk types (HPV 33, 35, 39, 51, 52, 56, 58, 59), and probably high-risk types (HPV 26, 53, 66, 68, 73, 82). Oncogenic HPV types 16, 18, 31, 33, and 35 are related to HPV(+) HNC, while HPV-16 is most frequently found in oropharyngeal cancer [91, 92]. Majority of the cases of HPV(+) HNC arise from the oro‐ pharyngeal region due to epithelial injury predisposition of its location and lack of protective

HPVs are circular, non-enveloped, epitheliotrophic, double-stranded DNA viruses belonging to family *Papovaviridae* with an approximately 8000 base pair-sized viral genome carrying early open-reading frame (ORF), late ORF, and noncoding control region between these two regions with histones within a 52–55-nm virion [94]. HPV genome encodes two structural capsid proteins (L1 and L2), two regulatory proteins (E1, E2), three oncoproteins (E5, E6, E7) [94]. The early region encodes the regulatory proteins, E1-E2, E4-E7, which are responsible for gene regulation, replication, and pathogenesis, while the late region encodes the two structural proteins, L1, L2, which form the viral capsid and have no known role in carcinogenesis but are substantial immune response targets to HPV infection. The E4 protein is thought to ease viral particle release into the surroundings and be responsible for G2 arrest in HPV-infected cells

The best-known relation between high-risk HPVs and cancer is established for the uterine cervix. However, HPV is also correlated with vaginal, vulvar, anal, and penile cancers, and especially since 2007, it has also been demonstrated to be a risk factor for oropharyngeal cancer [15, 95, 96]. HPV-16 and HPV-18 remain the main mediating factors in most HPV-related cancers. For instance, they are associated with 70% of cases with cervical cancer [94]. Approx‐

Oncoprotein detection Monoclonal antibodies

P16 overexpression detection Monoclonal antibodies

ISH

ISH

1 HPV endocytosis HPV episome HPV DNA integration

262 Human Papillomavirus - Research in a Global Perspective

2 E6 mRNA E7 mRNA

3 Protein E6 Protein E7

4 Rb:E2F P16

[95, 96].

**5. Carcinogenesis**

The palatine and lingual tonsils are predominant targets of HPV among all other potential sites of HNC. The basal cells are infected by HPV in the stratified squamous epithelium. The reticulated lymphoepithelium of tonsillar crypts expresses programmed death ligand 1 (PD-L1), which suppresses response of T-cells against HPV, and leads to an "immune-privileged" region for viral infection initiation and adaptation to immune resistance [97]. Virus enters through microinjuries in the epithelium, although the receptor and mechanism of this entry remain unknown. Prior to virus entry to the cell by endocytosis, heparin sulfate is considered to mediate the virus particle attachment to the cell [98]. HPV infection of epithelial cells causes different types of viral protein expression and production of 20 to 100 and thousands of viral DNA per cell in the basal layer and superficial layers, respectively [98]. While majority of other virus infections result in the production of progeny from the same target cell, the HPV-infected cells undergo mitosis and continue differentiation [99]. Thus, basal cells are not the only proliferating cells in the infected epithelium since infected suprabasal cells have active cell cycle and differentiation [100].

The three HPV oncoproteins E5, E6, and E7 promote uncontrolled cellular proliferation to lead viral amplification, initiate, and contribute to progression of cancer through the same mecha‐ nism and induce genomic instability [101–103]. As in cervical cancer, E6 and E7 oncoproteins are mainly responsible for the malignant transformation and progression in HNCs [101, 102]. Since silencing the E6 and E7 oncogene expression in HPV16(+) human oropharyngeal squamous cell lines led to p53 and Rb tumor suppressor activation and apoptosis induction, these two oncoproteins are thought to be required for maintaining malignant processes [104].

High-risk HPV E7 oncoproteins play a critical role in initiating DNA synthesis by binding and inactivating the Rb and its related pocket proteins p107 and p130 which are tumor suppressor genes by targeting them for degradation [98]. Rb, the most well-known pocket protein family member functions to prevent excessive cell growth by inhibiting cell cycle progression [105]. The Rb inactivation by E7 causes E2F transcription factor overexpression with the cell cycle gene upregulation, leading to the cell transition from G1 to S phase [106]. The inactivation of pRb increases levels of p16/CDKN2A which is an inhibitor of cdk4/cyclin D and cdk6/cyclin D and promotes aberrant cell proliferation [107]. Thus, increased levels of p16/CDKN2A expression correspond as a diagnostic biomarker for transcriptionally active HPV infection and virus-mediated deregulation of cell cycle [108]. E7 oncoproteins also have ability to affect gene transcription by influencing histone acetylation in regulatory regions via either histone acetyl transferases or histone deacetylases [109].

E7-mediated proliferation results in the activation of p53-dependent growth inhibition and apoptosis. To counteract this, HPV E6 causes p53 degradation which leads to apoptosis inhibition and uncontrolled cellular growth as a consequence [101]. E6 transcript produces a 19-kDa protein that binds with a ubiquitin protein ligase (E6AP) that will result in p53 tumor suppressor protein ubiquitination (posttranslational modification) and proteosomal degrada‐ tion [101]. The p53 regulates the cell cycle by controlling the transition from G1 to the S phase at checkpoint by inducing cyclin inhibitor (p16, p21, and p27) expression [110]. Hence, E6 oncoprotein deregulates cell cycle checkpoints both at G1/S and G2/M in the case of cellular stress such as DNA damage which results in genomic instability. Moreover, E6 protein has the ability to downregulate p53 function either by direct binding or by interacting with the histone acetyltransferases (ADA3) and histone acetyltransferase binding proteins (p300 and CREB) [111, 112]. In addition, it can associate with *ras* and E7 for *in vitro* cell transformation [113]. Furthermore, E6 oncoprotein can activate cellular telomerase by upregulating human telo‐ merase hTERT which leads to cellular immortalization [114]. E6 targets not only p53, but also Bak and Myc proteins which regulate apoptosis [115].

Additionally, the E5 protein works together with E6 and E7 to induce proliferation in infected cells and is considered to have a more minor role in host cell transformation. It also blocks apoptosis in the late process of HPV-induced carcinogenesis [103].

Several studies report that both E6 and E7 bind multiple complementary cooperators that perform oncogenic impacts other than p53 and pRb degradation. Despite the robust growth inducing functions of these HPV oncoproteins, extra oncogenic events are required for malignant development. Although high-risk E6 and E7 cause genomic instability [116], the mutation rate in HPV(+) tumors seem to be lower than in HPV(−) ones [59, 117]. E6 and E7 oncoproteins may lead to chromosomal segregation errors and aneuploidy development during mitosis [118]. Due to the E7 induction of CDK2 activation, multiple immature centrioles are formed which results in several centrosome synthesis rounds [119]. Eventually, E6 and E7 cause these cells with aberrant mitoses to proliferate by inhibiting G2-M checkpoint control and apoptosis [120].

It has been demonstrated that HPV-mediated carcinogenesis causes significantly fewer genomic changes than HPV-independent ones. Particularly, HPV(+) HNC is associated with less mutated p53, higher EGFR expression, and more chromosomal aberrations (3p, 9p, and 17p) [63, 121–123]. It has been confirmed by whole-exome sequencing of HNC that HPV(+) HNC has a distinct genetic entity from HPV(−) HNC which has more mutations compared with HPV(+) ones, independent of smoking [59, 117]. While HPV(+) tumors have no p53 mutation, p53 mutation rate was 78% in HPV(−) cancers [59]. These data indicate that HPVmediated oncogenesis is correlated with cellular dysregulation to a lesser degree which may be the underlying explanation of better treatment response. HPV(+) tumor differs from HPV(−) HNC not only biologically, but also clinically. They tend to present at earlier T stage with extensive nodal involvement. Despite the fact that distant metastasis may constitute a major problem in HPV(+) tumors, their prognosis is better, especially in locally advanced diseases [124].

### **6. Mechanisms of improved response to treatment**

The prognostic advantage of HPV positivity may be explained to some extent by the patient population features affected with younger age, higher performance status, fewer comorbid diseases, and less tobacco and alcohol exposure. However, after adjustment to these conditions in multivariate analyses, the improved survival of HPV(+) patients still maintains [16, 46]. Actually, these factors only account for approximately 9% of the survival difference between patients with HPV(+) and HPV(−) tumors, which means that the survival difference seems to be largely resulted by HPV status [16, 125]. It is clear that the higher response rate of HPV(+) tumor to therapy is caused by a basic biological difference between HPV(+) and HPV(−) HNCs. It is accepted that intrinsic factors of each individual tumor (e.g., mutations, HPV status) may modulate the microenvironment of tumors [126]. These alterations have the ability to influence immune response, stromal structure, and tumor vasculature [126]. Not only experimental studies but also several clinical studies have demonstrated that patients with HPV/p16(+) tumors had a much better prognosis compared with HPV/p16(−) ones [16].

oncoprotein deregulates cell cycle checkpoints both at G1/S and G2/M in the case of cellular stress such as DNA damage which results in genomic instability. Moreover, E6 protein has the ability to downregulate p53 function either by direct binding or by interacting with the histone acetyltransferases (ADA3) and histone acetyltransferase binding proteins (p300 and CREB) [111, 112]. In addition, it can associate with *ras* and E7 for *in vitro* cell transformation [113]. Furthermore, E6 oncoprotein can activate cellular telomerase by upregulating human telo‐ merase hTERT which leads to cellular immortalization [114]. E6 targets not only p53, but also

Additionally, the E5 protein works together with E6 and E7 to induce proliferation in infected cells and is considered to have a more minor role in host cell transformation. It also blocks

Several studies report that both E6 and E7 bind multiple complementary cooperators that perform oncogenic impacts other than p53 and pRb degradation. Despite the robust growth inducing functions of these HPV oncoproteins, extra oncogenic events are required for malignant development. Although high-risk E6 and E7 cause genomic instability [116], the mutation rate in HPV(+) tumors seem to be lower than in HPV(−) ones [59, 117]. E6 and E7 oncoproteins may lead to chromosomal segregation errors and aneuploidy development during mitosis [118]. Due to the E7 induction of CDK2 activation, multiple immature centrioles are formed which results in several centrosome synthesis rounds [119]. Eventually, E6 and E7 cause these cells with aberrant mitoses to proliferate by inhibiting G2-M checkpoint control

It has been demonstrated that HPV-mediated carcinogenesis causes significantly fewer genomic changes than HPV-independent ones. Particularly, HPV(+) HNC is associated with less mutated p53, higher EGFR expression, and more chromosomal aberrations (3p, 9p, and 17p) [63, 121–123]. It has been confirmed by whole-exome sequencing of HNC that HPV(+) HNC has a distinct genetic entity from HPV(−) HNC which has more mutations compared with HPV(+) ones, independent of smoking [59, 117]. While HPV(+) tumors have no p53 mutation, p53 mutation rate was 78% in HPV(−) cancers [59]. These data indicate that HPVmediated oncogenesis is correlated with cellular dysregulation to a lesser degree which may be the underlying explanation of better treatment response. HPV(+) tumor differs from HPV(−) HNC not only biologically, but also clinically. They tend to present at earlier T stage with extensive nodal involvement. Despite the fact that distant metastasis may constitute a major problem in HPV(+) tumors, their prognosis is better, especially in locally advanced

The prognostic advantage of HPV positivity may be explained to some extent by the patient population features affected with younger age, higher performance status, fewer comorbid diseases, and less tobacco and alcohol exposure. However, after adjustment to these conditions in multivariate analyses, the improved survival of HPV(+) patients still maintains [16, 46].

Bak and Myc proteins which regulate apoptosis [115].

264 Human Papillomavirus - Research in a Global Perspective

and apoptosis [120].

diseases [124].

apoptosis in the late process of HPV-induced carcinogenesis [103].

**6. Mechanisms of improved response to treatment**

It has been suggested that the immune system plays a significant role in rejection process of HPV(+) tumors because of viral protein expressions that reveal T-cell responses performing a long-term immunosurveillance. Accumulating evidence over the years appears to reinforce this hypothesis. Spanos et al. [127] showed superior tumor control in HPV(+) cell lines implanted into immunocompetent mice compared with immunocompromised mice [127]. HPV-specific circulating HPV16 E7-specific CD8+ T cells and IFN*γ*-producing T cells have been defined in patients with HPV(+) HNC [128]. A greater alteration from naive to effector and memory T cells has been shown in patients with HPV(+) compared with both healthy donors and patients with HPV(−) tumors which indicates higher tumor response in HPV(+) patients [129]. In addition, it has been reported that the programmed death-1 (PD-1) positive T-cell presence was associated with improved survival in HPV(+) HNC patients [130]. Moreover, circulating anti-HPV16 antibodies which are suggested to associate with clinical outcome have been detected in HPV(+) HNC patients [92]. Furthermore, radiation may induce expression loss of CD47 which is an important transmembrane cell surface marker in selfidentification in HPV(+) cell lines that explains the interaction between the immune system and radiotherapy [131].

Even though radiosensitivity is mainly based on the cell ability to detect DNA damage and repair it, tumor oxygenation status may also be a factor for radiotherapy response [132]. In this context, in the Danish Head and Neck Cancer Group (DAHANCA)-5 study, the ratio of patients with high plasma osteopontin (a marker of hypoxia) levels was greater in HPV(−) tumors than in HPV(+) ones, indicating more hypoxia in HPV(−) group [133]. On the contrary, neither IHC staining for carbonic anhydrase IX (upregulated in hypoxic conditions) nor pO2 level of tumor was found to associate with HPV status of tumor in another study [134].

In two prospective trials investigating the impact of hypoxic modification (using nimorazole or tirapazamine) in HNC, the use of hypoxic cell radiosensitizer provided a trend toward better locoregional control in HPV(−) tumors but had no significant benefit in HPV(+) tumors [53, 135]. Surprisingly, HPV/p16-positive tumors appeared to be insensible to hypoxic modifier and revealed no benefit from nimorazole after which was suggested that HPV/p16(+) HNCs are less hypoxic than HPV(−) ones, and this might contribute to the better prognosis [135]. However, in studies evaluating the hypoxia with imaging markers including (18)F-fluoroa‐ zomycin arabinoside positron emission tomography/computed tomography (FAZA PET/ CT)), dynamic contrast enhanced-MRI (DCE-MRI), and proton magnetic resonance spectro‐ scopy ((1)H-MRS), no correlation between HPV positivity and intratumoral hypoxia was detected [136, 137]. Additionally, recent *in vitro* evidence comparing radiation response of HPV/p16(+) and (−) cell lines under hypoxia showed no difference regarding gene regulation patterns, while oxygen enhancement ratio (OER) of HPV/p16(+) cells was found similar to HPV/p16(−) ones [138].

Finally, it has been hypothesized that viral oncoproteins play a substantial role in improved treatment sensitivity. It has been shown that low levels of residual wild-type p53 in HPV(+) cells may be activated by radiation, resulting in increased cell death [139]. Recently, Rieckman et al. [140] demonstrated decreased survival fraction, increased double strand breaks levels, and extensive G2 arrest pointing to compromised DNA repair capacity in HPV/p16(+) cell lines compared with HPV(−) cell lines after irradiation [140].

## **7. Clinical considerations**

### **7.1. Impact of HPV on prognosis (clinical studies)**

The changing epidemiology of HPV-positive oropharyngeal cancer formed a new patient population in the clinic that consists of individuals at younger ages without a heavy alcohol or smoking history and with more advanced neck diseases [141]. The natural history of HPVpositive HNC began to be written with many retrospective studies published in the late 1990s to early 2000s. In a study of 42 patients, HPV(+) tonsil cancer revealed higher survival rates compared with HPV(−) ones [142]. In addition, in a German study including 208 HNC sample patients with HPV(+) samples had better survival despite more adverse pathological results [143]. Furthermore, in a Swedish study with 60 patients, HPV(+) oropharyngeal cancer patients were found to have higher 5-year OS rates (53.5% vs. 31.5%) and reduced recurrence risk irrespective of gender, age, or disease stage [144]. Gillison et al. retrospectively analyzed 252 HNC patients in 2000, and patients with HPV(+) HNC from all sites had a 40% reduction in death risk (*p* = 0.07) and a 59% reduction in disease-related death risk (*p* = 0.02) after adjustment for age, nodal status, and alcohol consumption [63]. Finally, a meta-analysis of 37 studies analyzing HPV and HNC reported that HPV(+) oropharyngeal cancer patients had a 28% reduced death risk and a 49% lower disease-failure risk compared with HPV(−) ones in 2007 [145].

In the Danish DAHANCA-5 phase III clinical trial, samples from 156 patients of whom 74 were oropharyngeal cancer patients were collected prospectively. Of oropharyngeal cancer samples, 24 were p16(+) and had a locoregional control benefit (OR, 5.1) compared with those with p16(−) samples [146]. In a phase II prospective trial of 42 oropharyngeal cancer patients of whom pretreatment biopsy HPV-positivity rate was 67%, HPV titer was correlated with improved induction chemotherapy response (*p* = 0.001), chemoradiation response (*p* = 0.005), overall survival (*p* = 0.007), and disease-specific survival (DSS) (*p* = 0.008) [147]. Similarly, in the Trans-Tasman Radiation Oncology Group (TROG), 20.02 trial which retrospectively reviewed for HPV and p16 status, a higher 2-year overall survival rate (91% vs. 74%; HR, 0.36; *p* = 0.004) and failure-free survival (87% vs. 72%; HR, 0.39; *p* = 0.003) were detected in 57% of 185 oropharyngeal cancer patients with p16 positivity compared with HPV(−) patients [53].

Several studies have demonstrated the favorable effect of HPV positivity in oropharyngeal cancer patients [46, 53, 148]. First, in 2008, the Eastern Cooperative Oncology Group (ECOG) 2399 trial which was a phase II prospective study including stages III and IV, M0, oropharynx and larynx cancer patients treated with paclitaxel/carboplatin induction chemotherapy followed by concurrent paclitaxel and radiotherapy showed that patients with HPV(+) oropharyngeal cancer (HPV detection method was p16 IHC) had a 61% lower death risk (HR, 0.39; *p* = 0.06) and a 62% lower progression risk (HR, 0.38; *p* = 0.09) than patients with HPV(−) ones, after adjustments for age, ECOG performance status, and disease stage [46]. Following this, Ang et al. [16] reported the largest retrospective examination of Radiation Therapy Oncology Group (RTOG) 0129 study about HPV effect on survival in oropharyngeal cancer where HPV positivity was tested by ISH. The 3-year overall survival rates were 82.4% and 57.1% in the HPV(+) and HPV(−) subgroups, respectively, while the 3-year progression-free survival rates were similarly better in HPV(+) subgroup compared with HPV(−) ones (73.7% vs. 43.4%). In addition, HPV(+) patients had a 58% lower death risk (HR, 0.42; *p* < 0.001) and a 51% lower progression risk (HR, 0.49; *p* < 0.001). Furthermore, patients were classified into risk-of-death categories (low, moderate, high) based on a recursive partitioning analysis according to HPV status, tumor burden, and tobacco use. The low-risk group included patients with HPV(+) cancer with the exception of smokers with advanced nodal metastasis, while smoker patients with HPV(+) tumors and advanced nodal metastasis or nonsmoker patients HPV(−) tumors of stage T2 or T3 were considered to be at intermediate risk. On the contrary, nonsmoker patients with HPV(−) T4 tumors or smoker patients with HPV(−) tumors consisted of high-risk group while 3-year survival rates of low-, moderate-, and high-risk patients were 93%, 70.8%, and 46.2%, respectively. Importantly, smoking had a negative factor on prognosis, regardless of HPV status [16]. Similarly, in the retrospective analysis of TAX 324 trial, which investigated triple-agent versus double-agent induction chemotherapy confirmed the better prognosis of HPV(+) patients, 5-year overall survival rates were significantly higher in HPV(+) group compared with HPV(−) (82% vs. 35%, *p* < 0.0001). The HPV status was tested using E6/ E7 PCR methods in this study [47].

detected [136, 137]. Additionally, recent *in vitro* evidence comparing radiation response of HPV/p16(+) and (−) cell lines under hypoxia showed no difference regarding gene regulation patterns, while oxygen enhancement ratio (OER) of HPV/p16(+) cells was found similar to

Finally, it has been hypothesized that viral oncoproteins play a substantial role in improved treatment sensitivity. It has been shown that low levels of residual wild-type p53 in HPV(+) cells may be activated by radiation, resulting in increased cell death [139]. Recently, Rieckman et al. [140] demonstrated decreased survival fraction, increased double strand breaks levels, and extensive G2 arrest pointing to compromised DNA repair capacity in HPV/p16(+) cell lines

The changing epidemiology of HPV-positive oropharyngeal cancer formed a new patient population in the clinic that consists of individuals at younger ages without a heavy alcohol or smoking history and with more advanced neck diseases [141]. The natural history of HPVpositive HNC began to be written with many retrospective studies published in the late 1990s to early 2000s. In a study of 42 patients, HPV(+) tonsil cancer revealed higher survival rates compared with HPV(−) ones [142]. In addition, in a German study including 208 HNC sample patients with HPV(+) samples had better survival despite more adverse pathological results [143]. Furthermore, in a Swedish study with 60 patients, HPV(+) oropharyngeal cancer patients were found to have higher 5-year OS rates (53.5% vs. 31.5%) and reduced recurrence risk irrespective of gender, age, or disease stage [144]. Gillison et al. retrospectively analyzed 252 HNC patients in 2000, and patients with HPV(+) HNC from all sites had a 40% reduction in death risk (*p* = 0.07) and a 59% reduction in disease-related death risk (*p* = 0.02) after adjustment for age, nodal status, and alcohol consumption [63]. Finally, a meta-analysis of 37 studies analyzing HPV and HNC reported that HPV(+) oropharyngeal cancer patients had a 28% reduced death risk and a 49% lower disease-failure risk compared with HPV(−) ones in 2007

In the Danish DAHANCA-5 phase III clinical trial, samples from 156 patients of whom 74 were oropharyngeal cancer patients were collected prospectively. Of oropharyngeal cancer samples, 24 were p16(+) and had a locoregional control benefit (OR, 5.1) compared with those with p16(−) samples [146]. In a phase II prospective trial of 42 oropharyngeal cancer patients of whom pretreatment biopsy HPV-positivity rate was 67%, HPV titer was correlated with improved induction chemotherapy response (*p* = 0.001), chemoradiation response (*p* = 0.005), overall survival (*p* = 0.007), and disease-specific survival (DSS) (*p* = 0.008) [147]. Similarly, in the Trans-Tasman Radiation Oncology Group (TROG), 20.02 trial which retrospectively reviewed for HPV and p16 status, a higher 2-year overall survival rate (91% vs. 74%; HR, 0.36; *p* = 0.004) and failure-free survival (87% vs. 72%; HR, 0.39; *p* = 0.003) were detected in 57% of 185 oropharyngeal cancer patients with p16 positivity compared with HPV(−) patients [53].

HPV/p16(−) ones [138].

266 Human Papillomavirus - Research in a Global Perspective

**7. Clinical considerations**

[145].

compared with HPV(−) cell lines after irradiation [140].

**7.1. Impact of HPV on prognosis (clinical studies)**

The retrospective subanalysis of 190 patients with oropharyngeal cancer in the RTOG 9003 study, a 4-arm, phase III trial which compared different RT protocols, p16 positivity was found to correlate with T1 stage, better performance status, absence of anemia, and less tobacco consumption. Independently from assigned treatment, the p16(+) oropharyngeal cancer group had better 5-year overall and progression-free survival, lower 5-year locoregional failure but similar 5-year distant metastasis rates compared with p16(−) ones [149]. Additionally, a recently published retrospective analysis of RTOG 0129 and RTOG 0522 revealed that HPV(+)oropharyngeal cancer patients had better overall survival even after disease progres‐ sion compared with HPV(−) group. Moreover, salvage surgery was detected to provide significantly improved prognosis [150]. Another recent retrospective subanalysis of the phase III trial, IMCL-9815 Bonner trial [53, 54], which evaluated the impact of the role of cetuximab addition to radiotherapy in patients with locally advanced HNC, based on HPV status determined by p16 IHC, p16 positivity was confirmed as a powerful prognostic determinant for oropharyngeal cancer patients [151].

Likewise, surgical reports further confirmed the favorable effect of p16 positivity in orophar‐ yngeal cancer. Rich et al. [148] reported that in a cohort of 84 stage III or IV oropharyngeal cancers, patients who received transoral laser microsurgery (TLM) ± adjuvant therapy, p16 positivity was significantly associated with higher 5-year overall survival (90% vs. 25%, *p* < 0.0001) and disease-specific survival (DSS) rates (94% vs. 50%, *p* = 0.0078) [148]. Furthermore, two recent meta-analysis of clinical trial data of oropharyngeal cancer patients demonstrated hazard ratios for better overall survival of 0.49 and 0.47 correlated with HPV positivity [125, 152]. Overall, patients with HPV(+) tumors have a 60–80% reduced mortality rates, a finding that was confirmed by multiple trials.

The HPV(+) tumors had some unique features. The TAX324 trial analysis showed that HPV(+) patients had T1 or T2 tumors more commonly (49% vs. 20%) and better ECOG performance status (ECOG 0: 77% vs.49%) [47] which was in parallel with the results TROG 02.02 and ECOG 2399 trials [46, 53]. In addition, it has been shown that cystic lymph node metastases were associated with HPV(+) tonsil cancer in a surgical series of neck dissection [153]. Furthermore, Princess Margaret Hospital data of N2-3 HNC patients (*n* = 493) detected that HPV(+) lymph nodes (*n* = 257) were larger (2.9 vs. 2.5 cm), more commonly in cystic structure (38% vs. 6%), regressed more frequently after treatment (36% vs. 41%), and more likely to be eliminated after 36 weeks (90% vs. 0%) compared with HPV(−) ones [154].

### **7.2. The role of HPV in recurrent/metastatic HNC**

The characteristics of metastatic disease in HPV(+) patients differ from HPV(−) ones in terms of sites and time, since metastases is more likely to develop at sites other than lungs and may occur after 2 years following the initial treatment. Locally advanced diseases (T4 and N3-N2C) are the risk factors for metastasis development in HPV(+) disease [16]. Princess Margaret Hospital reviewed the distant metastasis rates in 457 HPV(+) and 167 HPV(−) oropharyngeal cancer cases and found that metastasis rates were similar at 3 years. HPV(+) tumors were more likely to develop metastases after a long interval, such that metastases occurred within 2 years in 24 of 25 HPV(−) cases, while metastasis development were detected 3 years post treatment in 13% of HPV(+) cases. In addition, dissemination pattern was more common in HPV(+) patients (33% vs. 0%). Nevertheless, post-metastases 2-year survival was significantly better in HPV(+) group compared with HPV(−) group (11% vs. 4%, *p* = 0.02) [155]. These findings were confirmed by additional recent retrospective reports showing similar unique metastatic spread patterns [53, 156–158].

Recently, the impact of HPV in recurrent-metastatic HNC was evaluated in large trials. In the phase III EXTREME randomized trial which assessed the benefit of cetuximab addition to platinum + 5-fluorouracil (5-FU) in patients with recurrent-metastatic HNC as first-line therapy, paired tissue samples were analyzed for p16 expression by IHC using a 70% expres‐ sion cutoff value and HPV via oligonucleotide hybridization test [159]. The p16 and HPV positivity was correlated with higher survival rates compared with p16 and HPV negativity in both cetuximab and control groups [159]. Since the predictive analyses indicated that cetuximab addition to chemotherapy improved survival rates independently from p16 or HPV status, these biomarkers did not have any significance in treatment efficacy prediction. By contrast, in the phase III SPECTRUM randomized trial which assessed the benefit of panitu‐ mumab addition to chemotherapy instead of cetuximab in recurrent-metastatic HNC, a nonsignificant difference between HPV(+) and HPV(−) groups was demonstrated. Further‐ more, the benefit of panitumumab was not detected in p16(+) group in contrast to p16(−) group [159].

The pooled analysis of patients with recurrent-metastatic HNC from E1395, a phase III trial comparing platinum + 5-fluorouracil with cisplatin + paclitaxel and E3301, a phase II trial evaluating irinotecan + docetaxel demonstrated that patients with HPV(+)/p16(+) disease had better overall survival than HPV(−)/p16(−) ones [160]. Taken together, these three studies indicate the positive impact of HPV positivity on improved survival. The efficiency of anti-EGFR antibodies on survival of HPV(+) and HPV(−) patients remains to be clarified. Hence, further studies including only oropharyngeal cancer patients are required in recurrentmetastatic setting since the impact of HPV positivity at other sides of HNC is not fully known.

### **7.3. Prognostic factors**

Likewise, surgical reports further confirmed the favorable effect of p16 positivity in orophar‐ yngeal cancer. Rich et al. [148] reported that in a cohort of 84 stage III or IV oropharyngeal cancers, patients who received transoral laser microsurgery (TLM) ± adjuvant therapy, p16 positivity was significantly associated with higher 5-year overall survival (90% vs. 25%, *p* < 0.0001) and disease-specific survival (DSS) rates (94% vs. 50%, *p* = 0.0078) [148]. Furthermore, two recent meta-analysis of clinical trial data of oropharyngeal cancer patients demonstrated hazard ratios for better overall survival of 0.49 and 0.47 correlated with HPV positivity [125, 152]. Overall, patients with HPV(+) tumors have a 60–80% reduced mortality rates, a finding

The HPV(+) tumors had some unique features. The TAX324 trial analysis showed that HPV(+) patients had T1 or T2 tumors more commonly (49% vs. 20%) and better ECOG performance status (ECOG 0: 77% vs.49%) [47] which was in parallel with the results TROG 02.02 and ECOG 2399 trials [46, 53]. In addition, it has been shown that cystic lymph node metastases were associated with HPV(+) tonsil cancer in a surgical series of neck dissection [153]. Furthermore, Princess Margaret Hospital data of N2-3 HNC patients (*n* = 493) detected that HPV(+) lymph nodes (*n* = 257) were larger (2.9 vs. 2.5 cm), more commonly in cystic structure (38% vs. 6%), regressed more frequently after treatment (36% vs. 41%), and more likely to be eliminated after

The characteristics of metastatic disease in HPV(+) patients differ from HPV(−) ones in terms of sites and time, since metastases is more likely to develop at sites other than lungs and may occur after 2 years following the initial treatment. Locally advanced diseases (T4 and N3-N2C) are the risk factors for metastasis development in HPV(+) disease [16]. Princess Margaret Hospital reviewed the distant metastasis rates in 457 HPV(+) and 167 HPV(−) oropharyngeal cancer cases and found that metastasis rates were similar at 3 years. HPV(+) tumors were more likely to develop metastases after a long interval, such that metastases occurred within 2 years in 24 of 25 HPV(−) cases, while metastasis development were detected 3 years post treatment in 13% of HPV(+) cases. In addition, dissemination pattern was more common in HPV(+) patients (33% vs. 0%). Nevertheless, post-metastases 2-year survival was significantly better in HPV(+) group compared with HPV(−) group (11% vs. 4%, *p* = 0.02) [155]. These findings were confirmed by additional recent retrospective reports showing similar unique metastatic

Recently, the impact of HPV in recurrent-metastatic HNC was evaluated in large trials. In the phase III EXTREME randomized trial which assessed the benefit of cetuximab addition to platinum + 5-fluorouracil (5-FU) in patients with recurrent-metastatic HNC as first-line therapy, paired tissue samples were analyzed for p16 expression by IHC using a 70% expres‐ sion cutoff value and HPV via oligonucleotide hybridization test [159]. The p16 and HPV positivity was correlated with higher survival rates compared with p16 and HPV negativity in both cetuximab and control groups [159]. Since the predictive analyses indicated that cetuximab addition to chemotherapy improved survival rates independently from p16 or HPV status, these biomarkers did not have any significance in treatment efficacy prediction. By

that was confirmed by multiple trials.

268 Human Papillomavirus - Research in a Global Perspective

36 weeks (90% vs. 0%) compared with HPV(−) ones [154].

**7.2. The role of HPV in recurrent/metastatic HNC**

spread patterns [53, 156–158].

Combined with being a nonsmoker, HPV DNA/RNA presence and p16 overexpression are strong prognostic markers [16]. Smoking, the most important risk factor for HNC overall, affects negative survival and response to treatment [161]. In addition, tobacco exposure has been shown to be a significant independent factor for prognosis in patients with HPV(+) oropharyngeal cancer, as it predicted progression and death risk in a dose-dependent pattern. Independently from HPV status and other factors, each pack-year of smoking increases the progression and death risk and the risk of second primary cancers of by 1% and 1.5%, respectively. Furthermore, the risk of death doubles if patients do not quit smoking during radiotherapy [161]. Based on the data of RTOG 0129 trial, a risk stratification for oropharyngeal cancer patients was developed using HPV status, history of smoking (>10 pack-years), and disease stage [16].

Other factors which are correlated with poor prognosis are local extension, disease stage at presentation, cervical node involvement, and rich vascular and lymphatic network [162]. Tumor thickness greater than 5 mm is significantly correlated with occult lymph node metastasis which is suggested to be a stronger determiner of prognosis than TNM staging by some authors. It has been shown that tumor differentiation, angiogenesis, extracapsular extension, and perineural invasion had significant role in prognosis determination [162]. Regarding treatment associated factors, cervical node dissection and disease free margins are important [162], since local relapse rates are 64–84% in patients with positive surgical margins [163].

Concerning the molecular markers, aberrations in chromosomes 3, 9, 11, 13, and 17 and tumor suppressor genes (p53 and pRb) are significant for prognosis [162]. In addition, cytokeratin 8/18 has been shown to be an independent factor correlated with poor prognosis [162, 163]. Furthermore, the absent/low expression of MHC class I, CD44, or CD98 has strong prognostic value for HPV(+) oropharyngeal cancer patients such that MHC class I staining absence defines 3-year disease-free and overall survival with 95–100% probability [164–166]. Moreover, higher CD8+ tumor infiltrating lymphocyte (TIL) counts is also a prognostic marker for patients with HPV(+) oropharyngeal cancer [167].

HPV16 E6 and E7 in serum were demonstrated to be the most predictive factor in determining the HNC prognosis among the several biomarkers. E6/E7 seropositivity was associated with improved survival, whereas their seronegativity was correlated with poor prognosis irrespec‐ tive of the DNA or p16 status [168]. Another suggested prognostic factor for survival is pretreatment tumor HPV copy number; higher tumor HPV copy number was correlated with better induction chemotherapy (*p* = 0.001) and chemoradiotherapy (*p* = 0.005) response, disease-specific (*p* = 0.004) and overall survival and (*p* = 0.008) after adjustment for other significant factors [147]. Similarly, higher viral load was shown to be correlated with improved recurrence-free (*p* = 0.0037) and overall (*p* = 0.028) survival in tonsil cancer patients [169]. Furthermore, the presence of both HPV16 and p16 indicated significantly improved prognosis compared with either p16 or HPV16 alone [170].

### **7.4. Deintensification of treatment in HPV(+) oropharyngeal cancer**

Three principal routes have been drawn in treatment deintensification: (1) EGFR inhibitor use (mostly Cetuximab) instead of cisplatin, concurrently with radiotherapy; (2) radiotherapy dose reduction with concurrent chemotherapy (based on induction chemotherapy response); and (3) minimally invasive transoral surgery use followed by reduced adjuvant treatment accord‐ ing to the histopathological properties of the excised tumor.

Since responses to treatment alter even between HPV(+) oropharyngeal cancer patients, defining appropriate patient subgroup is essential. It must be emphasized that all patients with HPV(+) oropharyngeal cancer are not suitable candidates for deintensification approaches. The clinical trial cohorts' analysis indicates some evidence. Some of them suggest that lifetime tobacco use history of >10 pack-years in HPV(+) patients with advanced regional nodal metastasis (N2b, N3) as well as smoking during radiotherapy decreases survival time (3-year overall survival rate of 70.8%) [16, 161]. The Italian study which reported that intermediaterisk patients are not convenient for deintensification trials confirmed these findings [171]. Furthermore, O'Sullivan et al. [27] demonstrated that the 3-year distant control rates of HPV(+) oropharyngeal cancer with advanced T stage (T4) or nodal (N3) was 72% and 78%, respectively [124]. Hence, late recurrences and relapses as distant metastasis are not rare in HPV(+) oropharyngeal cancer. These data indicate the presence of a limit of the biologic advantage provided by HPV positivity and may be helpful in determining patient subgroup that requires chemotherapy use in order to treat early micrometastasis. Both amount of smoking and locoregionally advanced disease have implications in designing deintensification trials for HPV(+) oropharyngeal cancer patients.

Many clinical trials have been planned to evaluate the efficiency of treatment deintensification in HNC patients and recently they have been reviewed by Masterson et al. [152] in detail. Enrolling patients into these trials to define optimum treatment of HPV(+) oropharyngeal cancer is encouraged. Nevertheless, besides as a clinical trial, there is not sufficient data for the treatment reduction or modification according to HPV positivity until we receive the results from these deintensification studies.

### **7.5. Prevention-vaccination**

CD8+ tumor infiltrating lymphocyte (TIL) counts is also a prognostic marker for patients with

HPV16 E6 and E7 in serum were demonstrated to be the most predictive factor in determining the HNC prognosis among the several biomarkers. E6/E7 seropositivity was associated with improved survival, whereas their seronegativity was correlated with poor prognosis irrespec‐ tive of the DNA or p16 status [168]. Another suggested prognostic factor for survival is pretreatment tumor HPV copy number; higher tumor HPV copy number was correlated with better induction chemotherapy (*p* = 0.001) and chemoradiotherapy (*p* = 0.005) response, disease-specific (*p* = 0.004) and overall survival and (*p* = 0.008) after adjustment for other significant factors [147]. Similarly, higher viral load was shown to be correlated with improved recurrence-free (*p* = 0.0037) and overall (*p* = 0.028) survival in tonsil cancer patients [169]. Furthermore, the presence of both HPV16 and p16 indicated significantly improved prognosis

Three principal routes have been drawn in treatment deintensification: (1) EGFR inhibitor use (mostly Cetuximab) instead of cisplatin, concurrently with radiotherapy; (2) radiotherapy dose reduction with concurrent chemotherapy (based on induction chemotherapy response); and (3) minimally invasive transoral surgery use followed by reduced adjuvant treatment accord‐

Since responses to treatment alter even between HPV(+) oropharyngeal cancer patients, defining appropriate patient subgroup is essential. It must be emphasized that all patients with HPV(+) oropharyngeal cancer are not suitable candidates for deintensification approaches. The clinical trial cohorts' analysis indicates some evidence. Some of them suggest that lifetime tobacco use history of >10 pack-years in HPV(+) patients with advanced regional nodal metastasis (N2b, N3) as well as smoking during radiotherapy decreases survival time (3-year overall survival rate of 70.8%) [16, 161]. The Italian study which reported that intermediaterisk patients are not convenient for deintensification trials confirmed these findings [171]. Furthermore, O'Sullivan et al. [27] demonstrated that the 3-year distant control rates of HPV(+) oropharyngeal cancer with advanced T stage (T4) or nodal (N3) was 72% and 78%, respectively [124]. Hence, late recurrences and relapses as distant metastasis are not rare in HPV(+) oropharyngeal cancer. These data indicate the presence of a limit of the biologic advantage provided by HPV positivity and may be helpful in determining patient subgroup that requires chemotherapy use in order to treat early micrometastasis. Both amount of smoking and locoregionally advanced disease have implications in designing deintensification trials for

Many clinical trials have been planned to evaluate the efficiency of treatment deintensification in HNC patients and recently they have been reviewed by Masterson et al. [152] in detail. Enrolling patients into these trials to define optimum treatment of HPV(+) oropharyngeal cancer is encouraged. Nevertheless, besides as a clinical trial, there is not sufficient data for the treatment reduction or modification according to HPV positivity until we receive the results

HPV(+) oropharyngeal cancer [167].

270 Human Papillomavirus - Research in a Global Perspective

compared with either p16 or HPV16 alone [170].

HPV(+) oropharyngeal cancer patients.

from these deintensification studies.

**7.4. Deintensification of treatment in HPV(+) oropharyngeal cancer**

ing to the histopathological properties of the excised tumor.

There are two prophylactic HPV vaccines available commercially; Gardasil® (Merck & Co., Whitehouse Station, NJ, USA) and Cervarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium). The virus-like proteins (VLPs) are used to create HPV major capsid protein L1 neutralizing antibodies. Gardasil is a quadrivalent L1 VLP recombinant vaccine protecting against HPV6, -11, -16, and -18, while Gardasil 9 provides protection against five additional HPV genotypes 16, 18, 31, 33, 45, 52, and 58. In contrast, Cervarix is a bivalent L1 VLP recombinant vaccine against HPV-16 and -18. Both HPV vaccines are highly effective in anogenital HPV infection prevention and consequent anal and cervical cancer development.

Despite the fact that the prophylactic value of vaccination for oropharyngeal cancer has not been proven in randomized trials yet, both the Cervarix and Gardasil vaccines are expected to prevent oropharyngeal HPV16 and -18 infections and consequent oropharyngeal cancer, since HPV16 is the most common type detected in oropharyngeal cancer [172]. The vaccination efficiency was estimated as 93.3% in 7466 women who were aged 18–25 years and randomized to HPV16/18 (Cervarix) vaccine versus hepatitis A vaccine as control for oral HPV 16/18 prevalence evaluation 4 years after vaccination [173]. In addition, cross-sectional study of US population as part of the National Health and Nutrition Examination Survey (NHANES) oral HPV infection prevalence was reduced significantly in 290 vaccinated women compared with 1985 unvaccinated women (0% vs. 0.5%) [54].

## **Author details**

Makbule Tambas1\*, Musa Altun2 and Deniz Tural3

\*Address all correspondence to: makbule\_tambas@hotmail.com

1 Department of Radiation Oncology, Okmeydani Training and Research Hospital, Istanbul, Turkey

2 Department of Radiation Oncology, Istanbul Faculty of Medicine, Istanbul University, Is‐ tanbul, Turkey

3 Department of Medical Oncology, Bakirkoy Dr. Sadi Konuk Education and Research Hos‐ pital, Istanbul, Turkey

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## **Preventive Strategies against Human Papillomaviruses**

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2013/07/23.10.1371/journal.pone.0068329

Naveed Shahzad, Muhammad Umer, Memoona Ramzan and Bilal Aslam

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62831

### **Abstract**

Human papillomavirus (HPV) infection is among the most common viral infections of the reproductive tract. Out of more than 100 different types of HPV identified so far, only a few (termed as "high-risk" subtypes) are associated with cervical cancer. On the other hand, "low-risk" subtypes are associated with genital warts and other benign changes in cervical and oral mucosa. Majority of the HPV infections usually clear up without any intervention within a few months. However, a fraction of HPV infections, such as those with types 16 and 18, can become persistent which may lead to the development of anogenital or cervical cancers. HPV subtypes 16 and 18 together are responsible for approximately 70% of all cervical cancer cases, the fourth major cause of cancer-related deaths in women. In the absence of any specific treatment options, preventive meas‐ ures are considered as cornerstone of strategies aimed at curbing the burden of this disease. This chapter presents a comprehensive review of strategies that can be employed to prevent and eradicate HPV infection. Minimizing the exposure to HPV risk factors such as unprotected sex, multiple sex partners, early age sex, and not being circumcised, can reduce the chances of getting HPV infection to a significant level. Mass screening programs have also been effective in HPV eradication. Nevertheless, immunization against HPV has proven to be the most promising strategy in fight against HPV. Virus-like particles based on bivalent, quadrivalent, and nonavalent anti-HPV vaccines have been licensed and are available in market under the trade names of Cervarix®, Gardasil®, and Gardasil9®, respectively. Various clinical trials and population-based studies have demonstrated high levels of efficacy for all the three vaccines in preventing typespecific malignancies.

**Keywords:** human papillomavirus (HPV), prevention of HPV, immunization, HPV vaccines, cervical cancer

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Although genital warts, papillomatous, and verrucous lesions of skin have been known to human beings since ancient times [1, 2], it was not until the dawn of twentieth century that infectious nature of these warts could be demonstrated [3]. Toward the end of twentieth century, data started to emerge that hinted toward a link between papillomavirus infection and cervical cancer [4]. It is now well established that this heterogeneous family of epithelio‐ tropic viruses is responsible for a spectrum of diseases that range from mild self-limiting anogenital warts and rare recurrent respiratory papillomatosis (RRP) to the penile, vaginal, vulvar, cervical, anal as well as pharyngeal carcinomas [5, 6].


**Table 1.** Diseases caused by HPV Infection and associated subtypes.

More than a 100 types of Human papillomaviruses (HPV) have so far been identified and are classified into five genera; alpha beta, gamma, mu, and nu [7]. HPV genus alpha, also known as genital or mucosal HPV, comprises of about 40 different subtypes and is the most important genus from medical point of view. These genital or mucosal HPV types are further categorized as low-risk (non-oncogenic) and high-risk (oncogenic) types [8] and infect epithelial cells of skin as well as mucosa of anogenital and upper aerodigestive tracts. There are about 15 highrisk subtypes associated with various cancers of anogenital tract as well as head and neck cancer [7]. Notable high-risk HPV genotypes include subtypes 16 and 18 which together account for more than 90% of cervical cancer cases worldwide [9]. Low-risk subtypes, such as subtypes 6 and 11, on the other hand, are responsible for benign changes in cervical tissue as well as genital warts [10]. The beta genus of HPV includes various cutaneous subtypes some of which might act as a cofactor in the development of non-melanoma skin cancer [11]. The genera gamma, mu, and nu also have cutaneous tropism and are rare [12]. **Table 1** summarizes the diseases caused by HPV infection and associated subtypes.

HPV is the most commonly occurring sexually transmitted infection [5] and around 300 million women worldwide are HPV carriers [13]. In USA, around 14 million people acquire HPV infection every year [14]. Young women are the most commonly affected with highest prevalence in <25 years age group [15, 16]. The incidence of HPV infection is directly correlated with the start of sexual activity as well as the number of sex partners. However, even the persons who remain monogamous for their whole lives are still at the risk of contracting this infection. The major route of HPV transmission is oro-genital and genital–genital contact, and it does not necessarily involve sexual intercourse [5]. More than 90% of HPV infections are cleared within 2 years without any major consequences on the health of patients [17]. A petite fraction of infections with certain types of HPV can persist and progress to cancer, however, this progression usually takes many years. Only persistent viral infection turns into tumors or cancer in the body [18].

Cervical cancer is the fourth major cause of cancer-related deaths in women, and more than 90% of cases are associated with high-risk HPV infection [19]. Infection with any of the 15 highrisk HPV types, particularly subtypes 16 and 18, is considered as necessary but not a sufficient cause of cervical cancer [20]. More than 500,000 new cases of cervical cancer are diagnosed every year out of which about 80% live in developing countries [10] and about 250,000 women die of this malignancy every year [15]. More than 90% of cervical cancer cases are curable with surgical and radiochemotherapeutic interventions if diagnosed at early stages.

Recent years have seen phenomenal success in fight against HPV infection and related cancers. However, the major focus of these efforts remained to be cancerous subtypes or at the best only a couple of warts causing subtypes like 6 and 11. Therefore, there is an urgent need to broaden the scope of preventive strategies to other clinically relevant subtypes as well. This chapter covers an expert commentary on various preventive as well as eradication strategies against HPV and methods being practiced routinely in developed and underdeveloped countries. Guidelines and bottlenecks established by WHO and other-related bodies in prevention and control of HPV will also be a part of this commentary in order to highlight strengths and shortcomings of prevention strategies currently in practice in various regions of the world. Since immunization has been proved the most promising method for HPV preven‐ tion, this chapter will focus mainly on the components of the immune system, passive and active immunity, mechanisms of vaccines for immune stimulation, and types of HPV vaccines available.

## **2. Preventive strategies for HPV**

**1. Introduction**

290 Human Papillomavirus - Research in a Global Perspective

**Cutaneous lesions**

**Mucosal lesions**

tract

Although genital warts, papillomatous, and verrucous lesions of skin have been known to human beings since ancient times [1, 2], it was not until the dawn of twentieth century that infectious nature of these warts could be demonstrated [3]. Toward the end of twentieth century, data started to emerge that hinted toward a link between papillomavirus infection and cervical cancer [4]. It is now well established that this heterogeneous family of epithelio‐ tropic viruses is responsible for a spectrum of diseases that range from mild self-limiting anogenital warts and rare recurrent respiratory papillomatosis (RRP) to the penile, vaginal,

**Disease Commonly associated HPV subtypes**

More than a 100 types of Human papillomaviruses (HPV) have so far been identified and are classified into five genera; alpha beta, gamma, mu, and nu [7]. HPV genus alpha, also known as genital or mucosal HPV, comprises of about 40 different subtypes and is the most important genus from medical point of view. These genital or mucosal HPV types are further categorized as low-risk (non-oncogenic) and high-risk (oncogenic) types [8] and infect epithelial cells of skin as well as mucosa of anogenital and upper aerodigestive tracts. There are about 15 highrisk subtypes associated with various cancers of anogenital tract as well as head and neck cancer [7]. Notable high-risk HPV genotypes include subtypes 16 and 18 which together account for more than 90% of cervical cancer cases worldwide [9]. Low-risk subtypes, such as

16

vulvar, cervical, anal as well as pharyngeal carcinomas [5, 6].

Verrucae vulgares, verrucae palmares et plantares 1, 2, 4 Bowen's disease 16 Butcher's warts 7 Verrucae planae 3, 10 EV-squamous cell carcinomas 5, 8 Epidermodysplasia verruciformis (EV) 3, 5, 8

Condylomataacuminata 6, 11 Laryngeal papillomatosis 6, 11 Buschke–Löwenstein tumor 6, 11 Bowenoidpapulosis, erythroplasia of Queyrat 16 Squamous intraepithelial neoplasias and invasive carcinomas of the anogenital

Heck's disease 13, 32

**Table 1.** Diseases caused by HPV Infection and associated subtypes.

As no specific treatments are available against HPV yet, therefore, more emphasis is put on the prevention rather than treating the infection. Many developed countries including USA, Australia, Canada, Brazil, Sweden, and United Kingdom have established national guidelines to defeat the HPV and associated cancers [21]. Notably, the established guidelines in men‐ tioned countries altogether focus mainly on the HPV-related cervical cancer in women only. There is no recommendation to screen men particularly and women under the age of 30 years. Similarly, only a small portion of management guidelines refer to other HPV-associated infections/cancers in both genders.

**Figure 1.** The possible ways for prevention and eradication of the HPV infections.

Despite millions of new HPV infections cases every year, only a few women infected even with hrHPV types manifest precancerous lesions and even fewer develop invasive cervical cancer. This difference in the ratio between HPV infections and HPV-related cancers clearly indicates the role of other risk factors that might be involved in the development of cervical cancer [22]. Current guidelines suggest that HPV infections can be avoided by minimizing the exposure to risk factors such as unprotected sex, multiple sex partners, early age sex, and not being circumcised. In addition to that, practice of mass screening in women aged 21–65 years has been declared a promising strategy to combat the burden of HPV [23]. Above all, vaccination against HPV in both men and women has proven to be the most criti‐ cal way in preventing the HPV infection. The possible way forward in handling the HPV infections are depicted in **Figure 1**.

### **2.1. Reduction of exposure to the risk factors**

Australia, Canada, Brazil, Sweden, and United Kingdom have established national guidelines to defeat the HPV and associated cancers [21]. Notably, the established guidelines in men‐ tioned countries altogether focus mainly on the HPV-related cervical cancer in women only. There is no recommendation to screen men particularly and women under the age of 30 years. Similarly, only a small portion of management guidelines refer to other HPV-associated

infections/cancers in both genders.

292 Human Papillomavirus - Research in a Global Perspective

**Figure 1.** The possible ways for prevention and eradication of the HPV infections.

Despite millions of new HPV infections cases every year, only a few women infected even with hrHPV types manifest precancerous lesions and even fewer develop invasive cervical cancer. This difference in the ratio between HPV infections and HPV-related cancers clearly indicates the role of other risk factors that might be involved in the development of cervical cancer [22]. Current guidelines suggest that HPV infections can be avoided by minimizing the exposure to risk factors such as unprotected sex, multiple sex partners, early age sex, and not being circumcised. In addition to that, practice of mass screening in women aged 21–65 years has been declared a promising strategy to combat the burden of HPV [23]. Almost all HPV infections are transmitted from skin to skin and sexual contact [5]. To the best of our knowledge, HPV transmission through body fluids and secretions has never been reported. However, HPV can be transferred vertically from infected mother to child during birth such as in papillomatosis [18, 22]. Risk factors associated with HPV infection and development of its long-term sequelae, particularly cancers, can be broadly categorized into two categories: those associated with HPV infection of mucosal layers lining the oral cavity and lungs and the second category that is associated with warts and cancers of anogenital tract.

Factors, such as smoking, alcoholism, drugs, and direct skin contact with infected person, can play a supportive role for initiating HPV infection, and thus cancer, in the mucosal layers lining the oral cavity and lungs. These types of cancers already constitute a smaller fraction among HPV-associated cancers. Nonetheless, oropharynx cancer has exhibited increasing trend in USA and other parts of the world since the last decade. The cancer of mucosal layers can be prevented by avoiding direct skin contact with infected person. Further, decrease of tobacco and alcohol can also be helpful in reducing head, neck, and oropharyngeal cancer [22].

Genital infections and ultimately cancers are commonly transmitted through sexual contact. Indeed, HPV is the most commonly occurring sexually transmitted virus. Different studies revealed that the sexual behavior including early age sex, multiple sexual partners, oral contraceptives, and co-infection with other sexually transmitted diseases predispose the HPV infection [5]. Data show that risk of HPV infection among women of 18–25 years of age with three life time sexual partners is more than double as compared to women of same age group with one life time partner [24]. Therefore, genital HPV infections can be prevented by reducing the number of sexual partners. Likewise, HPV burden is also linked with certain risk factor in male; for instance, circumcised males are associated with a lower risk of penile HPV infection [25]. Various cohort studies carried out for cervical cancer prevention has demonstrated that the use of alcohol and smoking along with poor and unhealthy sexual practices lead to cervical cancer in early ages [24, 26]. Vulvar, vaginal anal, and penile cancers are also attributed to same risk factors. Altogether, safe and ethical practices can reduce the spread of disease among population.

### **2.2. Screening methodologies**

Up till now, no precise guidelines have been put forward by medical organizations for the surveillance of all HPV types except for those associated with cancer. The available guidelines vary with the severity of pathogenesis and the level of gender involved. There is no require‐ ment of HPV screening for anogenital warts or papillomatosis. However, cervical cancer and precancerous lesions are strongly recommended to be screened at regular intervals for suspicion of cancer. In this regard, the mass screening is helpful in order to detect the virus at early stages before becoming drastic and uncontrollable. It is also helpful to diagnose the silent HPV infection where virus does not produce any disease symptoms. Common methods available for HPV screening are visual examination, cytology-based tests, and a few molecular assays. Although these methods are equally beneficial for detection of HPV in any part of the body, they are commonly practiced for the screening of cervical cancer only [27]. Researchers have also endorsed the implementation of these methods for the screening of HPV in anogen‐ ital warts and cancer, oropharyngeal cancer/infection, lung cancer, vaginal, and vulvar or penile cancer.

### *2.2.1. Cytology-based screening*

Since the most commonly performed HPV screening involves the cervical cancer, therefore, most of the tests and data are available in this regard. The Papanicolaou test usually known as Pap test is the most common method of cervical screening. This test is applied to detect abnormal cervical cells, precancerous lesions, or early stage cancerous lesions among women between ages of 30 and 65 years. Moreover, it is practiced equally for both non-HPV and HPVinfected cervix. Due to accuracy and ease of performance, this test has become the cornerstone of cervical HPV screening strategies [28]. Unfortunately, no Pap-like test is available for the screening of HPV among men [29]. Similarly, histopathological examination is the only method carried out for anogenital, vulvar, vaginal, or oropharyngeal cancers to detect the involvement of HPV in these cases.

#### *2.2.2. Visual examination*

Regularly repeated Pap smears followed by appropriate treatment has saved the lives of millions of women in developed countries [27]. But HPV infections and associated cancers still pose a burden in less developed countries where poor socioeconomic conditions prevail. Therefore, in such low resource set ups such as Africa, Asia, South and Central America visual examination is recommended in screening programs [14]. This paradigm shift in screening programs has occurred due to the moderate sensitivity of cytology-based tests. Moreover, quality assurance and high possibility of false positives has led to the evaluation of alternative methods such as visual examination and HPV DNA testing. Visual inspection with 3–5% acetic acid or Lugol's iodine is performed to observe abnormal lesions in HPV associated cervical and penile cancers. However, the application of acetic acid has been most widely evaluated as compared to visual inspection with iodine as most of the cohort and field studies in the areas of Africa, India, Bangladesh, Thailand, China, and Philippines, report the utilization of acetic acid before visual examination. Altogether, these studies have suggested visual screening as an effective, acceptable, safe, accurate, and cost-effective method for the screening of cervical cancer [14].

However, visual inspection is not feasible for the detection of HPV in oropharyngeal or anogenital cancers. But genital warts or other HPV warts can be identified by their peculiar characteristics on visual examination [30]. In addition to all the merits of visual exam, one needs to be sure for the HPV genotype involved in the infection. For this purpose, some tests with high accuracy and efficiency are required such as nucleic acid testing.

### *2.2.3. Molecular testing of HPV*

early stages before becoming drastic and uncontrollable. It is also helpful to diagnose the silent HPV infection where virus does not produce any disease symptoms. Common methods available for HPV screening are visual examination, cytology-based tests, and a few molecular assays. Although these methods are equally beneficial for detection of HPV in any part of the body, they are commonly practiced for the screening of cervical cancer only [27]. Researchers have also endorsed the implementation of these methods for the screening of HPV in anogen‐ ital warts and cancer, oropharyngeal cancer/infection, lung cancer, vaginal, and vulvar or

Since the most commonly performed HPV screening involves the cervical cancer, therefore, most of the tests and data are available in this regard. The Papanicolaou test usually known as Pap test is the most common method of cervical screening. This test is applied to detect abnormal cervical cells, precancerous lesions, or early stage cancerous lesions among women between ages of 30 and 65 years. Moreover, it is practiced equally for both non-HPV and HPVinfected cervix. Due to accuracy and ease of performance, this test has become the cornerstone of cervical HPV screening strategies [28]. Unfortunately, no Pap-like test is available for the screening of HPV among men [29]. Similarly, histopathological examination is the only method carried out for anogenital, vulvar, vaginal, or oropharyngeal cancers to detect the involvement

Regularly repeated Pap smears followed by appropriate treatment has saved the lives of millions of women in developed countries [27]. But HPV infections and associated cancers still pose a burden in less developed countries where poor socioeconomic conditions prevail. Therefore, in such low resource set ups such as Africa, Asia, South and Central America visual examination is recommended in screening programs [14]. This paradigm shift in screening programs has occurred due to the moderate sensitivity of cytology-based tests. Moreover, quality assurance and high possibility of false positives has led to the evaluation of alternative methods such as visual examination and HPV DNA testing. Visual inspection with 3–5% acetic acid or Lugol's iodine is performed to observe abnormal lesions in HPV associated cervical and penile cancers. However, the application of acetic acid has been most widely evaluated as compared to visual inspection with iodine as most of the cohort and field studies in the areas of Africa, India, Bangladesh, Thailand, China, and Philippines, report the utilization of acetic acid before visual examination. Altogether, these studies have suggested visual screening as an effective, acceptable, safe, accurate, and cost-effective method for the screening of cervical

However, visual inspection is not feasible for the detection of HPV in oropharyngeal or anogenital cancers. But genital warts or other HPV warts can be identified by their peculiar characteristics on visual examination [30]. In addition to all the merits of visual exam, one

penile cancer.

*2.2.1. Cytology-based screening*

294 Human Papillomavirus - Research in a Global Perspective

of HPV in these cases.

*2.2.2. Visual examination*

cancer [14].

Molecular tests offer more rapid and robust screening of HPV and its particular genotype involved in the infection. Based on the nucleic acid detection of virus in clinical specimen, these tests are helpful to detect the virus before the appearance of any cellular abnormalities [27]. Numerous molecular screening modalities have been developed for the detection of hrHPV and lrHPV types among the subjects showing abnormal Pap test. These tests include Hybrid Capture 2 assay, Cervista High Risk HPV assay, Cobas 4800 HPV test, Abbot real Time High Risk HPV test, Papillocheck HPV screening, APTIMA HPV assay, E6/E7 quantitative PCR, GP5+/6+ PCR, and Matrix-assisted laser desorption/ionization time-of-light (MALDI-TOF). Despite the availability of a large number of commercial assays, only Hybrid Capture 2 assay, GP5+/6+ PCR, Cobas 4800 HPV test and APTIMA HPV assay are commonly applied [9, 31]. These four tests were validated in various cohort studies and large randomized trials carried out for 8 years or more in different parts of the world. Moreover, FDA and WHO have recommended these tests to be used in first-line primary screening. They can be used both in adjunct to cytology assays or alone for screening purpose [32]. Other mentioned tests are in the process of validation on large and small cohorts but they still need approval from FDA and other relevant governing bodies.

Usually, HPV testing with above-mentioned assays is not practiced in mass screening of men where simple PCR is performed for the detection of HPV in penile and anal cancer. Most of the testing data constitutes the cross-sectional studies on patients with sexually transmitted diseases (STDs), men having HPV infected partner, military recruits, and few small-scale studies [5]. Similarly, no research or analytical data have been found in support of practicing nucleic acid based assays for the detection of HPV in other associated cancers. But there lies a great potential in these methods for the detection of HPV due to high sensitivity and specificity as compared to visual or cytological examination.

### **2.3. Immunization**

### *2.3.1. Immunity and principles of vaccine development*

A fair comprehension on the basic function of the immune system is absolutely necessary in order to understand the mechanism of vaccines preparation and the prescribed ways to use them. However, the detailed discussion is beyond the scope of this chapter. The immune system is a multifaceted system comprising of interacting cells, tissues, and organs whose prime purpose is to identify and protect the body from pathogens and other potentially damaging foreign objects known as antigens. It is generally divided into two categories: "Innate" and "Adaptive" Immune systems that interact with each other to provide an effective immune response. The Innate immune system is first line of defense against invading patho‐ gens and is equipped with physical and chemical barriers and some non-specific immune cells such as phagocytic leukocytes, dendritic cells, and natural killer cells which come into action immediately (within hours) after the manifestation of the antigens in the body [33]. Though non-specific but innate immunity plays a significant role in controlling infections until the initial adaptive response takes place [34], the adaptive immune response is composed of two arms: the humoral and cell-mediated response. The humoral response involves the production of antibodies by B-lymphocytes; whereas, cell-mediated response includes the specific cells known as T-lymphocytes which facilitate the elimination of foreign substances. The adaptive immune system provides a more versatile means of security as it manifests wonderful specificity for its target antigens and confers increased protection against subsequent reinfection with the same pathogen [30].

The active and passive ways are two basic mechanisms for acquiring the immunity. Active immunity emerges from the person's own immune response either as a result of exposure to a live pathogen or induced by the vaccine. It involves the production of antigen specific antibody or cellular response of T-lymphocytes. This kind of immunity is very long lasting, usually continues for life time in the form of immunologic memory mediated by memory B cells which survive in the blood after infection, and generate antibodies very quickly in case of re-exposure to the same antigen providing the rapid protection [35]. Some vaccines create the immune response analogous to natural infection without causing a disease signs and symptoms. Likewise, vaccine-mediated immune response also involve production of immu‐ nologic memory similar to the natural infection [36]. Unlike active immunity, passive immun‐ ity is a short-term immunization in which antibodies from another organism are transferred to the recipient. that is, antibodies are not generated by the immune cells of recipient. This type of immunity protects the host temporarily as the injected antibodies will be degraded over the short time span (weeks to months) leaving the host no more protected. Numerous host factors such as age, genetics, co infection of other disease, immune status, and nutritional factors may influence the response of passive immunization [37, 38].

#### *2.3.2. Classification of vaccines*

As a matter of fact, the immune response extremely diverges with antigenic variation. Therefore, a fundamental information of antigen properties; for instance, how it infect cells and what is the response of immune system to that antigen, must be considered for designing vaccines. The most efficient immune response is produced against live antigens. However, purified products from the microbes may also be used to formulate vaccines, though the immune response will not be much effective [39]. Likewise, recent developments in molecular biology enabled scientists to devise the alternative methods of vaccine production. Followings are different possibilities:


Greater part of the vaccines being used today is based on the use of whole virus, whether, live attenuated or killed. Live attenuated vaccines contain the laboratory prepared version of the viruses which are usually attenuated by passaging in cultures. The attenuated virus retains the replication ability inside the host and induces immunity but lacks pathogenecity. In fact, the live-attenuated vaccines generate nearly identical immune response to that of natural infection [35]. Conversely, inactivated vaccines consist of pathogens that are usually inacti‐ vated by the effect of heat or chemicals. Inactivated strains lack replication ability within the host and cannot produce disease even in the immunocompromised individuals. Unlike, liveattenuated vaccines, inactive vaccines produce only humoral but not the cellular response. The protection in case of inactive vaccine is for limited time period because the antibody titer declines after some time [37].

such as phagocytic leukocytes, dendritic cells, and natural killer cells which come into action immediately (within hours) after the manifestation of the antigens in the body [33]. Though non-specific but innate immunity plays a significant role in controlling infections until the initial adaptive response takes place [34], the adaptive immune response is composed of two arms: the humoral and cell-mediated response. The humoral response involves the production of antibodies by B-lymphocytes; whereas, cell-mediated response includes the specific cells known as T-lymphocytes which facilitate the elimination of foreign substances. The adaptive immune system provides a more versatile means of security as it manifests wonderful specificity for its target antigens and confers increased protection against subsequent re-

The active and passive ways are two basic mechanisms for acquiring the immunity. Active immunity emerges from the person's own immune response either as a result of exposure to a live pathogen or induced by the vaccine. It involves the production of antigen specific antibody or cellular response of T-lymphocytes. This kind of immunity is very long lasting, usually continues for life time in the form of immunologic memory mediated by memory B cells which survive in the blood after infection, and generate antibodies very quickly in case of re-exposure to the same antigen providing the rapid protection [35]. Some vaccines create the immune response analogous to natural infection without causing a disease signs and symptoms. Likewise, vaccine-mediated immune response also involve production of immu‐ nologic memory similar to the natural infection [36]. Unlike active immunity, passive immun‐ ity is a short-term immunization in which antibodies from another organism are transferred to the recipient. that is, antibodies are not generated by the immune cells of recipient. This type of immunity protects the host temporarily as the injected antibodies will be degraded over the short time span (weeks to months) leaving the host no more protected. Numerous host factors such as age, genetics, co infection of other disease, immune status, and nutritional factors may

As a matter of fact, the immune response extremely diverges with antigenic variation. Therefore, a fundamental information of antigen properties; for instance, how it infect cells and what is the response of immune system to that antigen, must be considered for designing vaccines. The most efficient immune response is produced against live antigens. However, purified products from the microbes may also be used to formulate vaccines, though the immune response will not be much effective [39]. Likewise, recent developments in molecular biology enabled scientists to devise the alternative methods of vaccine production. Followings

infection with the same pathogen [30].

296 Human Papillomavirus - Research in a Global Perspective

influence the response of passive immunization [37, 38].

**•** Whole organism vaccines (Live attenuated and inactivated vaccines)

**•** Subunit vaccines (Subvirion, toxoid, and capsule polysaccharides vaccines)

*2.3.2. Classification of vaccines*

are different possibilities:

**•** Recombinant vaccines

**•** DNA vaccines

Subunit vaccines include purified macromolecules (antigens) rather than the entire organism. More precisely, major antigenic sites of viral antigens that are recognized efficiently by antibodies or T cells are identified and subjected to purification. These purified molecules are often coupled to an immunogenic carrier protein or adjuvant, for instance, an aluminum salt in order to enhance their immunogenic potential. Immunologists obtain subunit vaccines either by breaking the microbes with chemicals in the laboratory or using recombinant DNA technology [40].

The development of DNA vaccines has ushered the immunization technology into a new exciting era. Precisely, DNA vaccines employ only the genes encoding the immunogenic antigen. Genes of interest are injected either alone (naked) or mixed with molecules that facilitate their entry into the cell, by taking up some cells which prepare the antigen under the instructions of foreign DNA. This way the host cells become vaccines making factories producing the antigens required to evoke the immune response [31]. The immune response to DNA vaccines is very strong and involves cellular and antibodies reaction. Some serious concerns are also linked with DNA vaccine, for instance, the integration of foreign DNA in host chromosome where it can manipulate the expression of onco- or tumor suppressor genes [41].

Only a handful of viral infections can be prevented using conventional live attenuated or killed vaccines. However, advances in recombinant DNA technology have opened up novel avenues for the development of vaccines against organisms for which development of conventional vaccines has so far proved unsuccessful. Virus-like particles (VLPs) are an efficient recombi‐ nant DNA technology-based tool which have been used as carriers of other organisms' genes. Immunogenic protein/s of a particular microorganism is introduced into harmless and weakened viruses which act as a vehicle to carry these proteins of interest to the desired site/ organ inside the body. Similarly, attenuated bacteria are used as a vector where they display the antigens of other microbes on their surface and induce a strong immune response [42]. Recombinant vaccines mimic the natural infection in producing the immune response and stimulate both humoral and cellular immunity [15]. Five genetically engineered vaccines including Human papillomavirus (HPV) vaccine are being used in USA these days. The pros and cons of all above discussed vaccine types are summarized in the **Table 2**.


**Table 2.** General features of various vaccines used for immunization against HPV.

### *2.3.3. HPV vaccines*

The HPV vaccines in use are based on recombinant DNA technology where the major capsid proteins L1 of HPV strains are synthesized and expressed in *in vitro* system. This protein is capable of self-assembling into HPV virus-like particles (VLPs) which display the morpho‐ logical and antigenic properties similar to HPV virion but lack the viral DNA, therefore not capable of producing cancer. These HPV VLPs are used to synthesize HPV subunit vaccines [43]. All HPV vaccines being used today contain an adjuvant but not a preservative. The VLPsbased vaccines are highly immunogenic and generate even stronger response than the natural HPV infection [44]. All HPV vaccines available today and some other viral vaccines for instance Hepatitis B vaccine are VLP based.

#### *2.3.4. Currently available HPV vaccines*

An explosion of interest has been observed in vaccine production against HPV in recent years. Unfortunately, after many scientific endeavors, vaccines are not available against all strains of HPV; however, scientists are manufacturing newer vaccines including more strains of HPV. Till now, three HPV vaccines have been licensed by Food and Drug Administration (FDA) and equally recommended by Advisory Committee on Immunization Practices (ACIP).

## *2.3.4.1. CervarixTM*

**Type of vaccines**

Subunit vaccines

Recombinant vaccines

*2.3.3. HPV vaccines*

Hepatitis B vaccine are VLP based.

*2.3.4. Currently available HPV vaccines*

Live attenuated vaccines

**Features Dose Booster shots**

298 Human Papillomavirus - Research in a Global Perspective

**Requirement of adjuvant**

Killed vaccines High Multiple Yes No Temporary Can be

DNA vaccines Low Single No No Long

Low Single No Possible Long

**Table 2.** General features of various vaccines used for immunization against HPV.

Low Single No Possible More than

**Virulence Duration of efficacy**

High Multiple Yes No Short Safe as compared

10 years

lasting

lasting

The HPV vaccines in use are based on recombinant DNA technology where the major capsid proteins L1 of HPV strains are synthesized and expressed in *in vitro* system. This protein is capable of self-assembling into HPV virus-like particles (VLPs) which display the morpho‐ logical and antigenic properties similar to HPV virion but lack the viral DNA, therefore not capable of producing cancer. These HPV VLPs are used to synthesize HPV subunit vaccines [43]. All HPV vaccines being used today contain an adjuvant but not a preservative. The VLPsbased vaccines are highly immunogenic and generate even stronger response than the natural HPV infection [44]. All HPV vaccines available today and some other viral vaccines for instance

An explosion of interest has been observed in vaccine production against HPV in recent years. Unfortunately, after many scientific endeavors, vaccines are not available against all strains of HPV; however, scientists are manufacturing newer vaccines including more strains of HPV.

 **Potential advantages**

like

to live

Produce immunity

natural infection

administered to immunecompromised patients

attenuated vaccines

Safe, cost-effective, no side effects

Cost-effective, easy production **Limitations**

Instable, heat labile

Can only activate humoral immune response

Sometimes may produce toxins, initiate hypersensitivity response

May trigger the expression of onco-

May cause contagious spread of virus

genes

The CervarixTM is a bivalent HPV vaccine marketed by GlaxoSmithKline Biologicals, Belgium, which protects the host from two most lethal types of HPV, 16 and 18, that are responsible for 70% cases of cervical cancer. These HPV types are also responsible for genital warts as well as head and neck cancer [21]. The CervarixTM contains L1 capsid proteins from HPV 16 and 18 in the form of VLPs and an adjuvant AS04 containing: 3-O-desacyl-4'-monophosphoryl lipid A. In fact, the L1 protein from HPV 16 and 18 strains are cloned in a baculovirus vector and expressed in Hi-5 Rix4446 cells that are derived from insect Trichoplusia. The VLPs for these strains are generated separately and then combined together. In addition to protection against HPV16 and 18, this vaccine has manifested cross reactivity with HPV 45 and 31. However, it does not provide protection in case the women have previously been exposed to one of the HPV strains. Clinical data in 2009 have shown that Cervarix TM was still affective after 7 years of vaccine administration showing that protection provided by this vaccine is long lasting [45].

### *2.3.4.2. Gardasil®*

The quadrivalent HPV vaccine Gardasil® is being marketed by Merck & Co. Inc, against 4 HPV types: 16, 18, 6, and 11. The HPV strains 6 and 11 altogether are responsible for 90% of genital warts burden [46]. The VLPs from L1 capsid protein of each strain are produced using a recombinant Saccharomyces Pombe vector and mixed with alum adjuvant for better delivery. In addition to contributing protection against mentioned HPV types, this vaccine manifested a fractional protection against some other HPV types which are responsible for anal, vulvar, and vaginal cancer as well as genital warts [47].

### *2.3.4.3. Gardasil 9®*

Very recently, in 2014, another HPV vaccine namely Gardsil9® was approved by US Food and Drug Administration. It is 9-valent recombinant vaccine which provides protection against wide range of HPV strains. It was recommend for the prevention of cervical, vulvar, anal, and vaginal cancers caused by HPV 16, 18, 31, 33, 45, 52, and 58, genital warts caused by HPV 6 and 11 and dysplastic lesions caused by HPV types: 16, 18, 31, 33, 45, 52, and 58 [12, 48]. Both Gardasil and Gardasil9 HPV vaccines are recommended for males also. In addition, both Gardasil and Gardasil 9 are recommended by FDA for males against the HPV-caused precan‐ cerous and cancerous lesions, and genital warts.

All three available HPV vaccines are administered into the body by a series of three intramus‐ cular shots during a period of 6 months. The first shot is followed by second and third shots after 2 and 6 months, respectively.

## **3. Effective implementation of HPV vaccines**

### **3.1. Age for HPV vaccination**

The Centers for Disease Control and Prevention (CDC) recommends the routine administra‐ tion of HPV vaccines in preteen boys and girls at the age of 11 or 12 before their first potential exposure to HPV [38]. Likewise, a more vigorous immune response is produced against vaccines at this age. However, if they are not fully vaccinated at this age, it is also recommended that women can get vaccinated at age 26 and boys and men at age 21. Recently, FDA has approved the Gardasil® and Gardsil®9 use in both male and female ages 9 through 26 [49]. Young homosexual men with weakened immune response may also get vaccine until they are 27. No vaccine is licensed yet in both male and female over the age of 27 years. However, the HPV vaccines can be given at the same age, similar to other age-specific vaccines for instance, tetanus toxoid, acellular pertussis vaccine, influenza vaccine, and hepatitis B vaccine. The HPV vaccine-targeted population is further enlisted in **Table 3**.


**Table 3.** List of possible candidates who may or may not be safely administered with HPV vaccines.

### **3.2. HPV vaccine efficacy**

The available HPV vaccines target the HPV types that most commonly cause cervical cancer and genital warts. Several studies have been conducted for bivalent and quadrivalent HPV vaccines to check their efficacy in young women of age between 15 and 25 years. These studies demonstrated that antibody response against the included types of HPV is generated approx‐ imately 1 month after the 3 shots of HPV vaccines in 99% of studied female population [38]. Clinical trials have demonstrated that the bivalent vaccine is 93% efficient in preventing cervical cancers caused by HPV 16 and 18 in women who had not been previously exposed to those strains [50]. The quadrivalent HPV vaccines have demonstrated more promising results as they were found 100% efficient in women for preventing cervical, vulvar, vaginal cancers along with genital warts due to HPV types 16, 18, 6, and 11 [48, 51]. The quadrivalent vaccine was equally effective in controlling genital warts and anal precancerous lesions of male. Besides that HPV vaccines have no therapeutic effects on HPV caused diseases and do not confer protection to the host already infected with those HPV types [52].

### **3.3. HPV vaccines safety**

**3. Effective implementation of HPV vaccines**

vaccine-targeted population is further enlisted in **Table 3**.

Lactating mothers Pregnant woman

Females with abnormal Pap test Patients with acute illness

**Persons who can receive HPV vaccine Persons who cannot receive HPV vaccine** Patients with HPV positive test Patients with history of hypersensitivity

**Table 3.** List of possible candidates who may or may not be safely administered with HPV vaccines.

confer protection to the host already infected with those HPV types [52].

Persons who may develop allergies to yeast, latex or any vaccine component

The available HPV vaccines target the HPV types that most commonly cause cervical cancer and genital warts. Several studies have been conducted for bivalent and quadrivalent HPV vaccines to check their efficacy in young women of age between 15 and 25 years. These studies demonstrated that antibody response against the included types of HPV is generated approx‐ imately 1 month after the 3 shots of HPV vaccines in 99% of studied female population [38]. Clinical trials have demonstrated that the bivalent vaccine is 93% efficient in preventing cervical cancers caused by HPV 16 and 18 in women who had not been previously exposed to those strains [50]. The quadrivalent HPV vaccines have demonstrated more promising results as they were found 100% efficient in women for preventing cervical, vulvar, vaginal cancers along with genital warts due to HPV types 16, 18, 6, and 11 [48, 51]. The quadrivalent vaccine was equally effective in controlling genital warts and anal precancerous lesions of male. Besides that HPV vaccines have no therapeutic effects on HPV caused diseases and do not

The Centers for Disease Control and Prevention (CDC) recommends the routine administra‐ tion of HPV vaccines in preteen boys and girls at the age of 11 or 12 before their first potential exposure to HPV [38]. Likewise, a more vigorous immune response is produced against vaccines at this age. However, if they are not fully vaccinated at this age, it is also recommended that women can get vaccinated at age 26 and boys and men at age 21. Recently, FDA has approved the Gardasil® and Gardsil®9 use in both male and female ages 9 through 26 [49]. Young homosexual men with weakened immune response may also get vaccine until they are 27. No vaccine is licensed yet in both male and female over the age of 27 years. However, the HPV vaccines can be given at the same age, similar to other age-specific vaccines for instance, tetanus toxoid, acellular pertussis vaccine, influenza vaccine, and hepatitis B vaccine. The HPV

**3.1. Age for HPV vaccination**

300 Human Papillomavirus - Research in a Global Perspective

Patients suffering from any mild

disease/immunocompromised

**3.2. HPV vaccine efficacy**

Large-scale clinical trials have confirmed the safety of vaccine [53]. However, common minor side effects such as pain, redness, fever, dizziness, and nausea could be observed. The medical procedure of injecting HPV vaccines may cause syncope (to faint) in teens or preteens such as other medical procedures. Being safe to use, 46 million doses of HPV vaccine have been distributed in United States as of June 2012 [54].

### **3.4. Impact of HPV vaccination**

In general, vaccines are considered the most victorious medical intervention because they have provided protections against various diseases axf nd saved the death of millions of people [55]. In fact, HPV vaccines have been proved to be an important strategy for a notable decrease in the global burden of cervical cancer and genital warts. According to an estimate, the common use of vaccine during last decade has reduced cervical cancer deaths by 50% [56]. In addition, some additional long-term benefits are also associated with HPV vaccination such as it shows marked reduction in the prevalence of high-grade lesions CIN grade 2 and 3 [57]. The reduction in percentage of cervical associated deaths are further anticipated to rise up to 70% by next few decades when more vaccines would be available against a wide range of HPV strains.

## **4. Public awareness**

Biomedical scientists have succeeded to develop reasonable approaches to cope with the obnoxious HPV infection. However, the success of these methods relies largely in creating awareness among general public about HPV infection and cervical cancer particularly in countries where inadequate attention is paid to the health problems. At first, the knowledge about HPV infection and its relation to anogenital as well as cervical cancer must be tailored in a very comprehensive and easily understandable way for general public in the form of booklets and brochures. The cultural and religious aspects should also be considered while devising an HPV prevention strategy. The dissemination of information should be ensured as much as possible through medical practitioners, teachers, and other sectors of the society. Likewise, the parents should be convinced and encouraged to get their child vaccinated at preteen ages. Both paper and electronic media should play a constructive role in spreading the information about HPV.

### **5. Conclusions**

HPV is the main sexually transmitted viral infection which is associated with the cancers of oral cavity and reproductive tract of both male and female. In the absence of particular treatment for obnoxious HPV infection, the prevention strategies have been centered upon. The prevention paradigm against HPV infection must be multipronged. Briefly, to fend off HPV infection systematically the armamentarium should include avoiding risk factors which support the establishment of HPV infection such as multiple sex partners, early age sex, and unprotected sex, regular screening for cervical cancer, and administration of HPV vaccines. Indeed, during last few years, it has been revealed that early and specific diagnosis in combi‐ nation with effective therapeutic intervention could be the pragmatic and preeminent choice to overcome HPV-related diseases. In addition to that, several molecular therapeutic strategies can prove to be the indispensable allies in this quest against HPV infection. However, vacci‐ nation at the age of 10–12 in both genders is even a better choice since it provides immunity even before the first exposure to HPV lethal strains. The bivalent, quadrivalent, and nanovalent HPV vaccines have been successfully used in developed countries during last decade and proved to be highly efficient and safe to use. Theses vaccines not only provided a significant protection against highly virulent HPV strains against which they were designed, but also showed considerable seroconversion rate and lowered the occurring of other HPV-related abnormalities such as CIN and genital warts.

### **6. Future perspectives**

Currently, no specific therapies are available for HPV infected patients. Therefore, there is an urgent need to invest our efforts in developing novel drugs against HPV. Moreover, the costs associated with HPV prevention and therapy is so far among the major hurdles in eradication of this problem. Considering the fact that more than 80% of HPV positive cases reside in lowand middle-income countries, accessibility and cost-effectiveness of any new drugs should also be kept in consideration. Any future HPV strategies must also take into account the cultural and religious stigma attached to vaccination in general and HPV in particular. Necessary measures should be devised and implemented in order to do away with these stigmas. There is also a need to devise and implement global anti-HPV vaccination campaigns for women of developing countries.

Development of new, sensitive, and cost-effective diagnostic tests is also one of the areas which demands high attention. To overcome this issue in developing countries, a sufficient advance‐ ment in diagnostics is mandatory. Although, screening and vaccination are being applied successfully in different parts of the world, HPV is still causing a significant number of deaths per year [58]. Likewise, quality control and assurance is another great hurdle towards the success of currently proposed modalities for elimination of HPV [20]. Keeping in view the given scenario, updated screening, and management guidelines are needed.

### **Author details**

Naveed Shahzad1\*, Muhammad Umer2 , Memoona Ramzan1 and Bilal Aslam3


### **References**

Indeed, during last few years, it has been revealed that early and specific diagnosis in combi‐ nation with effective therapeutic intervention could be the pragmatic and preeminent choice to overcome HPV-related diseases. In addition to that, several molecular therapeutic strategies can prove to be the indispensable allies in this quest against HPV infection. However, vacci‐ nation at the age of 10–12 in both genders is even a better choice since it provides immunity even before the first exposure to HPV lethal strains. The bivalent, quadrivalent, and nanovalent HPV vaccines have been successfully used in developed countries during last decade and proved to be highly efficient and safe to use. Theses vaccines not only provided a significant protection against highly virulent HPV strains against which they were designed, but also showed considerable seroconversion rate and lowered the occurring of other HPV-related

Currently, no specific therapies are available for HPV infected patients. Therefore, there is an urgent need to invest our efforts in developing novel drugs against HPV. Moreover, the costs associated with HPV prevention and therapy is so far among the major hurdles in eradication of this problem. Considering the fact that more than 80% of HPV positive cases reside in lowand middle-income countries, accessibility and cost-effectiveness of any new drugs should also be kept in consideration. Any future HPV strategies must also take into account the cultural and religious stigma attached to vaccination in general and HPV in particular. Necessary measures should be devised and implemented in order to do away with these stigmas. There is also a need to devise and implement global anti-HPV vaccination campaigns

Development of new, sensitive, and cost-effective diagnostic tests is also one of the areas which demands high attention. To overcome this issue in developing countries, a sufficient advance‐ ment in diagnostics is mandatory. Although, screening and vaccination are being applied successfully in different parts of the world, HPV is still causing a significant number of deaths per year [58]. Likewise, quality control and assurance is another great hurdle towards the success of currently proposed modalities for elimination of HPV [20]. Keeping in view the

, Memoona Ramzan1

and Bilal Aslam3

given scenario, updated screening, and management guidelines are needed.

1 School of Biological Sciences, University of the Punjab, Lahore, Pakistan

2 National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

3 Department of Microbiology, Government College University Faisalabad, Pakistan

\*Address all correspondence to: hnaveed.shahzad@gmail.com

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302 Human Papillomavirus - Research in a Global Perspective

**6. Future perspectives**

for women of developing countries.

Naveed Shahzad1\*, Muhammad Umer2

**Author details**


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## *Edited by Rajamanickam Rajkumar*

This book is a feast of knowledge, yet a balanced diet of healthy foods. There are high values of rich essential nutrients from top-quality medical research. But they are made easily digestible and absorbable, even by health care providers and planners, working in resource-limited settings, in all parts of the world, through social implications and community applications. All the chapters are value-added master pieces. The book would serve both as a scientific reference guide and a practical work manual. The authors, editor, and Intech publishers, together, are pleased to provide the readers a precious blend of scientific excellence and social relevance, for health empowerment, globally. We wish the readers great success, savoring science and sociology together.

Human Papillomavirus - Research in a Global Perspective

Human Papillomavirus

Research in a Global Perspective

*Edited by Rajamanickam Rajkumar*

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