Epidemiology and Pathogenesis of Human Papillomavirus

**19**

**Chapter 2**

**Abstract**

Human Papillomavirus and

Cervical cancer is by far the most common HPV-related disease. About 99.7% of cervical cancer are caused by persistent genital high-risk human papillomavirus (HPV) infection. Worldwide, cervical cancer is one of the most common cancer in women with an estimated 528,000 new cases reported in 2012. Most HPV infections clear spontaneously but persistent infection with the oncogenic or high-risk types may cause cancer of the oropharynx and anogenital regions. The virus usually infects the mucocutaneous epithelium and produces viral particles in matured epithelial cells and then causes a disruption in normal cell-cycle control and the promotion of uncontrolled cell division leading to the accumulation of genetic damage. There are currently two effective prophylactic vaccines against HPV infection in many developed countries and these comprise of HPV types 16 and 18, and HPV types 6, 11, 16 and 18 virus-like particles. HPV testing in the secondary prevention of cervical cancer is clinically valuable in triaging low-grade cytological abnormalities and is also more sensitive than cytology as a primary screening. If these prevention strategies can be implemented in both in developed and developing countries,

**Keywords:** cervical cancer, high-risk HPV, HPV vaccines, screening, triaging

Human papillomavirus (HPV) is the commonest viral infection of the reproductive tract and is one of the most common causes of sexually transmitted infection worldwide [1]. Even though it is sexually transmitted, HPV transmission does not require penetrative sexual intercourse. Skin-to-skin genital contact is a wellestablished mode of transmission. Over 70% of sexually active women and men will be infected at some point in their lives and some may even be infected on more than one occasion [2]. The peak period for acquiring HPV infection is shortly after becoming sexually active. The infection usually clears up spontaneously within a few months after the acquisition with about 90% clearing within 2 years. There are over 200 HPV types recognized based on DNA sequence data showing genomic differences, and many of these are harmless. HPV can infect basal epithelial cells of the mucocutaneous membrane, and it is associated with a variety of clinical conditions that range from innocuous lesions to cancer. Most of the infections are benign, causing lesions such as cutaneous warts on the hands, feet and anogenital regions. Warts are areas of hypertrophied skin filled with keratin and are mainly a cosmetic nuisance; generally, they resolve spontaneously within 1–5 years. Only a

Cervical Cancer

*Kehinde Sharafadeen Okunade*

many thousands of lives could be saved.

**1. Introduction**

### **Chapter 2**

## Human Papillomavirus and Cervical Cancer

*Kehinde Sharafadeen Okunade*

### **Abstract**

Cervical cancer is by far the most common HPV-related disease. About 99.7% of cervical cancer are caused by persistent genital high-risk human papillomavirus (HPV) infection. Worldwide, cervical cancer is one of the most common cancer in women with an estimated 528,000 new cases reported in 2012. Most HPV infections clear spontaneously but persistent infection with the oncogenic or high-risk types may cause cancer of the oropharynx and anogenital regions. The virus usually infects the mucocutaneous epithelium and produces viral particles in matured epithelial cells and then causes a disruption in normal cell-cycle control and the promotion of uncontrolled cell division leading to the accumulation of genetic damage. There are currently two effective prophylactic vaccines against HPV infection in many developed countries and these comprise of HPV types 16 and 18, and HPV types 6, 11, 16 and 18 virus-like particles. HPV testing in the secondary prevention of cervical cancer is clinically valuable in triaging low-grade cytological abnormalities and is also more sensitive than cytology as a primary screening. If these prevention strategies can be implemented in both in developed and developing countries, many thousands of lives could be saved.

**Keywords:** cervical cancer, high-risk HPV, HPV vaccines, screening, triaging

### **1. Introduction**

Human papillomavirus (HPV) is the commonest viral infection of the reproductive tract and is one of the most common causes of sexually transmitted infection worldwide [1]. Even though it is sexually transmitted, HPV transmission does not require penetrative sexual intercourse. Skin-to-skin genital contact is a wellestablished mode of transmission. Over 70% of sexually active women and men will be infected at some point in their lives and some may even be infected on more than one occasion [2]. The peak period for acquiring HPV infection is shortly after becoming sexually active. The infection usually clears up spontaneously within a few months after the acquisition with about 90% clearing within 2 years. There are over 200 HPV types recognized based on DNA sequence data showing genomic differences, and many of these are harmless. HPV can infect basal epithelial cells of the mucocutaneous membrane, and it is associated with a variety of clinical conditions that range from innocuous lesions to cancer. Most of the infections are benign, causing lesions such as cutaneous warts on the hands, feet and anogenital regions. Warts are areas of hypertrophied skin filled with keratin and are mainly a cosmetic nuisance; generally, they resolve spontaneously within 1–5 years. Only a

small proportion of infections with certain types of HPV can persist and progress to cancer such as oropharyngeal, cervical, vulvar, vaginal and penile cancer [1].

Cervical cancer is by far the most common HPV-related disease [1]. Nearly all cases of cervical cancer are due to persistent or chronic HPV infection. Cervical cancer is the fourth most common cancer in women worldwide and it accounts for an estimated 528,000 new cases [2]. About 85% of the global burden occurs in the less developed regions, where it accounts for almost 12% of all female malignancies. In 2012, an estimated 266,000 deaths were attributed to cervical cancer, accounting for 7.5% of all female cancer deaths with almost 90% these deaths occurring in the less developed regions [2]. In these developing countries, cervical cancer may constitute up to 25% of all female cancer cases [3] and is only preceded by breast cancer as the most common cause of cancer deaths in women worldwide [4].

### **2. Basic virology of HPV**

HPV is a member of the Papovaviridae family. It is a relatively small, nonenveloped virus of about 55 nm diameter. It has an icosahedral capsid with 72 capsomers and these contain at least two capsid proteins, L1 and L2. Each capsomer is a pentamer of the major capsid protein, L1 [5]. Each virion capsid contains about 12 copies of the minor capsid protein, L2 [6]. The HPV genome consists of a single molecule of double-stranded, circular DNA [7] with all Open Reading Frame (ORF) protein-coding sequences confined to one strand. There are three functional regions in the genome (**Figure 1**) [8]: The first is a "non-coding upstream regulatory region" also referred to as the long control region (LCR), or the upper regulatory region (URR). This region contains the highest degree of variation in the viral genome and contains the p97 core promoter along with enhancer and silencer sequences that control ORFs transcription in the regulation of DNA replication [9]. The second is called the "early region (E)" and it consists of ORFs E1, E2, E4, E5, E6, and E7, which are involved in viral replication and tumorigenesis. The third is referred to as the "late region (L)" and this encodes the L1 and L2 ORFs for the viral capsid. The E6, E7, and L1 ORFs of a new or unknown HPV type should be 90% or less homologous to the corresponding sequences of known HPV types [10].

**21**

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

**3. Epidemiology of genital HPV infection**

The worldwide prevalence of high-risk HPV infection is 10.4% [11] and it can be as high as 36.5% in some developing countries [12, 13]. Several epidemiologic studies have clearly shown that the risk of contracting genital high-risk HPV infection and cervical cancer is influenced by sexual activity [14, 15]. An individual is at increased risk of having HPV infection if he or she has had multiple sexual partners at any time or if he or she has a partner who has had multiple sexual partners. Having sexual activity at an early age as well as having a history of other sexually transmitted infections, genital warts, or cervical or penile cancer in an individual or sexual partner may also increase the risk of becoming infected with HPV. In addition to sexual activity, age is an important determinant of the risk of HPV infection [16, 17]. The infection is most common among sexually active young women between the age of 18 and 30 years with a sharp decline in prevalence after the age of 30 years. Although, cervical cancer is more common in older women of 35 years and above, thus suggesting that the infection occurs at a younger age with a slow progression to cancer at an older age. Persistence of HPV infection is commoner with the high-risk or oncogenic types and this plays an important role in the development of invasive cancer of the cervix [1]. Cervical cancer arises at the transformation zone, which is the region between the squamous epithelium of the ectocervix and the columnar epithelium of the endocervix, where continuous metaplastic changes occur. The period of greatest metaplastic activity coincides with the greatest risk of HPV infection and this occurs at puberty and the first pregnancy

and subsequently declines slowly after the occurrence of menopause.

**4. Link between genital HPV infections and cervical cancer**

In the past 3–4 decades, the natural history of cervical cancer has been well studied, and persistent infection of the cervix with certain types of HPV has been reported as a necessary causative factor for its occurrence [18]. The link between HPV and cervical squamous cell carcinoma has become well established since the early 1980. The magnitude of the association between HPV and squamous cell carcinoma of the cervix is higher than that for the association between smoking and lung cancer [19]. About 30 HPV types that are transmitted through sexual contact and infect primarily the cervix, vagina, vulva, penis, and anus have been identified. At least one of these HPV types has been implicated in 99.7% of cases of squamous cell carcinoma of the cervix [18]. HPV is a family of closely related viruses with each designated as a type based on their nucleic acid sequencing and then numbered in the order of discovery. More than 200 HPV types are known to exist [1, 20] with 15 types associated with cervical cancer. Genital HPV types can be grouped as highrisk (oncogenic) and low-risk (non-oncogenic) HPV types based on this association with cervical cancer and its precursor lesions. Low-risk or non-oncogenic HPV types include types 6, 11, 42, 43, and 44 while the high-risk or oncogenic HPV types include types 16, 18, 31, 33, 34, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 [18]. Lowrisk subtypes are also occasionally found in cervical carcinomas. The virus usually infects the mucocutaneous epithelium and produces viral particles in matured epithelial cells and then causes a disruption in normal cell-cycle control and the promotion of uncontrolled cell division leading to the accumulation of genetic damage [20]. Adenocarcinomas of the cervix are also less commonly related to HPV infection and is age dependent [21]. Almost 90% of adenocarcinoma of the cervix in women younger than 40 years of age are related to HPV infection, whereas it was observed in only 43% of adenocarcinomas in those aged 60 years and older. Most

**Figure 1.** *Genome organization of HPV [9].*

*Current Perspectives in Human Papillomavirus*

worldwide [4].

**2. Basic virology of HPV**

known HPV types [10].

small proportion of infections with certain types of HPV can persist and progress to

Cervical cancer is by far the most common HPV-related disease [1]. Nearly all cases of cervical cancer are due to persistent or chronic HPV infection. Cervical cancer is the fourth most common cancer in women worldwide and it accounts for an estimated 528,000 new cases [2]. About 85% of the global burden occurs in the less developed regions, where it accounts for almost 12% of all female malignancies. In 2012, an estimated 266,000 deaths were attributed to cervical cancer, accounting for 7.5% of all female cancer deaths with almost 90% these deaths occurring in the less developed regions [2]. In these developing countries, cervical cancer may constitute up to 25% of all female cancer cases [3] and is only preceded by breast cancer as the most common cause of cancer deaths in women

HPV is a member of the Papovaviridae family. It is a relatively small, nonenveloped virus of about 55 nm diameter. It has an icosahedral capsid with 72 capsomers and these contain at least two capsid proteins, L1 and L2. Each capsomer is a pentamer of the major capsid protein, L1 [5]. Each virion capsid contains about 12 copies of the minor capsid protein, L2 [6]. The HPV genome consists of a single molecule of double-stranded, circular DNA [7] with all Open Reading Frame (ORF) protein-coding sequences confined to one strand. There are three functional regions in the genome (**Figure 1**) [8]: The first is a "non-coding upstream regulatory region" also referred to as the long control region (LCR), or the upper regulatory region (URR). This region contains the highest degree of variation in the viral genome and contains the p97 core promoter along with enhancer and silencer sequences that control ORFs transcription in the regulation of DNA replication [9]. The second is called the "early region (E)" and it consists of ORFs E1, E2, E4, E5, E6, and E7, which are involved in viral replication and tumorigenesis. The third is referred to as the "late region (L)" and this encodes the L1 and L2 ORFs for the viral capsid. The E6, E7, and L1 ORFs of a new or unknown HPV type should be 90% or less homologous to the corresponding sequences of

cancer such as oropharyngeal, cervical, vulvar, vaginal and penile cancer [1].

**20**

**Figure 1.**

*Genome organization of HPV [9].*

### **3. Epidemiology of genital HPV infection**

The worldwide prevalence of high-risk HPV infection is 10.4% [11] and it can be as high as 36.5% in some developing countries [12, 13]. Several epidemiologic studies have clearly shown that the risk of contracting genital high-risk HPV infection and cervical cancer is influenced by sexual activity [14, 15]. An individual is at increased risk of having HPV infection if he or she has had multiple sexual partners at any time or if he or she has a partner who has had multiple sexual partners. Having sexual activity at an early age as well as having a history of other sexually transmitted infections, genital warts, or cervical or penile cancer in an individual or sexual partner may also increase the risk of becoming infected with HPV. In addition to sexual activity, age is an important determinant of the risk of HPV infection [16, 17]. The infection is most common among sexually active young women between the age of 18 and 30 years with a sharp decline in prevalence after the age of 30 years. Although, cervical cancer is more common in older women of 35 years and above, thus suggesting that the infection occurs at a younger age with a slow progression to cancer at an older age. Persistence of HPV infection is commoner with the high-risk or oncogenic types and this plays an important role in the development of invasive cancer of the cervix [1]. Cervical cancer arises at the transformation zone, which is the region between the squamous epithelium of the ectocervix and the columnar epithelium of the endocervix, where continuous metaplastic changes occur. The period of greatest metaplastic activity coincides with the greatest risk of HPV infection and this occurs at puberty and the first pregnancy and subsequently declines slowly after the occurrence of menopause.

### **4. Link between genital HPV infections and cervical cancer**

In the past 3–4 decades, the natural history of cervical cancer has been well studied, and persistent infection of the cervix with certain types of HPV has been reported as a necessary causative factor for its occurrence [18]. The link between HPV and cervical squamous cell carcinoma has become well established since the early 1980. The magnitude of the association between HPV and squamous cell carcinoma of the cervix is higher than that for the association between smoking and lung cancer [19]. About 30 HPV types that are transmitted through sexual contact and infect primarily the cervix, vagina, vulva, penis, and anus have been identified. At least one of these HPV types has been implicated in 99.7% of cases of squamous cell carcinoma of the cervix [18]. HPV is a family of closely related viruses with each designated as a type based on their nucleic acid sequencing and then numbered in the order of discovery. More than 200 HPV types are known to exist [1, 20] with 15 types associated with cervical cancer. Genital HPV types can be grouped as highrisk (oncogenic) and low-risk (non-oncogenic) HPV types based on this association with cervical cancer and its precursor lesions. Low-risk or non-oncogenic HPV types include types 6, 11, 42, 43, and 44 while the high-risk or oncogenic HPV types include types 16, 18, 31, 33, 34, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 [18]. Lowrisk subtypes are also occasionally found in cervical carcinomas. The virus usually infects the mucocutaneous epithelium and produces viral particles in matured epithelial cells and then causes a disruption in normal cell-cycle control and the promotion of uncontrolled cell division leading to the accumulation of genetic damage [20]. Adenocarcinomas of the cervix are also less commonly related to HPV infection and is age dependent [21]. Almost 90% of adenocarcinoma of the cervix in women younger than 40 years of age are related to HPV infection, whereas it was observed in only 43% of adenocarcinomas in those aged 60 years and older. Most

HPV-induced cervical changes are transient with 90% regressing spontaneously within 12–36 months [22–26]. However, various other factors such as the individual's genetic predisposition, genetic variation within different HPV type, coinfection with more than one type of HPV, frequency of reinfection, hormone levels, and immune response may alter an individual's ability to clear the infection. Therefore, detection of high-risk HPV is necessary but may not be enough for the development of cervical cancer. Whether a woman will develop cervical cancer depends on several factors that act in conjunction with oncogenic HPV types in a process that leads to cervical cancer. These factors or modifiers of HPV activities include:

### **4.1 Suppressed primary immune response**

Immune response to HPV infection is cell mediated and thus conditions that impair cell-mediated responses such as renal transplantation or HIV disease increase the risk of acquisition and progression of HPV [10, 27, 28]. Studies have consistently shown higher prevalence of HPV infection and cervical cancer precursors in HIV infected women [29–31].

### **4.2 Long-term use of oral contraceptives**

This is a significant risk factor for high-grade cervical disease according to some studies [16, 32]. This is because the upstream regulatory region of high-risk HPV contains sequences which are similar to the responsive elements of glucocorticoid that can be induced by steroid hormones such as progesterone which is the active component of oral contraceptives and dexamethasone.

### **4.3 Cigarette smoking**

The suppression of local immune response induced by smoking and the mutagenic activity of tobacco components have been demonstrated in cervical cells and this may contribute to HPV persistence or to malignant changes in the cervix [33–35]. It appears that smoking is the most important risk factor independent of HPV infection for high grade cervical disease [16]. Smoking shows little or no relationship to low grade cervical disease [1].

### **4.4 Increasing parity**

Having an increasing number of full-term pregnancies is a significant independent risk factor for persistent HPV infection and cervical cancer [36, 37]. The possible mechanisms proposed for this are the increased hormone levels and impaired immune response of pregnancies [38]. In multiparous women, the transformation zone remains longer on the ectocervix and this facilitates its direct exposure to the virus and other potential cofactors [39]. However, the most plausible mechanism is the local tissue damage occurring during vaginal childbirth or cellular oxidative stress with the increased likelihood of DNA damage and HPV integration [40, 41].

### **5. Prevention of HPV-associated cervical cancer**

The natural history of cervical cancer offers unique opportunities for prevention of the disease [42]. Conventionally, Pap smear and liquid-based cytology, combined with treatment of cervical pre-cancerous lesions and early-stage cancer, has been successful in preventing up to 80% of invasive cervical cancer cases in

**23**

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

**5.1 HPV testing**

*5.1.1 Hybrid screening*

*5.1.1.1 Repeat cervical cytology*

*5.1.1.2 Immediate colposcopy*

referral to colposcopy is recommended.

disease.

the developed world [43, 44]. Cervical cancer screening involves testing for HPV infection and cervical cancer precursor lesions among women who have no symptoms. When screening detects cervical pre-cancerous lesions, treatment can easily be instituted, and cancer avoided. Screening can also detect early stage cervical cancer at a time when treatment has a high potential for cure. Currently, primary approaches to HPV prevention include both risk reduction and development of vaccines again HPV. The risk of contracting HPV may be decreased with the use of latex condoms and spermicides. However, these are not totally reliable, since HPV infection may be transmitted through contact with other parts of the body, such as

This is a laboratory test in which cells from the cervix are tested for DNA from certain types of HPV that are known to cause cervical cancer. This may be done alone (primary HPV screening) or in combination with cervical cytology (hybrid HPV screening). These 2 screening strategies are meant to minimize unnecessary follow-up visits and invasive procedures without compromising the detection of

This test is usually done using the sample of cells removed during a Pap smear test or Liquid Based Cytology (LBC). It is done if the results of a Pap smear test show certain abnormal cervical cells (reflex testing). When both the HPV test and Pap test are done using cells from the sample removed during a Pap test, it is called a Pap Smear/ HPV co-testing. Large-scale studies to evaluate management options for women with abnormal Pap smear results have been conducted and these studies indicate the potential utility of HPV DNA testing in the management of women with Pap smear results of Atypical Squamous Cells of Undetermined significance (ASCUS) [45–47]. Based on the results of these studies, screening strategy options that include testing for high-risk HPV DNA as an adjunct to cytology have been developed to triage and monitor ASCUS patients. These improvements in cytologic screening through LBC as well as the introduction of HPV DNA testing greatly facilitate the identification of women at risk for cervical cancer. There are three recommended options in the

In this approach, ASCUS patients would undergo cytology at 4 to 6-month intervals until two negative results are obtained after which the patient can be returned to routine cytologic screening. If any repeat cytology shows ASCUS or greater,

If this is used, women with biopsy-confirmed CIN are treated as per standard protocol for management of cervical intraepithelial neoplasia (CIN) using excision or coagulation techniques. If biopsy is negative for CIN, patients will undergo repeat cytology at 12 months. In postmenopausal women who have ASCUS and clinical or cytologic evidence of atrophy, a 6-week course of intravaginal estrogen is recommended if there are no contraindications to estrogen use. A repeat cytology is performed after completion of the estrogen regimen and if this is negative, the test

the external genitalia, or anus, that are not protected by a condom [1].

management of women with ASCUS [47] and these include:

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

the developed world [43, 44]. Cervical cancer screening involves testing for HPV infection and cervical cancer precursor lesions among women who have no symptoms. When screening detects cervical pre-cancerous lesions, treatment can easily be instituted, and cancer avoided. Screening can also detect early stage cervical cancer at a time when treatment has a high potential for cure. Currently, primary approaches to HPV prevention include both risk reduction and development of vaccines again HPV. The risk of contracting HPV may be decreased with the use of latex condoms and spermicides. However, these are not totally reliable, since HPV infection may be transmitted through contact with other parts of the body, such as the external genitalia, or anus, that are not protected by a condom [1].

### **5.1 HPV testing**

*Current Perspectives in Human Papillomavirus*

**4.1 Suppressed primary immune response**

sors in HIV infected women [29–31].

**4.3 Cigarette smoking**

**4.4 Increasing parity**

**4.2 Long-term use of oral contraceptives**

relationship to low grade cervical disease [1].

**5. Prevention of HPV-associated cervical cancer**

component of oral contraceptives and dexamethasone.

HPV-induced cervical changes are transient with 90% regressing spontaneously within 12–36 months [22–26]. However, various other factors such as the individual's genetic predisposition, genetic variation within different HPV type, coinfection with more than one type of HPV, frequency of reinfection, hormone levels, and immune response may alter an individual's ability to clear the infection. Therefore, detection of high-risk HPV is necessary but may not be enough for the development of cervical cancer. Whether a woman will develop cervical cancer depends on several factors that act in conjunction with oncogenic HPV types in a process that leads to cervical cancer. These factors or modifiers of HPV activities include:

Immune response to HPV infection is cell mediated and thus conditions that impair cell-mediated responses such as renal transplantation or HIV disease increase the risk of acquisition and progression of HPV [10, 27, 28]. Studies have consistently shown higher prevalence of HPV infection and cervical cancer precur-

This is a significant risk factor for high-grade cervical disease according to some studies [16, 32]. This is because the upstream regulatory region of high-risk HPV contains sequences which are similar to the responsive elements of glucocorticoid that can be induced by steroid hormones such as progesterone which is the active

The suppression of local immune response induced by smoking and the mutagenic activity of tobacco components have been demonstrated in cervical cells and this may contribute to HPV persistence or to malignant changes in the cervix [33–35]. It appears that smoking is the most important risk factor independent of HPV infection for high grade cervical disease [16]. Smoking shows little or no

Having an increasing number of full-term pregnancies is a significant independent risk factor for persistent HPV infection and cervical cancer [36, 37]. The possible mechanisms proposed for this are the increased hormone levels and impaired immune response of pregnancies [38]. In multiparous women, the transformation zone remains longer on the ectocervix and this facilitates its direct exposure to the virus and other potential cofactors [39]. However, the most plausible mechanism is the local tissue damage occurring during vaginal childbirth or cellular oxidative stress with the increased likelihood of DNA damage and HPV integration [40, 41].

The natural history of cervical cancer offers unique opportunities for prevention of the disease [42]. Conventionally, Pap smear and liquid-based cytology, combined with treatment of cervical pre-cancerous lesions and early-stage cancer, has been successful in preventing up to 80% of invasive cervical cancer cases in

**22**

This is a laboratory test in which cells from the cervix are tested for DNA from certain types of HPV that are known to cause cervical cancer. This may be done alone (primary HPV screening) or in combination with cervical cytology (hybrid HPV screening). These 2 screening strategies are meant to minimize unnecessary follow-up visits and invasive procedures without compromising the detection of disease.

### *5.1.1 Hybrid screening*

This test is usually done using the sample of cells removed during a Pap smear test or Liquid Based Cytology (LBC). It is done if the results of a Pap smear test show certain abnormal cervical cells (reflex testing). When both the HPV test and Pap test are done using cells from the sample removed during a Pap test, it is called a Pap Smear/ HPV co-testing. Large-scale studies to evaluate management options for women with abnormal Pap smear results have been conducted and these studies indicate the potential utility of HPV DNA testing in the management of women with Pap smear results of Atypical Squamous Cells of Undetermined significance (ASCUS) [45–47]. Based on the results of these studies, screening strategy options that include testing for high-risk HPV DNA as an adjunct to cytology have been developed to triage and monitor ASCUS patients. These improvements in cytologic screening through LBC as well as the introduction of HPV DNA testing greatly facilitate the identification of women at risk for cervical cancer. There are three recommended options in the management of women with ASCUS [47] and these include:

### *5.1.1.1 Repeat cervical cytology*

In this approach, ASCUS patients would undergo cytology at 4 to 6-month intervals until two negative results are obtained after which the patient can be returned to routine cytologic screening. If any repeat cytology shows ASCUS or greater, referral to colposcopy is recommended.

### *5.1.1.2 Immediate colposcopy*

If this is used, women with biopsy-confirmed CIN are treated as per standard protocol for management of cervical intraepithelial neoplasia (CIN) using excision or coagulation techniques. If biopsy is negative for CIN, patients will undergo repeat cytology at 12 months. In postmenopausal women who have ASCUS and clinical or cytologic evidence of atrophy, a 6-week course of intravaginal estrogen is recommended if there are no contraindications to estrogen use. A repeat cytology is performed after completion of the estrogen regimen and if this is negative, the test

is repeated in 4 to 6 months. If the repeat test shows ASCUS or greater, the patient is referred to colposcopy. Immunosuppressed women with ASCUS should be referred directly to colposcopy.

### *5.1.1.3 HPV DNA testing*

This the most preferred approach especially if liquid-based cytology (LBC) is used or if specimens are co-collected for HPV DNA testing. If HPV DNA testing is negative for high-risk HPV types, the patient undergoes repeat cytology testing at 12 months. However, direct referral to colposcopy is recommended for women who test positive for any of the high-risk HPV types. If biopsy confirms CIN, patients are treated per standard protocol for management of CIN. If biopsy does not confirm CIN, then cytology should be repeated at 6 and 12 months with referral back to colposcopy if results show ASCUS or greater or repeat HPV DNA testing at 12 months with referral back to colposcopy if high-risk HPV types are detected.

### *5.1.2 Primary HPV screening*

HPV DNA testing alone without a Pap smear test may also be used for screening in women aged 25 years and older [48, 49]. It is as effective as a hybrid screening strategy that uses cytology in women aged 25–29 years and co-testing in those at 30 years or older [49]. However, HPV primary screening requires less screening frequency (every 5 years). This involves direct referral to colposcopy for women who test positive for HPV types 16/18 and cytology for those who test positive for any of the other high-risk HPV types [50]. The International Agency for Research on Cancer (IARC) and the World Health Organization (WHO) have endorsed HPV testing as the primary screening method for cervical cancer. Several developed countries are now changing to HPV primary screening [50–52].

### **5.2 HPV vaccination**

One of the major prevention strategies for cervical cancer is vaccination against HPV infection among adolescents prior to their first sexual exposure [15]. HPV vaccines are composed of virus-like particles (VLPs), which contains the major and minor HPV capsid antigens but lacking viral DNA. The vaccines are produced by expressing the L1 or L1 and L2 ORFs in eukaryotic cells. These proteins then selfassemble into VLPs which are highly immunogenic. There is no cross-protection among the HPV types due to the high level of antigenic specificity of HPV capsid antigens and thus protection against each HPV type requires vaccination with VLPs of that type. Optimal vaccines would contain a cocktail of VLPs of the most common high-risk HPV subtypes. There are currently 2 commonly used vaccines (Bivalent and Quadrivalent) which protect against both HPV 16 and 18, which are known to cause at least 70% of cervical cancers. In addition, the quadrivalent vaccine also protects against HPV types 6 and 11 which cause anogenital warts. Both vaccines are more effective if administered prior to exposure to HPV and thus, it is preferable to administer them before first sexual activity. The WHO recommends vaccination for girls aged 9–13 years as this is the most cost-effective public health measure against cervical cancer [1, 53, 54]. Some countries have started to vaccinate boys as the vaccination prevents genital cancers in males as well as females, and the quadrivalent vaccine also prevents genital warts in males and females. These vaccines may provide some cross-protection against other less common HPV types which cause invasive cervical cancer. At present, vaccination against HPV is not recommended as a replacement for cervical cancer screening and in countries where

**25**

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

smoking.

**5.3 Future perspectives**

*5.3.2 Therapeutic HPV vaccines*

**6. Executive summary/conclusions**

cervical cancer.

*5.3.1 Measurement of HPV oncoprotein levels*

the vaccine is introduced, cervical screenings still need to be developed or further strengthened [53]. However, in most developing countries, there is still a generally low level of awareness of the existence and availability of these HPV vaccines [55] compared to the developed countries with well-organized cervical cancer screening and HPV vaccination programs. Recently, a nonavalent vaccine against HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58, which has shown a better impact compared to the bivalent and quadrivalent vaccine, has been approved by the US Food and Drug

Other recommended preventive interventions against HPV infections that are appropriate for both boys and girls are education about safe sexual practices including delayed onset of sexual activity; promotion and provision of condoms for those already engaged in sexual activity; male circumcision; and warnings about tobacco

Measuring the levels of HPV E6/E7 oncoproteins is now a potential biomarker for high-risk HPV infection and this may have a role in the future screening of women for high-risk HPV especially type 16 which accounts for more than 50% of all cervical cancer cases [57–59]. The E6/E7 oncoproteins are overexpressed after HPV invasion into the host cervical cells in the form of HPV DNA or viral integration into the host's genome and are closely related to the development of cervical cancers [60]. In a recent pilot study, HPV16 E6/E7 oncoprotein test has a satisfactory diagnostic value for cervical cancer screening and demonstrated a better sensitivity than cytological test and a better specificity than HPV DNA testing [61].

There are currently no approved therapeutic vaccines against HPV in humans. However, there are many recent studies that have generated promising vaccine candidates tested in clinical trials [62–64]. Despite the success of these vaccine candidates, there still remains the concern that conventional expression methods when fully developed might result in very expensive products [65, 66] that will be inaccessible to the resource-constraint countries who have the highest incidences of

Molecular and epidemiologic studies have solidified the association between high-risk strains of genital HPV and squamous cell carcinoma of the cervix. The incidence of cervical cancer and its associated mortality have declined in recent years, largely due to the widespread implementation of screening programs. Screening for cervical cancer remains an important public health and economic concern throughout the world. Large-scale studies to evaluate management options for women with abnormal Pap smear results have been conducted and these studies highlighted the potential utilization of HPV DNA testing in the management of women with ASCUS Pap smear results. From these studies, screening strategies that include testing for high-risk HPV DNA as an adjunct to cytology have been developed for the triage and surveillance of women with ASCUS. Several other studies, such as the ATHENA study [45], have also examined and confirmed the role of HPV

Administration (FDA) and is now commercially available [56].

### *Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

*Current Perspectives in Human Papillomavirus*

directly to colposcopy.

*5.1.1.3 HPV DNA testing*

*5.1.2 Primary HPV screening*

**5.2 HPV vaccination**

is repeated in 4 to 6 months. If the repeat test shows ASCUS or greater, the patient is referred to colposcopy. Immunosuppressed women with ASCUS should be referred

This the most preferred approach especially if liquid-based cytology (LBC) is used or if specimens are co-collected for HPV DNA testing. If HPV DNA testing is negative for high-risk HPV types, the patient undergoes repeat cytology testing at 12 months. However, direct referral to colposcopy is recommended for women who test positive for any of the high-risk HPV types. If biopsy confirms CIN, patients are treated per standard protocol for management of CIN. If biopsy does not confirm CIN, then cytology should be repeated at 6 and 12 months with referral back to colposcopy if results show ASCUS or greater or repeat HPV DNA testing at 12 months with referral back to colposcopy if high-risk HPV types are detected.

HPV DNA testing alone without a Pap smear test may also be used for screening in women aged 25 years and older [48, 49]. It is as effective as a hybrid screening strategy that uses cytology in women aged 25–29 years and co-testing in those at 30 years or older [49]. However, HPV primary screening requires less screening frequency (every 5 years). This involves direct referral to colposcopy for women who test positive for HPV types 16/18 and cytology for those who test positive for any of the other high-risk HPV types [50]. The International Agency for Research on Cancer (IARC) and the World Health Organization (WHO) have endorsed HPV testing as the primary screening method for cervical cancer. Several developed

One of the major prevention strategies for cervical cancer is vaccination against HPV infection among adolescents prior to their first sexual exposure [15]. HPV vaccines are composed of virus-like particles (VLPs), which contains the major and minor HPV capsid antigens but lacking viral DNA. The vaccines are produced by expressing the L1 or L1 and L2 ORFs in eukaryotic cells. These proteins then selfassemble into VLPs which are highly immunogenic. There is no cross-protection among the HPV types due to the high level of antigenic specificity of HPV capsid antigens and thus protection against each HPV type requires vaccination with VLPs of that type. Optimal vaccines would contain a cocktail of VLPs of the most common high-risk HPV subtypes. There are currently 2 commonly used vaccines (Bivalent and Quadrivalent) which protect against both HPV 16 and 18, which are known to cause at least 70% of cervical cancers. In addition, the quadrivalent vaccine also protects against HPV types 6 and 11 which cause anogenital warts. Both vaccines are more effective if administered prior to exposure to HPV and thus, it is preferable to administer them before first sexual activity. The WHO recommends vaccination for girls aged 9–13 years as this is the most cost-effective public health measure against cervical cancer [1, 53, 54]. Some countries have started to vaccinate boys as the vaccination prevents genital cancers in males as well as females, and the quadrivalent vaccine also prevents genital warts in males and females. These vaccines may provide some cross-protection against other less common HPV types which cause invasive cervical cancer. At present, vaccination against HPV is not recommended as a replacement for cervical cancer screening and in countries where

countries are now changing to HPV primary screening [50–52].

**24**

the vaccine is introduced, cervical screenings still need to be developed or further strengthened [53]. However, in most developing countries, there is still a generally low level of awareness of the existence and availability of these HPV vaccines [55] compared to the developed countries with well-organized cervical cancer screening and HPV vaccination programs. Recently, a nonavalent vaccine against HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58, which has shown a better impact compared to the bivalent and quadrivalent vaccine, has been approved by the US Food and Drug Administration (FDA) and is now commercially available [56].

Other recommended preventive interventions against HPV infections that are appropriate for both boys and girls are education about safe sexual practices including delayed onset of sexual activity; promotion and provision of condoms for those already engaged in sexual activity; male circumcision; and warnings about tobacco smoking.

### **5.3 Future perspectives**

### *5.3.1 Measurement of HPV oncoprotein levels*

Measuring the levels of HPV E6/E7 oncoproteins is now a potential biomarker for high-risk HPV infection and this may have a role in the future screening of women for high-risk HPV especially type 16 which accounts for more than 50% of all cervical cancer cases [57–59]. The E6/E7 oncoproteins are overexpressed after HPV invasion into the host cervical cells in the form of HPV DNA or viral integration into the host's genome and are closely related to the development of cervical cancers [60]. In a recent pilot study, HPV16 E6/E7 oncoprotein test has a satisfactory diagnostic value for cervical cancer screening and demonstrated a better sensitivity than cytological test and a better specificity than HPV DNA testing [61].

### *5.3.2 Therapeutic HPV vaccines*

There are currently no approved therapeutic vaccines against HPV in humans. However, there are many recent studies that have generated promising vaccine candidates tested in clinical trials [62–64]. Despite the success of these vaccine candidates, there still remains the concern that conventional expression methods when fully developed might result in very expensive products [65, 66] that will be inaccessible to the resource-constraint countries who have the highest incidences of cervical cancer.

### **6. Executive summary/conclusions**

Molecular and epidemiologic studies have solidified the association between high-risk strains of genital HPV and squamous cell carcinoma of the cervix. The incidence of cervical cancer and its associated mortality have declined in recent years, largely due to the widespread implementation of screening programs. Screening for cervical cancer remains an important public health and economic concern throughout the world. Large-scale studies to evaluate management options for women with abnormal Pap smear results have been conducted and these studies highlighted the potential utilization of HPV DNA testing in the management of women with ASCUS Pap smear results. From these studies, screening strategies that include testing for high-risk HPV DNA as an adjunct to cytology have been developed for the triage and surveillance of women with ASCUS. Several other studies, such as the ATHENA study [45], have also examined and confirmed the role of HPV DNA testing as a primary screening for cervical precursor lesions. In addition to the changes in screening strategies, HPV 16 testing through measurement of HPV E6/ E7 oncoprotein levels and effective therapeutic HPV vaccines that have the potential to contribute significantly to the control and prevention of cervical cancer are also currently being developed for future use.

### **Acknowledgements**

The author acknowledges the mentorship provided by Prof. Folasade Ogunsola, Deputy Vice-Chancellor, Development Services of the University of Lagos, Lagos, Nigeria, Prof Rose Anorlu, Head of Department of Obstetrics and Gynecology, College of Medicine of the University of Lagos and Phyllis Kanki, Harvard University School of Public Health, Boston, MA, USA, throughout the preparation of this manuscript. The work reported in this publication was supported by the Fogarty International Center and National Institute of Mental Health, of the National Institutes of Health under Award Numbers D43TW010543 and D43TW010134. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

### **Conflict of interest**

The author declared no conflict of interest.

### **Author details**

Kehinde Sharafadeen Okunade Department of Obstetrics and Gynaecology, College of Medicine, University of Lagos/Lagos University Teaching Hospital, Lagos, Nigeria

\*Address all correspondence to: sokunade@unilag.edu.ng

© 2019 The Author(s). Licensee IntechOpen. 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.

**27**

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

[1] Burd EM. Human papillomavirus and cervical cancer. Clinical Microbiology

[9] Stanley MA, Pett MR, Coleman N. HPV: From infection to cancer. Biochemical Society Transactions.

[10] Torrisi A, Del Mistro A, Onnis GL, Merlin F, Bertorelle R, Minucci D. Colposcopy, cytology and HPV testing in HIV-positive and HIVnegative women. European Journal of Gynaecological Oncology.

[11] Sanjose de S, Diaz M, Castellsague X, Clifford G, Bruni L, Munoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: A meta-analysis. The Lancet Infectious Diseases.

[12] Okunade KS, Nwogu CM, Oluwole AA, Anorlu RI. Prevalence and risk factors for genital high-risk human papillomavirus infection among women attending the out-patient clinics of a university teaching hospital in Lagos, Nigeria. The Pan African Medical

[13] Bao YP, Li N, Smith JS, Qiao YL.

papillomavirus type distribution in women from Asia: A meta-analysis. International Journal of Gynecological

[14] Erickson BK, Alvarez RD, Huh WK. Human papillomavirus: What every provider should know. American Journal of Obstetrics and Gynecology.

[15] Human Papillomavirus Vaccination. The American College of Obstetricians and Gynaecologists Committee Opinion No. 588. Obstetrics & Gynecology.

[16] Adam E, Berkova Z, Daxnerova Z, Icenogle J, Reeves WC, Kaufman RH.

ACCPAB members: Human

Cancer. 2008;**18**(1):71-79

2013;**208**(3):169-175

2014;**123**:712-718

2007;**35**(6):1456-1460

2000;**21**:168-172

2003;**7**(7):453-459

Journal. 2017;**28**:227

[2] WHO: International Agency for Research on Cancer. Cervical cancer: Estimated incidence, mortality and prevalence worldwide in 2012. In: The GLOBOCAN 2012 Database. Lyon, France: International Agency for Research on Cancer; [Accessed: July 15, 2018]

[3] Harro CD, Pang Y-YS, Roden RBS, Hildesheim A, Wang Z, Reynolds MJ, et al. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. Journal of the National Cancer

[4] Jin XW, Cash J, Kennedy AW. Human papillomavirus typing and the reduction of cervical cancer risk. Cleveland Clinic Journal of Medicine. 1999;**66**:533-539

[5] Baker TS, Newcomb WW, Olson NH, Cowsert LM, Olson C, Brown JC. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and threedimensional image reconstruction. Biophysical Journal. 1991;**60**:1445-1456

[6] Sapp M, Volpers C, Muller M, Streck RE. Organization of the major and minor capsid proteins in human papillomavirus type 33 virus-like particles. The Journal of General Virology. 1995;**76**:2407-2412

[7] Favre M. Structural polypeptides of rabbit, bovine, and human

papillomaviruses. Journal of Virology.

[8] Apt D, Watts RM, Suske G, Bernard U. High Sp1/Sp3 ratios in epithelial cells during epithelial differentiation and cellular transcription correlate with the activation of the HPV-16 promoter.

1975;**15**:1239-1247

Virology. 1996;**224**:281-291

Institute. 2001;**93**:284-292

**References**

Reviews. 2003;**16**(1):1-17

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

### **References**

*Current Perspectives in Human Papillomavirus*

currently being developed for future use.

**Acknowledgements**

**Conflict of interest**

The author declared no conflict of interest.

DNA testing as a primary screening for cervical precursor lesions. In addition to the changes in screening strategies, HPV 16 testing through measurement of HPV E6/ E7 oncoprotein levels and effective therapeutic HPV vaccines that have the potential to contribute significantly to the control and prevention of cervical cancer are also

The author acknowledges the mentorship provided by Prof. Folasade Ogunsola,

Deputy Vice-Chancellor, Development Services of the University of Lagos, Lagos, Nigeria, Prof Rose Anorlu, Head of Department of Obstetrics and Gynecology, College of Medicine of the University of Lagos and Phyllis Kanki, Harvard University School of Public Health, Boston, MA, USA, throughout the preparation of this manuscript. The work reported in this publication was supported by the Fogarty International Center and National Institute of Mental Health, of the National Institutes of Health under Award Numbers D43TW010543 and D43TW010134. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

**26**

**Author details**

Kehinde Sharafadeen Okunade

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. 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,

Department of Obstetrics and Gynaecology, College of Medicine, University of

Lagos/Lagos University Teaching Hospital, Lagos, Nigeria

\*Address all correspondence to: sokunade@unilag.edu.ng

[1] Burd EM. Human papillomavirus and cervical cancer. Clinical Microbiology Reviews. 2003;**16**(1):1-17

[2] WHO: International Agency for Research on Cancer. Cervical cancer: Estimated incidence, mortality and prevalence worldwide in 2012. In: The GLOBOCAN 2012 Database. Lyon, France: International Agency for Research on Cancer; [Accessed: July 15, 2018]

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[4] Jin XW, Cash J, Kennedy AW. Human papillomavirus typing and the reduction of cervical cancer risk. Cleveland Clinic Journal of Medicine. 1999;**66**:533-539

[5] Baker TS, Newcomb WW, Olson NH, Cowsert LM, Olson C, Brown JC. Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and threedimensional image reconstruction. Biophysical Journal. 1991;**60**:1445-1456

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[7] Favre M. Structural polypeptides of rabbit, bovine, and human papillomaviruses. Journal of Virology. 1975;**15**:1239-1247

[8] Apt D, Watts RM, Suske G, Bernard U. High Sp1/Sp3 ratios in epithelial cells during epithelial differentiation and cellular transcription correlate with the activation of the HPV-16 promoter. Virology. 1996;**224**:281-291

[9] Stanley MA, Pett MR, Coleman N. HPV: From infection to cancer. Biochemical Society Transactions. 2007;**35**(6):1456-1460

[10] Torrisi A, Del Mistro A, Onnis GL, Merlin F, Bertorelle R, Minucci D. Colposcopy, cytology and HPV testing in HIV-positive and HIVnegative women. European Journal of Gynaecological Oncology. 2000;**21**:168-172

[11] Sanjose de S, Diaz M, Castellsague X, Clifford G, Bruni L, Munoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: A meta-analysis. The Lancet Infectious Diseases. 2003;**7**(7):453-459

[12] Okunade KS, Nwogu CM, Oluwole AA, Anorlu RI. Prevalence and risk factors for genital high-risk human papillomavirus infection among women attending the out-patient clinics of a university teaching hospital in Lagos, Nigeria. The Pan African Medical Journal. 2017;**28**:227

[13] Bao YP, Li N, Smith JS, Qiao YL. ACCPAB members: Human papillomavirus type distribution in women from Asia: A meta-analysis. International Journal of Gynecological Cancer. 2008;**18**(1):71-79

[14] Erickson BK, Alvarez RD, Huh WK. Human papillomavirus: What every provider should know. American Journal of Obstetrics and Gynecology. 2013;**208**(3):169-175

[15] Human Papillomavirus Vaccination. The American College of Obstetricians and Gynaecologists Committee Opinion No. 588. Obstetrics & Gynecology. 2014;**123**:712-718

[16] Adam E, Berkova Z, Daxnerova Z, Icenogle J, Reeves WC, Kaufman RH.

Papillomavirus detection: Demographic and behavioral characteristics influencing the identification of cervical disease. American Journal of Obstetrics and Gynecology. 2000;**182**:257-264

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[18] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Journal of Pathology. 1999;**189**:12-19

[19] Franco EL. Cancer causes revisited: Human papillomavirus and cervical neoplasia. Journal of the National Cancer Institute. 1995;**87**:779-780

[20] Unger ER, Eliav B. Human papillomavirus and cervical cancer. Emerging Infectious Diseases. 2004;**10**(11):2031-2032

[21] Andersson S, Rylander E, Larsson B, Strand A, Silfversvard C, Wilander E. The role of human papillomavirus in cervical adenocarcinoma carcinogenesis. European Journal of Cancer. 2001;**37**:246-250

[22] Chua KL, Hjerpe A. Persistence of human papillomavirus (HPV) infections preceding cervical carcinoma. Cancer. 1996;**77**:121-127

[23] Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. The New England Journal of Medicine. 1998;**338**:413-428

[24] Moscicki AB, Palefsky J, Smith G, Siboshski S, Schoolnik G. Variability of human papillomavirus DNA testing in a longitudinal cohort of young women. Obstetrics & Gynecology. 1993;**82**:578-585

[25] Ostor AG. Natural history of cervical intraepithelial neoplasia: A critical review. International Journal of Gynecological Pathology. 1993;**12**:186-192

[26] Syrjänen KJ. Natural history of genital human papillomavirus infections. In: Lacey C, editor. Papillomavirus Reviews. Leeds, United Kingdom: Leeds University Press; 1996. pp. 189-206

[27] Calore EE, Pereira SMM, Cavaliere MJ. Progression of cervical lesions in HIV-seropositive women: A cytological study. Diagnostic Cytopathology. 2001;**24**:117-119

[28] Cubie HA, Seagar AL, Beattie GJ, Monaghan S, Williams ARW. A longitudinal study if HPV detection and cervical pathology in HIV infected women. Sexually Transmitted Infections. 2000;**76**:257-261

[29] Conely LK, Ellerbrock TV, Bush TJ, Chiasson MA, Sawo D, Wright TC. HIV-1 infection and risk of vulvovaginal and perianal condylomata acuminate and intraepithelial neoplasia; a prospective cohort study. Lancet. 2002;**359**(9301):108-113

[30] Harris TG, Burk RD, Palesky JM, Massad S, Bang JY, Anastos K, et al. Incidence of cervical squamous intraepithelial lesions associated with HIV serostatus, CD4 cell counts, and human papillomavirus test results. Journal of the American Medical Association. 2005;**293**(12):1471-1476

[31] Singh DK, Anastos K, Hoover DR, Burk RD, Shi Q, Ngendahayo L, et al. Human papillomavirus infection and cervical cytology in HIV-infected and HIV-uninfected Rwandan women. The Journal of Infectious Diseases. 2009;**199**(12):1851-1861

**29**

2006;**119**:1108-1124

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*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

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[34] Villa LL. Human papillomaviruses and cervical cancer. Advances in Cancer Research. 1996;**71**:321-341

[35] Yang X, Jin G, Nakao Y, Rahimtula M, Pater MM, Pater A. Malignant transformation of HPV-16 immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis. International Journal of Cancer. 1996;**65**:338-344

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[37] Juneja A, Sehgal A, Mitra AB, Pandey A. A survey on risk factors associated with cervical cancer. Indian Journal of Cancer. 2003;**40**(1):15-22

[38] Appleby P, Beral V, Berrington de Gonzáles A, Colin D, Franceschi S, Green J, et al. Cervical carcinoma and reproductive factors: Collaborative reanalysis of individual data on 16,563 women with cervical carcinoma and 33,542 women without cervical carcinoma from 25 epidemiological studies. International Journal of Cancer. 2006;**119**:1108-1124

[39] Autier P, Coibion M, Huet F, Grivegnee AR. Transformation zone location and intraepithelial neoplasia of the cervix uteri. British Journal of Cancer. 1996;**74**:488-490

[40] Castle PE. Beyond human papillomavirus: The cervix, exogenous secondary factors, and the development of cervical precancer and cancer. Journal of Lower Genital Tract Disease. 2004;**8**:224-230

[41] Williams VM, Filippova M, Soto U, Duerksen-Hughes PJ. HPV-DNA integration and carcinogenesis: Putative roles for inflammation and oxidative stress. Future Virology. 2011;**6**:45-57

[42] Denny L. Cervical cancer: Prevention and treatment. Discovery Medicine. 2012;**14**(75):125-131

[43] Gichangi P, Estambale B, Bwayo J, Rogo K, Ojwang S, Opiyo A, et al. Knowledge and practice about cervical cancer and pap smear testing among patients at Kenyatta National Hospital, Nairobi, Kenya. International Journal of Gynecological Cancer. 2003;**13**(6):827-833

[44] Kivistic A, Lang K, Baili P, Anttila A, Veerus P. Women's knowledge about cervical cancer risk factors, screening, and reasons for non-participation in cervical cancer screening programme in Estonia. BMC Women's Health. 2011;**11**:43

[45] Saslow D, Solomon D, Lawson HW, Killackey M, Kulasingam SL, Cain J, et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology Screening Guidelines for the prevention and early detection of cervical cancer. American Journal of Clinical Pathology. 2012;**137**:516-542

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**28**

*Current Perspectives in Human Papillomavirus*

in a longitudinal cohort of young women. Obstetrics & Gynecology.

[25] Ostor AG. Natural history of cervical intraepithelial neoplasia: A critical review. International Journal of Gynecological Pathology.

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Papillomavirus Reviews. Leeds, United Kingdom: Leeds University Press; 1996.

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[28] Cubie HA, Seagar AL, Beattie GJ, Monaghan S, Williams ARW. A longitudinal study if HPV detection and cervical pathology in HIV

Infections. 2000;**76**:257-261

2002;**359**(9301):108-113

2009;**199**(12):1851-1861

[30] Harris TG, Burk RD, Palesky JM, Massad S, Bang JY, Anastos K, et al. Incidence of cervical squamous intraepithelial lesions associated with HIV serostatus, CD4 cell counts, and human papillomavirus test results. Journal of the American Medical Association. 2005;**293**(12):1471-1476

[31] Singh DK, Anastos K, Hoover DR, Burk RD, Shi Q, Ngendahayo L, et al. Human papillomavirus infection and cervical cytology in HIV-infected and HIV-uninfected Rwandan women. The Journal of Infectious Diseases.

infected women. Sexually Transmitted

[29] Conely LK, Ellerbrock TV, Bush TJ, Chiasson MA, Sawo D, Wright TC. HIV-1 infection and risk of

vulvovaginal and perianal condylomata acuminate and intraepithelial neoplasia; a prospective cohort study. Lancet.

1993;**82**:578-585

1993;**12**:186-192

pp. 189-206

2001;**24**:117-119

Papillomavirus detection: Demographic and behavioral characteristics influencing the identification of cervical disease. American Journal of Obstetrics and Gynecology. 2000;**182**:257-264

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[18] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Journal of

[19] Franco EL. Cancer causes revisited: Human papillomavirus and cervical neoplasia. Journal of the National Cancer Institute. 1995;**87**:779-780

Diseases. 1996;**23**:333-341

Pathology. 1999;**189**:12-19

[20] Unger ER, Eliav B. Human papillomavirus and cervical cancer. Emerging Infectious Diseases.

[21] Andersson S, Rylander E, Larsson B, Strand A, Silfversvard C, Wilander E. The role of human papillomavirus

carcinogenesis. European Journal of

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[23] Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. The New England Journal of Medicine. 1998;**338**:413-428

[24] Moscicki AB, Palefsky J, Smith G, Siboshski S, Schoolnik G. Variability of human papillomavirus DNA testing

2004;**10**(11):2031-2032

in cervical adenocarcinoma

Cancer. 2001;**37**:246-250

1996;**77**:121-127

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[49] Wright TC, Stoler MH, Behrens CM, Sharma A, Zhang G, Wright TL. Primary cervical cancer screening with human papillomavirus: End of study results from the ATHENA study using HPV as the first-line screening test. Gynecologic Oncology. 2015;**136**(2):189-197

[50] Huh WK, Ault KA, Chelmow D. Use of primary high-risk human papillomavirus testing for cervical cancer screening: Interim clinical guidance. Journal of Lower Genital Tract Disease. 2015;**19**:91-96

[51] Leinonen MK, Nieminen P, Lönnberg S, Malila N, Hakama M, Pokhrel A, et al. Detection rates of precancerous and cancerous cervical lesions within one screening round of primary human papillomavirus DNA testing: Prospective randomised trial in Finland. British Medical Journal. 2012;**345**:e7789

[52] Ronco G, Dillner J, Elfström KM, Tunesi S, Snijders PJ, Arbyn M, et al. Efficacy of HPV-based screening for prevention of invasive cervical cancer: Follow-up four European randomised controlled trials. The Lancet. 2013;**383**(9916):524-532

[53] WHO. World Health Organization human papillomavirus vaccines

WHO position paper. The Weekly Epidemiological Record. 2009;**15**(84):117-132

[54] Guideline Development Group. HPV vaccination. In: Comprehensive Cervical Cancer Control—A Guide to Essential Practice. 2nd ed. Geneva, Switzerland: World Health Organization; 2014. pp. 110-128

[55] Okunade KS, Sunmonu O, Osanyin GE, Oluwole AA. Knowledge and acceptability of human papillomavirus vaccination among women attending the gynaecological outpatient clinics of a university teaching Hospital in Lagos, Nigeria. Journal of Tropical Medicine. 2017;**2017**:8586459

[56] Capra G, Giovannelli L, Matranga D, Bellavia C, Guarneri MF, Fasciana T, et al. Potential impact of a nonavalent HPV vaccine on HPV related low-and high-grade cervical intraepithelial lesions: A referral hospital-based study in Sicily. Human Vaccines & Immunotherapeutics. 2017;**13**(8):1839-1843

[57] Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet. 2007;**370**:890-907

[58] Li N, Franceschi S, Howell-Jones R, Snijders PJ, Clifford GM. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication. International Journal of Cancer. 2011;**128**:927-935

[59] Schiffman M, Wentzensen N. Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiology, Biomarkers & Prevention. 2013;**22**:553-560

[60] Munagala R, Kausar H, Munjal C, Gupta RC. Withaferin A induces

**31**

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells. Carcinogenesis.

[61] Zhang J-J, Cao X-C, Zheng X-Y, Wang H-Y, Li Y-W. Feasibility study of a human papillomavirus E6 and E7 oncoprotein test for the diagnosis of cervical precancer and cancer. The Journal of International Medical Research. 2018;**46**(3):1033-1042

[62] Vici P. Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: Hope or reality from clinical studies. Expert Review of Vaccines.

[63] Yang A. Perspectives for therapeutic HPV vaccine development. Journal of Biomedical Science. 2016;**23**(1):75

[64] Kim HJ, Kim H-J. Current status and future prospects for human papillomavirus vaccines. Arch Pharm

[65] Giorgi C, Franconi R, Rybicki EP. Human papillomavirus vaccines in plants. Expert Review of Vaccines.

[66] Rybicki EP. Plant-based vaccines against viruses. Virology Journal.

Res. 2017;**40**(9):1050-1063

2010;**9**(8):913-924

2014;**11**(1):205

2016;**15**(10):1327-1336

2011;**32**:1697-1705

*Human Papillomavirus and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.81581*

*Current Perspectives in Human Papillomavirus*

WHO position paper. The Weekly Epidemiological Record.

[54] Guideline Development Group. HPV vaccination. In: Comprehensive Cervical Cancer Control—A Guide to Essential Practice. 2nd ed. Geneva, Switzerland: World Health Organization; 2014. pp. 110-128

[55] Okunade KS, Sunmonu O, Osanyin GE, Oluwole AA. Knowledge and acceptability of human papillomavirus vaccination among women attending the gynaecological outpatient clinics of a university teaching Hospital in Lagos, Nigeria. Journal of Tropical Medicine.

[56] Capra G, Giovannelli L, Matranga D, Bellavia C, Guarneri MF, Fasciana

[57] Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer.

[58] Li N, Franceschi S, Howell-Jones R, Snijders PJ, Clifford GM. Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: Variation by geographical region, histological type and year of publication. International Journal of

T, et al. Potential impact of a nonavalent HPV vaccine on HPV related low-and high-grade cervical intraepithelial lesions: A referral hospital-based study in Sicily. Human Vaccines & Immunotherapeutics.

2009;**15**(84):117-132

2017;**2017**:8586459

2017;**13**(8):1839-1843

Lancet. 2007;**370**:890-907

Cancer. 2011;**128**:927-935

Biomarkers & Prevention.

2013;**22**:553-560

[59] Schiffman M, Wentzensen N. Human papillomavirus infection and the multistage carcinogenesis of cervical cancer. Cancer Epidemiology,

[60] Munagala R, Kausar H, Munjal C, Gupta RC. Withaferin A induces

[48] Qiao YL, Sellors JW, Eder PS, Bao YP, Lim JM, Zhao FH, et al. A new HPV-DNA test for cervical-cancer screening

[49] Wright TC, Stoler MH, Behrens CM, Sharma A, Zhang G, Wright TL. Primary cervical cancer screening with human papillomavirus: End of study results from the ATHENA study using HPV as the first-line screening test. Gynecologic Oncology.

[50] Huh WK, Ault KA, Chelmow D. Use of primary high-risk human papillomavirus testing for cervical cancer screening: Interim clinical guidance. Journal of Lower Genital Tract Disease. 2015;**19**:91-96

[51] Leinonen MK, Nieminen P, Lönnberg S, Malila N, Hakama M, Pokhrel A, et al. Detection rates of precancerous and cancerous cervical lesions within one screening round of primary human papillomavirus DNA testing: Prospective randomised trial in Finland. British Medical Journal.

[52] Ronco G, Dillner J, Elfström KM, Tunesi S, Snijders PJ, Arbyn M, et al. Efficacy of HPV-based screening for prevention of invasive cervical cancer: Follow-up four European randomised

[53] WHO. World Health Organization human papillomavirus vaccines

controlled trials. The Lancet. 2013;**383**(9916):524-532

in developing regions: A crosssectional study of clinical accuracy in rural China. The Lancet Oncology.

2008;**9**(10):929-936

2015;**136**(2):189-197

2012;**345**:e7789

[47] Massad LS, Einstein MH, Huh WK, Katki HA, Kinney WK, Schiffman M, et al. 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. Obstetrics and Gynecology. 2013;**121**:829-846

**30**

p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells. Carcinogenesis. 2011;**32**:1697-1705

[61] Zhang J-J, Cao X-C, Zheng X-Y, Wang H-Y, Li Y-W. Feasibility study of a human papillomavirus E6 and E7 oncoprotein test for the diagnosis of cervical precancer and cancer. The Journal of International Medical Research. 2018;**46**(3):1033-1042

[62] Vici P. Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: Hope or reality from clinical studies. Expert Review of Vaccines. 2016;**15**(10):1327-1336

[63] Yang A. Perspectives for therapeutic HPV vaccine development. Journal of Biomedical Science. 2016;**23**(1):75

[64] Kim HJ, Kim H-J. Current status and future prospects for human papillomavirus vaccines. Arch Pharm Res. 2017;**40**(9):1050-1063

[65] Giorgi C, Franconi R, Rybicki EP. Human papillomavirus vaccines in plants. Expert Review of Vaccines. 2010;**9**(8):913-924

[66] Rybicki EP. Plant-based vaccines against viruses. Virology Journal. 2014;**11**(1):205

**33**

**Chapter 3**

**Abstract**

disease

**1. Introduction**

options [3, 4].

*and Nicolae Suciu*

HPV Infection and Vulvar Cancer

*Nicolae Bacalbasa, Irina Balescu, Ioan Suciu, Simona Dima* 

Although the strong association between human papilloma virus (HPV) and cervical cancer has been widely demonstrated, it seems that uterine cervix cancer is not the only gynecologic malignancy induced by this pathogenic agent. It has been shown that HPV infection plays a central role in the development of vulvar cancer too, HPV 16 and 18 being the most frequently reported genotypes that might induce this kind of lesions. This aspect presents a particular importation, patients diagnosed with HPV-related vulvar cancer reporting a more favorable trend in regard with the long-term outcome. The current chapter aims to describe the pathogenesis as well as the therapeutic options and the long-term outcomes of patients in which

**Keywords:** HPV, infection, squamous cell carcinoma, vulvar cancer, preneoplastic

Discovering human papilloma virus (HPV) represented a crucial step in understanding and preventing the apparition of cervical cancer in women worldwide, the most frequently incriminated carcinogenic subtypes including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 [1]. However, it has been demonstrated that HPV infection plays a central role in the development of other malignancies such as vulvar,

vaginal or anal cancer in women and anal or penile cancer in men [2].

inflammatory condition, lichen sclerosus [7, 8].

**2. The role of HPV in the development of premalignant vulvar lesions**

Due to the fact that the incidence of vulvar squamous cell carcinoma has reported a continuous increase in the last decades, attention was focused on determining the pathogenesis of this lesion as well as on improving the therapeutic

The main premalignant vulvar lesion consists of vulvar intraepithelial neoplasia (VIN), with an increasing incidence in the last decades; moreover, it seems that the age at diagnosis of this pathological feature has been consistently dropping in the last period of time, especially due to the relative increase of HPV infections [5, 6]. However, it seems that there are two different pathways leading to the apparition of this premalignant lesion; the first one is mainly related to type 16 HPV infection, while the second one is rather related to the presence of a nonneoplastic chronic

association between HPV and vulvar cancer can be assessed.

### **Chapter 3**

## HPV Infection and Vulvar Cancer

*Nicolae Bacalbasa, Irina Balescu, Ioan Suciu, Simona Dima and Nicolae Suciu*

### **Abstract**

Although the strong association between human papilloma virus (HPV) and cervical cancer has been widely demonstrated, it seems that uterine cervix cancer is not the only gynecologic malignancy induced by this pathogenic agent. It has been shown that HPV infection plays a central role in the development of vulvar cancer too, HPV 16 and 18 being the most frequently reported genotypes that might induce this kind of lesions. This aspect presents a particular importation, patients diagnosed with HPV-related vulvar cancer reporting a more favorable trend in regard with the long-term outcome. The current chapter aims to describe the pathogenesis as well as the therapeutic options and the long-term outcomes of patients in which association between HPV and vulvar cancer can be assessed.

**Keywords:** HPV, infection, squamous cell carcinoma, vulvar cancer, preneoplastic disease

### **1. Introduction**

Discovering human papilloma virus (HPV) represented a crucial step in understanding and preventing the apparition of cervical cancer in women worldwide, the most frequently incriminated carcinogenic subtypes including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 [1]. However, it has been demonstrated that HPV infection plays a central role in the development of other malignancies such as vulvar, vaginal or anal cancer in women and anal or penile cancer in men [2].

### **2. The role of HPV in the development of premalignant vulvar lesions**

Due to the fact that the incidence of vulvar squamous cell carcinoma has reported a continuous increase in the last decades, attention was focused on determining the pathogenesis of this lesion as well as on improving the therapeutic options [3, 4].

The main premalignant vulvar lesion consists of vulvar intraepithelial neoplasia (VIN), with an increasing incidence in the last decades; moreover, it seems that the age at diagnosis of this pathological feature has been consistently dropping in the last period of time, especially due to the relative increase of HPV infections [5, 6]. However, it seems that there are two different pathways leading to the apparition of this premalignant lesion; the first one is mainly related to type 16 HPV infection, while the second one is rather related to the presence of a nonneoplastic chronic inflammatory condition, lichen sclerosus [7, 8].

Squamous cell carcinomas represent more than 90% of all vulvar cancer and are associated with several histopathological subtypes such as keratinized, basaloid warty or verrucous lesions; moreover, it seems that basaloid and warty lesions are more commonly seen in younger women, being usually associated with HPV DNA positivity. Contrarily, keratinized lesions that usually develop from chronic dermatoses such as lichen sclerosus are not associated with HPV infection and develop in older patients [9]. In cases associated with lichen sclerosus, the premalignant disease is usually referred as differentiated VIN (dVIN) [10]. A third category has been also proposed, comprising the VIN lesions, which could not be classified in either of the two abovementioned classes. This category is referred as VIN, unclassified type [11].

In order to study the clinicopathological characteristics of lichen-related vulvar carcinomas, Regauer et al. conducted a study on 38 patients diagnosed with this pathology [12]. Among these cases, 32 patients presented solitary lesions, while the remaining 6 patients presented multifocal lesions, all cases being HPV and p16 negative. As for the stage of disease, inguinal metastases were present in 42% of cases at the time of presentation. When it comes to the performed therapy, radical surgery (consisting of radical resection with negative margins) was performed in 36 cases, while the remaining 2 cases were submitted to radiochemotherapy. However, 14 of the 36 surgically treated patients developed recurrent disease on the residual mucosa, 68% of them being diagnosed within the first year. Moreover, 14 of the 38 patients died of the disease. In this way, the study came to demonstrate the strong association between the presence of lichen, the absence of HPV and a relatively poorer outcome of this class of patients [12].

In HPV-related lesions, it seems that the immune system of the host encounters a failure in order to produce an effective response to HPV; the longer time the HPV infection persists, the longer certain oncoproteins such as E6 or E7 will interfere with the cyclic cellular mechanisms, inducing cellular escape from the apoptotic process, and therefore malignant transformation [13, 14].

As for the VIN grading system, the first classification was proposed in 1986 by the International Society for the Study of Vulvo-Vaginal Diseases and included the division of VIN in three grades [15]; two decades, later the same organization decided to change the grading system. Lesions that had been previously considered as VIN 1 have been regarded since that moment as warts or HPV infections, while VIN 2 and 3 have been generally referred as VIN [7, 15].

More recently, in order to provide a more specific classification, the Lower Anogenital Squamous Terminology Committee graded all the HPV-related tumors of the anogenital tract into two categories depending on the degree of differentiation: the first category, LSIL (low-grade squamous intraepithelial lesions) refers to lesions presenting a lower grade of pathological transformation, while the second type of lesions HSIL (high-grade squamous intraepithelial lesions) refers to lesions with a higher grade of pathological transformation [16].

When it comes to the HPV-related VIN development, it seems that HPV DNA integration into the host cell genome plays a crucial role [17, 18]. An interesting study conducted on this theme was published by Peter Hillemanns and Xiuli Wang in Gynecologic Oncology in 2006 [19]. The study included 30 patients diagnosed with VIN at the University of Munich-Grosshaderm, Germany; among the 30 patients, HPV DNA was detected in 25 women, the main identified subtypes including HPV 16 and HPV-18. The presence of HPV-16 or HPV-18 DNA was reported in eight cases, all of them being diagnosed with multicentric lesions of VIN, one of them also associating areas of vulvar carcinoma. Therefore, the authors concluded that the integration of HPV-DNA in the hosts presents a central role when it comes to the progression of the vulvar lesions to advanced or multifocal VIN lesions or even vulvar carcinomas [19].

**35**

subtypes [23, 24].

*HPV Infection and Vulvar Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.80601*

body mass index higher than 30 kg/m2

patients with HPV-related diseases [20].

better response to radiochemotherapy [23].

**3. Epidemiology of HPV-related vulvar neoplasms**

A recent study regarding the epidemiology of HPV-related vulvar neoplasms originates from the National Cancer Institute, Bethesda MD, United States of America, and was conducted by Brinton et al. [20]. The study was conducted on 201,469 women, 370 of them being diagnosed with vulvar neoplasms including 198 cases with grade 3 vulvar intraepithelial neoplasms. The mean age at the time of conducting the study was 61.8 years; among these cases, most patients were white, married, with a high level of education and parous. Moreover, a significant number of cases had been previously submitted to hysterectomy or reported a chronic usage of oral contraceptives or menopausal hormones, while more than one quarter of patients were obese. After a mean follow-up interval of 13.8 years, 170 cases developed vulvar cancer (the mean age at diagnosis being 71 years), while 198 cases developed grade 3 vulvar intraepithelial neoplasia (at a mean age of 67.5 years). Demographic risk factors included nonwhite women, as well as the marital status (divorced or separated women reporting a significantly higher risk of development of the disease); however, the educational status did not influence the risk of the development of any kind of vulvar lesions. As for the parity status, the risk of vulvar lesions was significantly decreased among women who had delivered. Moreover, patients with a previous history of hysterectomy reported a trend to a higher risk of vulvar neoplasm development, although this fact was not statistically significant. However, this difference was not found among cases submitted to a prior oophorectomy. When it comes to the relationship between the body mass index and the risk of developing vulvar lesions, obese patients (defined through a

ing invasive vulvar cancer; however, this relationship could not be established for patients with vulvar intraepithelial neoplasms. As for other lifestyle factors, diabetes or alcohol consumption did not influence the risk of vulvar neoplasm development, but smoking constituted in a significant risk factor. In the meantime, administration of hormonal therapies or oral contraceptives was only associated with the risk of development of intraepithelial lesions and not with the apparition of vulvar invasive cancer. Moreover, smoking was a risk factor especially among

**4. The influence of HPV infection on the overall prognostic in patients** 

Although vulvar squamous cell carcinoma is not a common malignancy, its estimated incidence ranging between 3 and 5% of all gynecological malignancies, it seems that HPV infection plays a central role in its development; therefore, it is estimated that up to 70% of cases diagnosed with this malignancy present in fact HPV-related lesions [21, 22]. However, the rate of correlation between HPV infection and vulvar squamous cell carcinoma widely varies between different studies depending on the detection method and the included tumoral histopathological

Due to the fact that other related HPV infection malignancies (such as head or neck tumors) are associated with a significantly better outcome when compared to HPV-negative lesions, attention was focused on determining whether a similar relationship could be established between this type of infection and the overall survival in vulvar cancer patients. The improved outcome of patients diagnosed with HPV-related head or neck tumors seems to be explained especially through a

**diagnosed with vulvar squamous cell carcinoma**

) had a significantly higher risk of develop-

*Current Perspectives in Human Papillomavirus*

poorer outcome of this class of patients [12].

process, and therefore malignant transformation [13, 14].

VIN 2 and 3 have been generally referred as VIN [7, 15].

with a higher grade of pathological transformation [16].

VIN lesions or even vulvar carcinomas [19].

Squamous cell carcinomas represent more than 90% of all vulvar cancer and are associated with several histopathological subtypes such as keratinized, basaloid warty or verrucous lesions; moreover, it seems that basaloid and warty lesions are more commonly seen in younger women, being usually associated with HPV DNA positivity. Contrarily, keratinized lesions that usually develop from chronic dermatoses such as lichen sclerosus are not associated with HPV infection and develop in older patients [9]. In cases associated with lichen sclerosus, the premalignant disease is usually referred as differentiated VIN (dVIN) [10]. A third category has been also proposed, comprising the VIN lesions, which could not be classified in either of the two above-

In order to study the clinicopathological characteristics of lichen-related vulvar carcinomas, Regauer et al. conducted a study on 38 patients diagnosed with this pathology [12]. Among these cases, 32 patients presented solitary lesions, while the remaining 6 patients presented multifocal lesions, all cases being HPV and p16 negative. As for the stage of disease, inguinal metastases were present in 42% of cases at the time of presentation. When it comes to the performed therapy, radical surgery (consisting of radical resection with negative margins) was performed in 36 cases, while the remaining 2 cases were submitted to radiochemotherapy. However, 14 of the 36 surgically treated patients developed recurrent disease on the residual mucosa, 68% of them being diagnosed within the first year. Moreover, 14 of the 38 patients died of the disease. In this way, the study came to demonstrate the strong association between the presence of lichen, the absence of HPV and a relatively

In HPV-related lesions, it seems that the immune system of the host encounters a failure in order to produce an effective response to HPV; the longer time the HPV infection persists, the longer certain oncoproteins such as E6 or E7 will interfere with the cyclic cellular mechanisms, inducing cellular escape from the apoptotic

As for the VIN grading system, the first classification was proposed in 1986 by the International Society for the Study of Vulvo-Vaginal Diseases and included the division of VIN in three grades [15]; two decades, later the same organization decided to change the grading system. Lesions that had been previously considered as VIN 1 have been regarded since that moment as warts or HPV infections, while

More recently, in order to provide a more specific classification, the Lower Anogenital Squamous Terminology Committee graded all the HPV-related tumors of the anogenital tract into two categories depending on the degree of differentiation: the first category, LSIL (low-grade squamous intraepithelial lesions) refers to lesions presenting a lower grade of pathological transformation, while the second type of lesions HSIL (high-grade squamous intraepithelial lesions) refers to lesions

When it comes to the HPV-related VIN development, it seems that HPV DNA integration into the host cell genome plays a crucial role [17, 18]. An interesting study conducted on this theme was published by Peter Hillemanns and Xiuli Wang in Gynecologic Oncology in 2006 [19]. The study included 30 patients diagnosed with VIN at the University of Munich-Grosshaderm, Germany; among the 30 patients, HPV DNA was detected in 25 women, the main identified subtypes including HPV 16 and HPV-18. The presence of HPV-16 or HPV-18 DNA was reported in eight cases, all of them being diagnosed with multicentric lesions of VIN, one of them also associating areas of vulvar carcinoma. Therefore, the authors concluded that the integration of HPV-DNA in the hosts presents a central role when it comes to the progression of the vulvar lesions to advanced or multifocal

mentioned classes. This category is referred as VIN, unclassified type [11].

**34**

### **3. Epidemiology of HPV-related vulvar neoplasms**

A recent study regarding the epidemiology of HPV-related vulvar neoplasms originates from the National Cancer Institute, Bethesda MD, United States of America, and was conducted by Brinton et al. [20]. The study was conducted on 201,469 women, 370 of them being diagnosed with vulvar neoplasms including 198 cases with grade 3 vulvar intraepithelial neoplasms. The mean age at the time of conducting the study was 61.8 years; among these cases, most patients were white, married, with a high level of education and parous. Moreover, a significant number of cases had been previously submitted to hysterectomy or reported a chronic usage of oral contraceptives or menopausal hormones, while more than one quarter of patients were obese. After a mean follow-up interval of 13.8 years, 170 cases developed vulvar cancer (the mean age at diagnosis being 71 years), while 198 cases developed grade 3 vulvar intraepithelial neoplasia (at a mean age of 67.5 years). Demographic risk factors included nonwhite women, as well as the marital status (divorced or separated women reporting a significantly higher risk of development of the disease); however, the educational status did not influence the risk of the development of any kind of vulvar lesions. As for the parity status, the risk of vulvar lesions was significantly decreased among women who had delivered. Moreover, patients with a previous history of hysterectomy reported a trend to a higher risk of vulvar neoplasm development, although this fact was not statistically significant. However, this difference was not found among cases submitted to a prior oophorectomy. When it comes to the relationship between the body mass index and the risk of developing vulvar lesions, obese patients (defined through a body mass index higher than 30 kg/m2 ) had a significantly higher risk of developing invasive vulvar cancer; however, this relationship could not be established for patients with vulvar intraepithelial neoplasms. As for other lifestyle factors, diabetes or alcohol consumption did not influence the risk of vulvar neoplasm development, but smoking constituted in a significant risk factor. In the meantime, administration of hormonal therapies or oral contraceptives was only associated with the risk of development of intraepithelial lesions and not with the apparition of vulvar invasive cancer. Moreover, smoking was a risk factor especially among patients with HPV-related diseases [20].

### **4. The influence of HPV infection on the overall prognostic in patients diagnosed with vulvar squamous cell carcinoma**

Although vulvar squamous cell carcinoma is not a common malignancy, its estimated incidence ranging between 3 and 5% of all gynecological malignancies, it seems that HPV infection plays a central role in its development; therefore, it is estimated that up to 70% of cases diagnosed with this malignancy present in fact HPV-related lesions [21, 22]. However, the rate of correlation between HPV infection and vulvar squamous cell carcinoma widely varies between different studies depending on the detection method and the included tumoral histopathological subtypes [23, 24].

Due to the fact that other related HPV infection malignancies (such as head or neck tumors) are associated with a significantly better outcome when compared to HPV-negative lesions, attention was focused on determining whether a similar relationship could be established between this type of infection and the overall survival in vulvar cancer patients. The improved outcome of patients diagnosed with HPV-related head or neck tumors seems to be explained especially through a better response to radiochemotherapy [23].

However, scarce data have been reported so far. An interesting study conducted on this theme was published by Alonso et al. in Gynecologic Oncology Journal in 2011 [23]. The study included 98 patients diagnosed with vulvar squamous cell carcinoma between 1995 and 2009 in which the authors studied the presence of HPV DNA; among these cases, 19 patients were diagnosed with HPV-associated infection, HPV-16 being the most prevalently detected subtype. Therefore, HPV-16 subtype was reported in 14 cases, one of these cases presenting in the meantime HPV 56 co-infection; HPV-33 was found in two patients, whereas HPV-31, 51 and 52 were each reported in 1 patient. When it comes to the clinical characteristics for the two subgroups, patients presenting with HPV-related tumors were significantly younger when compared to those in whom the presence of the infection could not be demonstrated (68 years versus 78 years, *p* = 0.005). As for the other clinical factors that had been studied (such as FIGO stage at diagnostic, the median dimension of the tumor, association of ulceration, invasion depth and lymph node metastases) as well as for the type of performed therapeutic strategy (resection or radiochemotherapy), there was no significant difference between patients with HPV-related tumors when compared to those in whom HPV infection had not been observed [23].

The most relevant studies that focused on the correlation between HPV status and clinicopathological findings of patients with vulvar cancer are summarized in **Table 1**.

When it comes to the long-term outcomes, both disease-free and overall survival were significantly influenced by the FIGO stage at diagnosis, while the association of HPV infection showed no significant influence. Moreover, no significant difference was reported between the association of radiotherapy, HPV infection and overall survival; however, cases in which radiotherapy was associated reported a higher morbidity rate. In univariate analysis, the most important factors associating with the risk of disease progression and mortality were represented by the age over 78 years, FIGO stages III–IV, tumor size larger than 20 mm, ulceration, invasion depth and the presence of lymph node metastases; however, in multivariate


### **Table 1.**

*Correlation between HPV status and clinicopathological findings in patients with vulvar cancer.*

**37**

*HPV Infection and Vulvar Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.80601*

analysis, only the association of lymph node metastases was still significantly associated with the mortality risk. When it comes to the influence of HPV infection, a significant association could not be seen even after adjusting for age. These data come to suggest that a supplementary mechanism might be involved in HPV-related vulvar neoplasms when compared to head and neck HPV-related neoplasms [23]. However, these results were not sustained by more recent studies conducted on the theme of the prognostic significance of HPV infection in patients diagnosed with vulvar squamous cell carcinoma and submitted to radiotherapy. In the article published by Lee in the same journal in 2016, contradictory results were found [29]. The study included 57 patients diagnosed with this pathological entity between 1985 and 2011 in Brigham and Women's Hospital and Dana Farber Cancer Institute who were treated with postoperative radiotherapy with radical intent or as part of the salvage setting. In all cases the presence of the following genotypes was studied: 6, 11, 16, 18, 26, 31, 33, 35, 40, 45, 51, 52, 56 and 59; similar to Alonso's study, HPV-16 genotype was the most commonly encountered subtype. When it comes to the long-term outcomes, patients with p16-positive tumors reported a significantly better five-year progression-free and overall survival rates. Moreover, in univariate analysis, older age at diagnosis as well as higher FIGO stage and development of recurrent disease were associated with increased risk of progressive disease and mortality-related disease; however, association of chemotherapy did not significantly impact on the overall survival. When a multivariate analysis was performed, the presence of p16 staining was associated with higher progression-free survival rates as well as with lower rates of recurrence [29]. The reported results of this study were similar to those regarding head and neck HPV-induced malignancies, the presence of HPV infection being associated with a better response to radiotherapy. A recent study that was conducted by Hinten et al. that will be published in 2018 in Gynecologic Oncology Journal demonstrated that in fact HPV-positive and negative vulvar cancer represent in fact two different pathologic entities with different localization and different prognosis. The study was conducted between March 1988 and January 2015 and included 318 patients. Among these cases, HPVrelated disease was reported in 55 cases, while the remaining 263 had non–HPVrelated vulvar neoplastic lesions [25]. The authors demonstrated that HPV-related lesions were more often localized on the perineum when compared to non-HPV lesions. When it comes to the long-term outcomes, the authors demonstrated that patients with HPV-induced lesions reported a better outcome in terms of both disease-free survival and overall survival when compared to non-HPV lesions; therefore, the 5-year and total disease-free survival were 76 versus 46%, and 28 versus 13% in HPV-related lesions versus non–HPV-related lesions. In the meantime, the 5-year and total overall survival rates were 85 versus 57% in HPV-related lesions, and only 16% in non-HPV lesions. Another important prognostic factor that significantly influenced survival was the site of the lesions; therefore, even among patients with HPV-related lesions, cases presenting with perineal development reported a significantly better prognosis when compared to nonperineal HPV-induced lesions; the difference remained significant in terms of both diseasefree and overall survival. Moreover, among patients presenting with perineal vulvar cancer, HPV-induced malignancies reported a more favorable outcome when compared to non–HPV-induced lesions. In the meantime, disease-free survival was also significantly influenced by FIGO stage and diameter of the tumor, while the overall survival was significantly influenced by age at primary treatment, stage at diagnosis, tumor diameter and relapse as well as by the perineal localization of the lesions. Moreover, the 10-year survival rate was significantly influenced by age at the time of initial treatment, FIGO stage at diagnosis, tumoral diameter, p16 expression and perineal localization of the lesions. In terms of histopathological characteristics,

### *HPV Infection and Vulvar Cancer DOI: http://dx.doi.org/10.5772/intechopen.80601*

*Current Perspectives in Human Papillomavirus*

observed [23].

**Name, year No of** 

Hinten, 2018 [25]

Brinton, 2017 [20]

Rasmussen, 2018 [27]

Monk, 1995 [28]

**cases (total)** **HPVrelated cases**

**Non– HPVrelated cases**

Yap, 2018 [26] 40 14 26 Lower risk of recurrence

*Correlation between HPV status and clinicopathological findings in patients with vulvar cancer.*

370 — — Smoking, age, obesity

**infection**

318 55 263 Patients' age, smoking status, immune status,

1638 541 1097 Higher overall survival rate in HPV-related lesions

55 33 22 Patients' age, smoking status, histopathological subtype

**Factors significantly associated with HPV** 

history of lichen sclerosus, diameter of the tumor, lympho-vascular space invasion, FIGO



stage, risk of recurrence

*survival interval, overall survival*

**Table 1**.

However, scarce data have been reported so far. An interesting study conducted on this theme was published by Alonso et al. in Gynecologic Oncology Journal in 2011 [23]. The study included 98 patients diagnosed with vulvar squamous cell carcinoma between 1995 and 2009 in which the authors studied the presence of HPV DNA; among these cases, 19 patients were diagnosed with HPV-associated infection, HPV-16 being the most prevalently detected subtype. Therefore, HPV-16 subtype was reported in 14 cases, one of these cases presenting in the meantime HPV 56 co-infection; HPV-33 was found in two patients, whereas HPV-31, 51 and 52 were each reported in 1 patient. When it comes to the clinical characteristics for the two subgroups, patients presenting with HPV-related tumors were significantly younger when compared to those in whom the presence of the infection could not be demonstrated (68 years versus 78 years, *p* = 0.005). As for the other clinical factors that had been studied (such as FIGO stage at diagnostic, the median dimension of the tumor, association of ulceration, invasion depth and lymph node metastases) as well as for the type of performed therapeutic strategy (resection or radiochemotherapy), there was no significant difference between patients with HPV-related tumors when compared to those in whom HPV infection had not been

The most relevant studies that focused on the correlation between HPV status and clinicopathological findings of patients with vulvar cancer are summarized in

When it comes to the long-term outcomes, both disease-free and overall survival were significantly influenced by the FIGO stage at diagnosis, while the association of HPV infection showed no significant influence. Moreover, no significant difference was reported between the association of radiotherapy, HPV infection and overall survival; however, cases in which radiotherapy was associated reported a higher morbidity rate. In univariate analysis, the most important factors associating with the risk of disease progression and mortality were represented by the age over 78 years, FIGO stages III–IV, tumor size larger than 20 mm, ulceration, invasion depth and the presence of lymph node metastases; however, in multivariate

**36**

**Table 1.**

analysis, only the association of lymph node metastases was still significantly associated with the mortality risk. When it comes to the influence of HPV infection, a significant association could not be seen even after adjusting for age. These data come to suggest that a supplementary mechanism might be involved in HPV-related vulvar neoplasms when compared to head and neck HPV-related neoplasms [23].

However, these results were not sustained by more recent studies conducted on the theme of the prognostic significance of HPV infection in patients diagnosed with vulvar squamous cell carcinoma and submitted to radiotherapy. In the article published by Lee in the same journal in 2016, contradictory results were found [29]. The study included 57 patients diagnosed with this pathological entity between 1985 and 2011 in Brigham and Women's Hospital and Dana Farber Cancer Institute who were treated with postoperative radiotherapy with radical intent or as part of the salvage setting. In all cases the presence of the following genotypes was studied: 6, 11, 16, 18, 26, 31, 33, 35, 40, 45, 51, 52, 56 and 59; similar to Alonso's study, HPV-16 genotype was the most commonly encountered subtype. When it comes to the long-term outcomes, patients with p16-positive tumors reported a significantly better five-year progression-free and overall survival rates. Moreover, in univariate analysis, older age at diagnosis as well as higher FIGO stage and development of recurrent disease were associated with increased risk of progressive disease and mortality-related disease; however, association of chemotherapy did not significantly impact on the overall survival. When a multivariate analysis was performed, the presence of p16 staining was associated with higher progression-free survival rates as well as with lower rates of recurrence [29]. The reported results of this study were similar to those regarding head and neck HPV-induced malignancies, the presence of HPV infection being associated with a better response to radiotherapy.

A recent study that was conducted by Hinten et al. that will be published in 2018 in Gynecologic Oncology Journal demonstrated that in fact HPV-positive and negative vulvar cancer represent in fact two different pathologic entities with different localization and different prognosis. The study was conducted between March 1988 and January 2015 and included 318 patients. Among these cases, HPVrelated disease was reported in 55 cases, while the remaining 263 had non–HPVrelated vulvar neoplastic lesions [25]. The authors demonstrated that HPV-related lesions were more often localized on the perineum when compared to non-HPV lesions. When it comes to the long-term outcomes, the authors demonstrated that patients with HPV-induced lesions reported a better outcome in terms of both disease-free survival and overall survival when compared to non-HPV lesions; therefore, the 5-year and total disease-free survival were 76 versus 46%, and 28 versus 13% in HPV-related lesions versus non–HPV-related lesions. In the meantime, the 5-year and total overall survival rates were 85 versus 57% in HPV-related lesions, and only 16% in non-HPV lesions. Another important prognostic factor that significantly influenced survival was the site of the lesions; therefore, even among patients with HPV-related lesions, cases presenting with perineal development reported a significantly better prognosis when compared to nonperineal HPV-induced lesions; the difference remained significant in terms of both diseasefree and overall survival. Moreover, among patients presenting with perineal vulvar cancer, HPV-induced malignancies reported a more favorable outcome when compared to non–HPV-induced lesions. In the meantime, disease-free survival was also significantly influenced by FIGO stage and diameter of the tumor, while the overall survival was significantly influenced by age at primary treatment, stage at diagnosis, tumor diameter and relapse as well as by the perineal localization of the lesions. Moreover, the 10-year survival rate was significantly influenced by age at the time of initial treatment, FIGO stage at diagnosis, tumoral diameter, p16 expression and perineal localization of the lesions. In terms of histopathological characteristics,

non–HPV-related lesions presented a larger diameter and were associated with a deeper invasion, more frequent metastases at the level of the lymphatic nodes and, therefore, a more frequent association of adjuvant radiotherapy. This different outcome could be explained by a more aggressive biological behavior of non–HPVrelated lesions as well as by the older age at diagnosis, elderly patients feeling most often ashamed to address to the gynecologist for such lesions [25]. All these data enabled the authors to conclude that most probably HPV and non–HPV-related lesions are in fact two different entities with different pathogenesis and different outcomes. Another possible explanation is related to the p53 status, non-HPV lesions being most commonly associated with a higher level of p53, and, in consequence, with a more aggressive biology of the tumor [30].

Another recent study that focused on determining the prognostic significance of human papilloma virus and p16 expression in patients with vulvar squamous cell carcinoma submitted to radiotherapy was conducted by Yap et al. and has been recently published in Clinical Oncology Journal [26].

### **5. Factors influencing relapse in patients with premalignant or malignant diseases**

Starting from the observation that patients with similar stages of disease who receive similar treatment strategies had a very different evolution, the researchers tried to identify the potential factors that influenced this evolution.

### **5.1 The influence of DNMT expression in development of recurrent vulvar cancer**

DNMT (DNA methyltransferases), the enzyme that dictates and maintains DNA methylation patterns through the genome, seems to have significant differences in terms of expression in patients with vulvar carcinomas. Among the general name of DNMT, there are in fact three enzymes with various influences on the methylation process. DNMT1 is directly involved in the methylation process in normal cells; however, it seems that it plays a certain role in tumorigenesis too. DNMT3A and DNMT3B represent two other enzymes that present low expression levels in adult cells; however, these molecules seem to be overexpressed in several epithelial tumors [31, 32]. Moreover, their expression is associated with poor prognosis in patients with such epithelial tumors. A recent study conducted on this theme was published in Gynecologic Oncology Journal in 2016 and included patients treated for vulvar squamous cell carcinomas at the Pan Birmingham Gynecological Cancer Center between 2001 and 2008 [33]. The authors demonstrated the overexpression of DNMT1 in 83% of cases and of DNMT3A in 44% of cases, while the overexpression of DNMT3B was present in 42% of patients. After determining these parameters, the authors studied their influence on the risk of recurrence. DNMT3A was associated with a 4.5 fold increased risk of developing recurrent vulvar cancer; in multivariate analysis, the overexpression of this enzyme was also significantly correlated with a higher risk of local recurrence. Moreover, the authors tested the patients with overexpression of DNMT3A for CDKN2A, an indicator of HPV-induced dysplasia, and demonstrated that among patients with negative staining for CDKN2A, the overexpression of DNMT3A was significantly higher. Similar to DNMT3A, a higher level of DNMT3B was significantly associated with the risk of recurrence; however, the levels of this enzyme could not be correlated with CDKN2A expression. As for DNMT1 levels, there was no significant

**39**

*HPV Infection and Vulvar Cancer*

CDKN2A expression [33].

**patients with VIN**

*DOI: http://dx.doi.org/10.5772/intechopen.80601*

with a more rapid pattern of growth [34].

correlation between this parameter and the risk of vulvar cancer recurrence; similar to DNMT3B, no significant correlation could be found between DNMT1 levels and

Another interesting topic in regard to the influence of HPV on the overall prognostic in patients with premalignant or malignant lesions is related to the effect of various viral subtypes on the long-term outcomes of these patients. A recent study conducted in Milan by Bogani et al. and published in 2017 in the European Journal of Obstetrics and Gynecology and Reproductive Biology included 64 patients diagnosed with high-grade VIN [34]. Among these cases, 41 patients had a previous history of HPV infection, the most commonly incriminated subtypes being HPV 16, 18, 31 and 33. As for the performed procedures, most often it consisted of LASER ablation, excision or diathermocoagulation. After a mean follow-up of 56.7 months, 10 patients were diagnosed with VIN2+ persistence or relapse, the mean diseasefree survival being 51.7 months; the authors demonstrated that a pretreatment infection with HPV 31 or HPV 33 subtype was associated with an increased risk of developing recurrent or persistent disease. Moreover, patients submitted to surgical excision followed by LASER ablation experienced a lower rate of relapse when compared with other types of therapies. These facts were explained through two mechanisms: the first one is related to the fact that HPV16, as well as HPV 31 and 33, was associated with multifocal lesions, while multifocal lesions usually associate with a higher risk of persistent/recurrent disease; the second mechanism is probably related to the fact that HPV 31- and HPV33-induced lesions usually associate

When it comes to the influence of HPV infection on the risk of recurrence of VIN, a recent study published by Satmary et al. in Gynecologic Oncology Journal in 2018 comes to demonstrate a significant relationship between these two entities [35]. The study included 784 patients with histopathological diagnostic of vulvar intraepithelial neoplasia which were treated with curative intent; however, 26,3% of cases developed recurrent intraepithelial neoplasia while 2,2% of these cases developed vulvar cancer. Among these cases, 25.9% of patients were 40 years of age or less, 23.9% were aged between 41 and 50 years, 24.6% were aged between 51 and 60 years, while the remaining 25.6% of patients were aged over 60 years. As for the immunity status, immunosuppression was reported in 189 cases and was caused by immune suppressant therapies (such as prednisone or methylprednisolone) in all but two patients (who were known to have HIV infection). When it comes to the performed therapy, it consisted of local excision in 54.8% of cases, laser therapy in 19.3% of cases and topical medication as single therapy or in association with excision or with laser in the remaining patients; in 17% of cases, data regarding therapy were not reported. However, cases in which the initial therapeutic option was not known were excluded from further study regarding recurrent disease. Among the 650 patients who benefited from any kind of treatment recurrence occurred in 171 cases, after a median diseasefree survival interval of 16.9 months, while the median follow-up period was 89 months. Moreover, it seems that 75% of cases recurred within 43.1 months. When analyzed according to the age at the time of diagnosis, recurrence rate was significantly higher among patients over the age of 50 (*p* = 0.0031) when compared to younger patients. In univariate analysis, a significant association was also found between the risk of recurrence and the immunity system of the

**5.2 The influence of pretreatment subtype of HPV on the risk of relapse in** 

*Current Perspectives in Human Papillomavirus*

quence, with a more aggressive biology of the tumor [30].

recently published in Clinical Oncology Journal [26].

**malignant diseases**

**cancer**

non–HPV-related lesions presented a larger diameter and were associated with a deeper invasion, more frequent metastases at the level of the lymphatic nodes and, therefore, a more frequent association of adjuvant radiotherapy. This different outcome could be explained by a more aggressive biological behavior of non–HPVrelated lesions as well as by the older age at diagnosis, elderly patients feeling most often ashamed to address to the gynecologist for such lesions [25]. All these data enabled the authors to conclude that most probably HPV and non–HPV-related lesions are in fact two different entities with different pathogenesis and different outcomes. Another possible explanation is related to the p53 status, non-HPV lesions being most commonly associated with a higher level of p53, and, in conse-

Another recent study that focused on determining the prognostic significance of human papilloma virus and p16 expression in patients with vulvar squamous cell carcinoma submitted to radiotherapy was conducted by Yap et al. and has been

Starting from the observation that patients with similar stages of disease who receive similar treatment strategies had a very different evolution, the researchers

DNMT (DNA methyltransferases), the enzyme that dictates and maintains DNA methylation patterns through the genome, seems to have significant differences in terms of expression in patients with vulvar carcinomas. Among the general name of DNMT, there are in fact three enzymes with various influences on the methylation process. DNMT1 is directly involved in the methylation process in normal cells; however, it seems that it plays a certain role in tumorigenesis too. DNMT3A and DNMT3B represent two other enzymes that present low expression levels in adult cells; however, these molecules seem to be overexpressed in several epithelial tumors [31, 32]. Moreover, their expression is associated with poor prognosis in patients with such epithelial tumors. A recent study conducted on this theme was published in Gynecologic Oncology Journal in 2016 and included patients treated for vulvar squamous cell carcinomas at the Pan Birmingham Gynecological Cancer Center between 2001 and 2008 [33]. The authors demonstrated the overexpression of DNMT1 in 83% of cases and of DNMT3A in 44% of cases, while the overexpression of DNMT3B was present in 42% of patients. After determining these parameters, the authors studied their influence on the risk of recurrence. DNMT3A was associated with a 4.5 fold increased risk of developing recurrent vulvar cancer; in multivariate analysis, the overexpression of this enzyme was also significantly correlated with a higher risk of local recurrence. Moreover, the authors tested the patients with overexpression of DNMT3A for CDKN2A, an indicator of HPV-induced dysplasia, and demonstrated that among patients with negative staining for CDKN2A, the overexpression of DNMT3A was significantly higher. Similar to DNMT3A, a higher level of DNMT3B was significantly associated with the risk of recurrence; however, the levels of this enzyme could not be correlated with CDKN2A expression. As for DNMT1 levels, there was no significant

**5.1 The influence of DNMT expression in development of recurrent vulvar** 

**5. Factors influencing relapse in patients with premalignant or** 

tried to identify the potential factors that influenced this evolution.

**38**

correlation between this parameter and the risk of vulvar cancer recurrence; similar to DNMT3B, no significant correlation could be found between DNMT1 levels and CDKN2A expression [33].

### **5.2 The influence of pretreatment subtype of HPV on the risk of relapse in patients with VIN**

Another interesting topic in regard to the influence of HPV on the overall prognostic in patients with premalignant or malignant lesions is related to the effect of various viral subtypes on the long-term outcomes of these patients. A recent study conducted in Milan by Bogani et al. and published in 2017 in the European Journal of Obstetrics and Gynecology and Reproductive Biology included 64 patients diagnosed with high-grade VIN [34]. Among these cases, 41 patients had a previous history of HPV infection, the most commonly incriminated subtypes being HPV 16, 18, 31 and 33. As for the performed procedures, most often it consisted of LASER ablation, excision or diathermocoagulation. After a mean follow-up of 56.7 months, 10 patients were diagnosed with VIN2+ persistence or relapse, the mean diseasefree survival being 51.7 months; the authors demonstrated that a pretreatment infection with HPV 31 or HPV 33 subtype was associated with an increased risk of developing recurrent or persistent disease. Moreover, patients submitted to surgical excision followed by LASER ablation experienced a lower rate of relapse when compared with other types of therapies. These facts were explained through two mechanisms: the first one is related to the fact that HPV16, as well as HPV 31 and 33, was associated with multifocal lesions, while multifocal lesions usually associate with a higher risk of persistent/recurrent disease; the second mechanism is probably related to the fact that HPV 31- and HPV33-induced lesions usually associate with a more rapid pattern of growth [34].

When it comes to the influence of HPV infection on the risk of recurrence of VIN, a recent study published by Satmary et al. in Gynecologic Oncology Journal in 2018 comes to demonstrate a significant relationship between these two entities [35]. The study included 784 patients with histopathological diagnostic of vulvar intraepithelial neoplasia which were treated with curative intent; however, 26,3% of cases developed recurrent intraepithelial neoplasia while 2,2% of these cases developed vulvar cancer. Among these cases, 25.9% of patients were 40 years of age or less, 23.9% were aged between 41 and 50 years, 24.6% were aged between 51 and 60 years, while the remaining 25.6% of patients were aged over 60 years. As for the immunity status, immunosuppression was reported in 189 cases and was caused by immune suppressant therapies (such as prednisone or methylprednisolone) in all but two patients (who were known to have HIV infection). When it comes to the performed therapy, it consisted of local excision in 54.8% of cases, laser therapy in 19.3% of cases and topical medication as single therapy or in association with excision or with laser in the remaining patients; in 17% of cases, data regarding therapy were not reported. However, cases in which the initial therapeutic option was not known were excluded from further study regarding recurrent disease. Among the 650 patients who benefited from any kind of treatment recurrence occurred in 171 cases, after a median diseasefree survival interval of 16.9 months, while the median follow-up period was 89 months. Moreover, it seems that 75% of cases recurred within 43.1 months. When analyzed according to the age at the time of diagnosis, recurrence rate was significantly higher among patients over the age of 50 (*p* = 0.0031) when compared to younger patients. In univariate analysis, a significant association was also found between the risk of recurrence and the immunity system of the

patient, association of cervical intraepithelial neoplasia and increased BMI; in multivariate analysis, only age over 50 years, immunity status and association of cervical intraepithelial neoplasia were significantly associated with the risk of recurrence. When it comes to the influence of the type of treatment, in multivariate analysis, a trend toward a higher rate of recurrence was reported in cases submitted to nonexcisional therapies. As for the cases in which progression to vulvar cancer was encountered, the median time to progression to malignancy was 36.2 months. When studying only the patients who developed recurrences, the authors demonstrated that the relapse was significantly associated with increased age (patients over 50 years of age reporting a higher risk of recurrence), immunosuppression, positive resection margins and adjacent areas of lichen sclerosus or HPV infection [35].

### **5.3 The influence of TP53 gene on the risk of recurrence of vulvar cancer**

Another factor that seems to influence the evolution of patients with vulvar cancer is represented by TP53 expression. Moreover, it has been widely demonstrated that antitumor agents activating the TP53 tumor suppression gene can be safely used as adjuvant therapy for cases exhibiting this gene. When it comes to the association between HPV-related infection and TP53 expression, in cases diagnosed with head and neck malignancies, disruptive TP53 mutations were exclusively seen in HPV-negative tumors; moreover, these lesions were associated with poorer outcomes when compared with HPV-positive lesions [36].

### **6. The potential preventive role of HPV vaccination against HPVinduced vulvar cancer**

In order to diminish the risk of development of HPV-related malignancies, the quadrivalent and the two-valent HPV vaccines were approved to be used in both males and females in the European Union since 2006 and 2007, respectively [37, 38]. The main viral subtypes that are controlled by these two vaccines are HPV-16 and HPV-18, while the quadrivalent vaccine also contains proteins derived from HPV6 and 11 [37]. Studies have demonstrated that using HPV vaccines in HPV-naïve persons protects against both benign and malignant conditions such as condylomas, perineal and anal neoplasia in men as well as cervical cancer in women [38]. Therefore, routine HPV vaccination has been recommended in Europe in 12-year-old girls since 2009, in order to decrease the risk of development of such pathologies [39]. Due to the fact that there is a strong relationship between HPV infection and certain cases of vulvar cancer, a decreased incidence of this pathology is to be expected in the next decades, once the HPV vaccination has been widely implemented [40].

In order to maximize the protective effect of vaccination against HPV-related diseases, a nine-valent second generation of HPV vaccine was proposed; this ninevalent HPV vaccine is expected to offer protection against the seven high-risk HPV subtypes (HPV 16/18/31/33/45/52/58) as well as against low-risk subtypes such as HPV 6/11. In this way, it is expected to provide a significant degree of protection against the main nine subtypes of HPV, which are responsible for up to 90% of all genital warts. The nine-valent vaccine proved to be effective in order to prevent 97% of all high-grade premalignant lesions of cervix, vulva and vagina. In the study conducted by Hartwig et al. and published in 2015, which included all HPV-related malignancies reported in Europe in the year 2013, the authors demonstrated the efficacy of the second-generation HPV vaccine [2].

**41**

*HPV Infection and Vulvar Cancer*

**7. Future perspectives**

therapeutic role [41].

**Acknowledgements**

PN-III-P1-1.2-PCCDI2017-0833.

**8. Conclusion**

*DOI: http://dx.doi.org/10.5772/intechopen.80601*

needed in order to clearly state this aspect.

Once the benefits of vaccination in terms of prevention of HPV-related malignancies are widely demonstrated, another problem is reported, the one of the vaccine's costs. It seems that, especially in the developing countries, where the incidence of HPV-related malignancies is higher, the accessibility to HPV vaccines is lower due to its price. Therefore, one of the future perspectives in regard to HPV-related diseases is lowering the price of the vaccine and increasing in this way the general accessibility to these products. Moreover, if we take into consideration the fact that a significant number of women self-refer to the gynecologist when neoplastic disease is already present, it seems that an important future perspective should refer to the development of a vaccine that could also have a

HPV infection seems to play a central role in developing premalignant or malignant vulvar lesions. Patients diagnosed with HPV-related lesions tend to have a younger age at diagnosis, especially due to the association with the presence of this virus. However, HPV-induced vulvar neoplastic lesions seem to have a better outcome in terms of both disease-free survival and overall survival when compared with non–HPV-related lesions. When it comes to the prevention of these lesions, it seems that anti-HPV vaccination might play a role; however, more studies are still

This work was supported by the project entitled "Multidisciplinary Consortium

for Supporting the Research Skills in Diagnosing, Treating and Identifying Predictive Factors of Malignant Gynecologic Disorders," project number

### **7. Future perspectives**

*Current Perspectives in Human Papillomavirus*

lichen sclerosus or HPV infection [35].

**induced vulvar cancer**

patient, association of cervical intraepithelial neoplasia and increased BMI; in multivariate analysis, only age over 50 years, immunity status and association of cervical intraepithelial neoplasia were significantly associated with the risk of recurrence. When it comes to the influence of the type of treatment, in multivariate analysis, a trend toward a higher rate of recurrence was reported in cases submitted to nonexcisional therapies. As for the cases in which progression to vulvar cancer was encountered, the median time to progression to malignancy was 36.2 months. When studying only the patients who developed recurrences, the authors demonstrated that the relapse was significantly associated with increased age (patients over 50 years of age reporting a higher risk of recurrence), immunosuppression, positive resection margins and adjacent areas of

**5.3 The influence of TP53 gene on the risk of recurrence of vulvar cancer**

**6. The potential preventive role of HPV vaccination against HPV-**

next decades, once the HPV vaccination has been widely implemented [40].

efficacy of the second-generation HPV vaccine [2].

In order to maximize the protective effect of vaccination against HPV-related diseases, a nine-valent second generation of HPV vaccine was proposed; this ninevalent HPV vaccine is expected to offer protection against the seven high-risk HPV subtypes (HPV 16/18/31/33/45/52/58) as well as against low-risk subtypes such as HPV 6/11. In this way, it is expected to provide a significant degree of protection against the main nine subtypes of HPV, which are responsible for up to 90% of all genital warts. The nine-valent vaccine proved to be effective in order to prevent 97% of all high-grade premalignant lesions of cervix, vulva and vagina. In the study conducted by Hartwig et al. and published in 2015, which included all HPV-related malignancies reported in Europe in the year 2013, the authors demonstrated the

In order to diminish the risk of development of HPV-related malignancies, the quadrivalent and the two-valent HPV vaccines were approved to be used in both males and females in the European Union since 2006 and 2007, respectively [37, 38]. The main viral subtypes that are controlled by these two vaccines are HPV-16 and HPV-18, while the quadrivalent vaccine also contains proteins derived from HPV6 and 11 [37]. Studies have demonstrated that using HPV vaccines in HPV-naïve persons protects against both benign and malignant conditions such as condylomas, perineal and anal neoplasia in men as well as cervical cancer in women [38]. Therefore, routine HPV vaccination has been recommended in Europe in 12-year-old girls since 2009, in order to decrease the risk of development of such pathologies [39]. Due to the fact that there is a strong relationship between HPV infection and certain cases of vulvar cancer, a decreased incidence of this pathology is to be expected in the

outcomes when compared with HPV-positive lesions [36].

Another factor that seems to influence the evolution of patients with vulvar cancer is represented by TP53 expression. Moreover, it has been widely demonstrated that antitumor agents activating the TP53 tumor suppression gene can be safely used as adjuvant therapy for cases exhibiting this gene. When it comes to the association between HPV-related infection and TP53 expression, in cases diagnosed with head and neck malignancies, disruptive TP53 mutations were exclusively seen in HPV-negative tumors; moreover, these lesions were associated with poorer

**40**

Once the benefits of vaccination in terms of prevention of HPV-related malignancies are widely demonstrated, another problem is reported, the one of the vaccine's costs. It seems that, especially in the developing countries, where the incidence of HPV-related malignancies is higher, the accessibility to HPV vaccines is lower due to its price. Therefore, one of the future perspectives in regard to HPV-related diseases is lowering the price of the vaccine and increasing in this way the general accessibility to these products. Moreover, if we take into consideration the fact that a significant number of women self-refer to the gynecologist when neoplastic disease is already present, it seems that an important future perspective should refer to the development of a vaccine that could also have a therapeutic role [41].

### **8. Conclusion**

HPV infection seems to play a central role in developing premalignant or malignant vulvar lesions. Patients diagnosed with HPV-related lesions tend to have a younger age at diagnosis, especially due to the association with the presence of this virus. However, HPV-induced vulvar neoplastic lesions seem to have a better outcome in terms of both disease-free survival and overall survival when compared with non–HPV-related lesions. When it comes to the prevention of these lesions, it seems that anti-HPV vaccination might play a role; however, more studies are still needed in order to clearly state this aspect.

### **Acknowledgements**

This work was supported by the project entitled "Multidisciplinary Consortium for Supporting the Research Skills in Diagnosing, Treating and Identifying Predictive Factors of Malignant Gynecologic Disorders," project number PN-III-P1-1.2-PCCDI2017-0833.

### **Author details**

Nicolae Bacalbasa1,2\*, Irina Balescu3 , Ioan Suciu4 , Simona Dima5 and Nicolae Suciu1,6

1 "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

2 Center of Excellence in Translational Medicine, Fundeni Clinical Institute, Bucharest, Romania

3 Ponderas Academic Hospital, Bucharest, Romania

4 "Floreasca" Clinical Emergency Hospital, Bucharest, Romania

5 "Dan Setlacec" Center of Gastrointestinal Disease and Liver Transplantation, Fundeni Clinical Institute, Bucharest, Romania

6 "Alessandrescu-Rusescu" Institute for Mother and Child Care, Bucharest, Romania

\*Address all correspondence to: nicolae\_bacalbasa@yahoo.ro

© 2018 The Author(s). Licensee IntechOpen. 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.

**43**

2005;**50**:807-810

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**42**

Romania

**Author details**

Bucharest, Romania

Nicolae Bacalbasa1,2\*, Irina Balescu3

3 Ponderas Academic Hospital, Bucharest, Romania

Fundeni Clinical Institute, Bucharest, Romania

4 "Floreasca" Clinical Emergency Hospital, Bucharest, Romania

\*Address all correspondence to: nicolae\_bacalbasa@yahoo.ro

provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. 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,

, Ioan Suciu4

1 "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

2 Center of Excellence in Translational Medicine, Fundeni Clinical Institute,

5 "Dan Setlacec" Center of Gastrointestinal Disease and Liver Transplantation,

6 "Alessandrescu-Rusescu" Institute for Mother and Child Care, Bucharest,

, Simona Dima5

and Nicolae Suciu1,6

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[2] Hartwig S, Baldauf JJ, Dominiak-Felden G, Simondon F, Alemany L, de Sanjosé S, Castellsagué X. Estimation of the epidemiologic alburden of HPVrelated anogenital cancers, precancerous lesions, and genital warts in women and men in Europe: Potential additional benefit of a nine-valent second generation HPV vaccine compared to first generation HPV vaccines. Papillomavirus Research. 2015;**1**:90-100

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[7] Sideri M, Jones RW, Wilkinson EJ, Preti M, Heller DS, Scurry J, Haefner H, Neill S. Squamous vulvar intraepithelial neoplasia: 2004 modified terminology, ISSVD Vulvar Oncology Subcommittee. The Journal of Reproductive Medicine. 2005;**50**:807-810

[8] van de Nieuwenhof HP, Bulten J, Hollema H, Dommerholt RG, Massuger LF, van der Zee AG, de Hullu JA, van Kempen LC. Differentiated vulvar intraepithelial neoplasia is often found in lesions, previously diagnosed as lichen sclerosus, which have progressed to vulvar squamous cell carcinoma. Modern Pathology. 2011;**24**:297-305

[9] de Sanjose S, Bruni L, Alemany L. HPV in genital cancers (at the exception of cervical cancer) and anal cancers. Presse Médicale. 2014;**43**:e423-e428

[10] Preti M, Igidbashian S, Costa S, Cristoforoni P, Mariani L, Origoni M, Sandri MT, Boveri S, Spolti N, Spinaci L, Sanvito F, Preti EP, Falasca A, Radici G, Micheletti L. VIN usual type—From the past to the future. Ecancermedicalscience. 2015;**9**:531

[11] Preti M, Scurry J, Marchitelli CE, Micheletti L. Vulvar intraepithelial neoplasia. Best Practice & Research. Clinical Obstetrics & Gynaecology. 2014;**28**:1051-1062

[12] Regauer S, Reich O, Eberz B. Vulvar cancers in women with vulvar lichen planus: A clinicopathological study. Journal of the American Academy of Dermatology. 2014;**71**:698-707

[13] van der Avoort I, Shirango H, Hoevenaars BM, Grefte JM, de Hullu JA, de Wilde PC, Bulten J, Melchers WJ, Massuger LF. Vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways. International Journal of Gynecological Pathology. 2006;**25**:22-29

[14] Hoevenaars BM, van der Avoort I, de Wilde PC, Massuger LF, Melchers WJ, de Hullu JA, Bulten J. A panel of p16(INK4A), MIB1 and p53 proteins can distinguish between the 2 pathways leading to vulvar squamous cell

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[15] Wilkinson EJ, Kneale BL, Lynch FW. Report of the ISSVD Terminology Committee: VIN. The Journal of Reproductive Medicine. 1986;**31**:973-974

[16] Darragh TM, Colgan TJ, Cox JT, Heller DS, Henry MR, Luff RD, McCalmont T, Nayar R, Palefsky JM, Stoler MH, Wilkinson EJ, Zaino RJ, Wilbur DC. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: Background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Archives of Pathology and Laboratory Medicine. 2012;**136**:1266-1297

[17] van Beurden M, ten Kate FJ, Smits HL, Berkhout RJ, de Craen AJ, van der Vange N, Lammes FB, ter Schegget J. Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus. Cancer. 1995;**75**:2879-2884

[18] Remmink AJ, Walboomers JM, Helmerhorst TJ, Voorhorst FJ, Rozendaal L, Risse EK, Meijer CJ, Kenemans P. The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: Natural history up to 36 months. International Journal of Cancer. 1995;**61**:306-311

[19] Hillemanns P, Wang X. Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia. Gynecologic Oncology. 2006;**100**:276-282

[20] Brinton LA, Thistle JE, Liao LM, Trabert B. Epidemiology of vulvar neoplasia in the NIH-AARP Study. Gynecologic Oncology. 2017;**145**:298-304

[21] Pinto AP, Schlecht NF, Pintos J, Kaiano J, Franco EL, Crum CP, Villa LL. Prognostic significance of lymph node variables and human papillomavirus DNA in invasive vulvar carcinoma. Gynecologic Oncology. 2004;**92**:856-865

[22] Sutton BC, Allen RA, Moore WE, Dunn ST. Distribution of human papillomavirus genotypes in invasive squamous carcinoma of the vulva. Modern Pathology. 2008;**21**:345-354

[23] Alonso I, Fuste V, del Pino M, Castillo P, Torne A, Fuste P, Rios J, Pahisa J, Balasch J, Ordi J. Does human papillomavirus infection imply a different prognosis in vulvar squamous cell carcinoma? Gynecologic Oncology. 2011;**122**:509-514

[24] Pinto AP, Signorello LB, Crum CP, Harlow BL, Abrao F, Villa LL. Squamous cell carcinoma of the vulva in Brazil: Prognostic importance of host and viral variables. Gynecologic Oncology. 1999;**74**:61-67

[25] Hinten F, Molijn A, Eckhardt L, Massuger LFAG, Quint W, Bult P, Bulten J, Melchers WJG, de Hullu JA. Vulvar cancer: Two pathways with different localization and prognosis. Gynecologic Oncology. 2018;**149**:310-317

[26] Yap ML, Allo G, Cuartero J, Pintilie M, Kamel-Reid S, Murphy J, Mackay H, Clarke B, Fyles A, Milosevic M. Prognostic significance of human papilloma virus and p16 expression in patients with vulvar squamous cell carcinoma who received radiotherapy. Clinical Oncology (Royal College of Radiologists (Great Britain)). 2018;**30**:254-261

[27] Rasmussen CL, Sand FL, Hoffmann FM, Kaae AK, Kjaer SK. Does HPV status influence survival after vulvar cancer? International Journal of Cancer. 2018;**142**:1158-1165

**45**

*HPV Infection and Vulvar Cancer*

2016;**142**:293-298

2016;**20**:252-256

2014;**7**:7597-7609

2012;**14**:116-124

2016;**143**:414-420

*DOI: http://dx.doi.org/10.5772/intechopen.80601*

[28] Monk BJ, Burger RA, Lin F, Parham G, Vasilev SA, Wilczynski SP. Prognostic significance of human papillomavirus DNA in vulvar carcinoma. Obstetrics and Gynecology. 1995;**85**:709-715

D, Scaffa C, Perotto S, Sabatucci I, Indini A, Lorusso D, Raspagliesi F. The association of pre-treatment HPV subtypes with recurrence of VIN. European Journal of Obstetrics, Gynecology, and Reproductive Biology.

[35] Satmary W, Holschneider CH, Brunette LL, Natarajan S. Vulvar intraepithelial neoplasia: Risk factors for recurrence. Gynecologic Oncology.

[36] Westra WH, Taube JM, Poeta ML, Begum S, Sidransky D, Koch WM. Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck. Clinical Cancer Research.

[37] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, Tous S, Felix A, Bravo LE, Shin HR, Vallejos CS, de Ruiz PA, Lima MA, Guimera N, Clavero O, Alejo M, Llombart-Bosch A, Cheng-Yang C, Tatti SA, Kasamatsu E, Iljazovic E, Odida M, Prado R, Seoud M, Grce M, Usubutun A, Jain A, Suarez GA, Lombardi LE, Banjo A, Menendez C, Domingo EJ, Velasco J, Nessa A, Chichareon SC, Qiao YL, Lerma E, Garland SM, Sasagawa T, Ferrera A, Hammouda D, Mariani L, Pelayo A, Steiner I, Oliva E, Meijer CJ, Al Jassar WF, Cruz E, Wright TC, Puras A, Llave CL, Tzardi M, Agorastos T, Garcia-Barriola V, Clavel C, Ordi J, Andujar M, Castellsague X, Sanchez GI, Nowakowski AM, Bornstein J, Munoz N, Bosch FX. Human papillomavirus genotype attribution in invasive cervical cancer: A retrospective cross-sectional worldwide study. Lancet Oncoloy.

2017;**211**:37-41

2018;**148**:126-131

2008;**14**:366-369

2010;**11**:1048-1056

[38] Garland SM, Hernandez-Avila M, Wheeler CM, Perez G, Harper DM, Leodolter S, Tang GW, Ferris DG, Steben M, Bryan J, Taddeo FJ, Railkar R, Esser MT, Sings HL,

[29] Lee LJ, Howitt B, Catalano P, Tanaka C, Murphy R, Cimbak N, DeMaria R, Bu P, Crum C, Horowitz N, Matulonis U, Viswanathan AN. Prognostic importance of human papillomavirus (HPV) and p16 positivity in squamous cell carcinoma of the vulva treated with radiotherapy. Gynecologic Oncology.

[30] Hay CM, Lachance JA, Lucas FL, Smith KA, Jones MA. Biomarkers p16, human papillomavirus and p53 predict recurrence and survival in early stage squamous cell carcinoma of the vulva. Journal of Lower Genital Tract Disease.

[31] Li M, Wang Y, Song Y, Bu R, Yin B, Fei X, Guo Q, Wu B. Expression profiling and clinicopathological significance of DNA methyltransferase 1, 3A and 3B in sporadic human renal cell carcinoma. International Journal of Clinical and Experimental Pathology.

[32] Zhang JJ, Zhu Y, Zhu Y, Wu JL, Liang WB, Zhu R, Xu ZK, Du Q, Miao Y. Association of increased DNA methyltransferase expression with carcinogenesis and poor prognosis in pancreatic ductal adenocarcinoma. Clinical and Translational Oncology.

[33] Leonard S, Pereira M, Fox R, Gordon N, Yap J, Kehoe S, Luesley D, Woodman C, Ganesan R. Overexpression of DNMT3A predicts the risk of recurrent vulvar squamous cell carcinomas. Gynecologic Oncology.

[34] Bogani G, Martinelli F, Ditto A, Signorelli M, Taverna F, Lombardo C, Chiappa V, Leone Roberti MU, Recalcati

### *HPV Infection and Vulvar Cancer DOI: http://dx.doi.org/10.5772/intechopen.80601*

*Current Perspectives in Human Papillomavirus*

[21] Pinto AP, Schlecht NF, Pintos J, Kaiano J, Franco EL, Crum CP, Villa LL. Prognostic significance of lymph node variables and human papillomavirus DNA in invasive vulvar carcinoma. Gynecologic Oncology.

[22] Sutton BC, Allen RA, Moore WE, Dunn ST. Distribution of human papillomavirus genotypes in invasive squamous carcinoma of the vulva. Modern Pathology. 2008;**21**:345-354

[23] Alonso I, Fuste V, del Pino M, Castillo P, Torne A, Fuste P, Rios J, Pahisa J, Balasch J, Ordi J. Does human papillomavirus infection imply a different prognosis in vulvar squamous cell carcinoma? Gynecologic Oncology.

[24] Pinto AP, Signorello LB, Crum CP, Harlow BL, Abrao F, Villa LL. Squamous cell carcinoma of the vulva in Brazil: Prognostic importance of host and viral variables. Gynecologic Oncology.

[25] Hinten F, Molijn A, Eckhardt L, Massuger LFAG, Quint W, Bult P, Bulten J, Melchers WJG, de Hullu JA. Vulvar cancer: Two pathways with different localization and prognosis. Gynecologic Oncology.

[26] Yap ML, Allo G, Cuartero J, Pintilie M, Kamel-Reid S, Murphy J, Mackay H, Clarke B, Fyles A, Milosevic M. Prognostic significance of human papilloma virus and p16 expression in patients with vulvar squamous cell carcinoma who received radiotherapy. Clinical Oncology (Royal College of Radiologists (Great Britain)).

[27] Rasmussen CL, Sand FL, Hoffmann FM, Kaae AK, Kjaer SK. Does HPV status influence survival after vulvar cancer? International Journal of Cancer.

2004;**92**:856-865

2011;**122**:509-514

1999;**74**:61-67

2018;**149**:310-317

2018;**30**:254-261

2018;**142**:1158-1165

carcinoma. International Journal of

[15] Wilkinson EJ, Kneale BL, Lynch FW. Report of the ISSVD Terminology Committee: VIN. The Journal of

[16] Darragh TM, Colgan TJ, Cox JT, Heller DS, Henry MR, Luff RD, McCalmont T, Nayar R, Palefsky JM, Stoler MH, Wilkinson EJ, Zaino RJ, Wilbur DC. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: Background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Archives of Pathology and Laboratory Medicine.

2012;**136**:1266-1297

1995;**75**:2879-2884

1995;**61**:306-311

2017;**145**:298-304

Reproductive Medicine. 1986;**31**:973-974

[17] van Beurden M, ten Kate FJ, Smits HL, Berkhout RJ, de Craen AJ, van der Vange N, Lammes FB, ter Schegget J. Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus. Cancer.

[18] Remmink AJ, Walboomers JM, Helmerhorst TJ, Voorhorst FJ, Rozendaal L, Risse EK, Meijer CJ, Kenemans P. The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: Natural history up to 36

months. International Journal of Cancer.

[19] Hillemanns P, Wang X. Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia. Gynecologic

Oncology. 2006;**100**:276-282

[20] Brinton LA, Thistle JE, Liao LM, Trabert B. Epidemiology of vulvar neoplasia in the NIH-AARP Study. Gynecologic Oncology.

Cancer. 2008;**123**:2767-2773

**44**

[28] Monk BJ, Burger RA, Lin F, Parham G, Vasilev SA, Wilczynski SP. Prognostic significance of human papillomavirus DNA in vulvar carcinoma. Obstetrics and Gynecology. 1995;**85**:709-715

[29] Lee LJ, Howitt B, Catalano P, Tanaka C, Murphy R, Cimbak N, DeMaria R, Bu P, Crum C, Horowitz N, Matulonis U, Viswanathan AN. Prognostic importance of human papillomavirus (HPV) and p16 positivity in squamous cell carcinoma of the vulva treated with radiotherapy. Gynecologic Oncology. 2016;**142**:293-298

[30] Hay CM, Lachance JA, Lucas FL, Smith KA, Jones MA. Biomarkers p16, human papillomavirus and p53 predict recurrence and survival in early stage squamous cell carcinoma of the vulva. Journal of Lower Genital Tract Disease. 2016;**20**:252-256

[31] Li M, Wang Y, Song Y, Bu R, Yin B, Fei X, Guo Q, Wu B. Expression profiling and clinicopathological significance of DNA methyltransferase 1, 3A and 3B in sporadic human renal cell carcinoma. International Journal of Clinical and Experimental Pathology. 2014;**7**:7597-7609

[32] Zhang JJ, Zhu Y, Zhu Y, Wu JL, Liang WB, Zhu R, Xu ZK, Du Q, Miao Y. Association of increased DNA methyltransferase expression with carcinogenesis and poor prognosis in pancreatic ductal adenocarcinoma. Clinical and Translational Oncology. 2012;**14**:116-124

[33] Leonard S, Pereira M, Fox R, Gordon N, Yap J, Kehoe S, Luesley D, Woodman C, Ganesan R. Overexpression of DNMT3A predicts the risk of recurrent vulvar squamous cell carcinomas. Gynecologic Oncology. 2016;**143**:414-420

[34] Bogani G, Martinelli F, Ditto A, Signorelli M, Taverna F, Lombardo C, Chiappa V, Leone Roberti MU, Recalcati D, Scaffa C, Perotto S, Sabatucci I, Indini A, Lorusso D, Raspagliesi F. The association of pre-treatment HPV subtypes with recurrence of VIN. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 2017;**211**:37-41

[35] Satmary W, Holschneider CH, Brunette LL, Natarajan S. Vulvar intraepithelial neoplasia: Risk factors for recurrence. Gynecologic Oncology. 2018;**148**:126-131

[36] Westra WH, Taube JM, Poeta ML, Begum S, Sidransky D, Koch WM. Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck. Clinical Cancer Research. 2008;**14**:366-369

[37] de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, Tous S, Felix A, Bravo LE, Shin HR, Vallejos CS, de Ruiz PA, Lima MA, Guimera N, Clavero O, Alejo M, Llombart-Bosch A, Cheng-Yang C, Tatti SA, Kasamatsu E, Iljazovic E, Odida M, Prado R, Seoud M, Grce M, Usubutun A, Jain A, Suarez GA, Lombardi LE, Banjo A, Menendez C, Domingo EJ, Velasco J, Nessa A, Chichareon SC, Qiao YL, Lerma E, Garland SM, Sasagawa T, Ferrera A, Hammouda D, Mariani L, Pelayo A, Steiner I, Oliva E, Meijer CJ, Al Jassar WF, Cruz E, Wright TC, Puras A, Llave CL, Tzardi M, Agorastos T, Garcia-Barriola V, Clavel C, Ordi J, Andujar M, Castellsague X, Sanchez GI, Nowakowski AM, Bornstein J, Munoz N, Bosch FX. Human papillomavirus genotype attribution in invasive cervical cancer: A retrospective cross-sectional worldwide study. Lancet Oncoloy. 2010;**11**:1048-1056

[38] Garland SM, Hernandez-Avila M, Wheeler CM, Perez G, Harper DM, Leodolter S, Tang GW, Ferris DG, Steben M, Bryan J, Taddeo FJ, Railkar R, Esser MT, Sings HL,

Nelson M, Boslego J, Sattler C, Barr E, Koutsky LA. Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. New England Journal of Medicine. 2007;**356**:1928-1943

[39] Lynge E, Rygaard C, Baillet MV, Dugue PA, Sander BB, Bonde J, Rebolj M. Cervical cancer screening at crossroads. APMIS. 2014;**122**:667-673

[40] Skorstengaard M, Thamsborg LH, Lynge E. Burden of HPV-caused cancers in Denmark and the potential effect of HPV-vaccination. Vaccine. 2017;**35**:5939-5945

[41] Bolhassani A. Future prospects in HPV prevention and treatment. In: Azam Bolhassani. HPV Infections: Diagnosis, Prevention, and Treatment. Bentham Science Publishers; 2018. p. 220-226. DOI: 10.2174/97816810861701180101

**47**

**Chapter 4**

**Abstract**

broadly reviewed.

**1. Introduction**

and Lung Cancer

*Oscar Arrieta and Rafael Rosell*

Human Papillomavirus Infection

*Andrés F. Cardona, Alejandro Ruiz-Patiño, Luisa Ricaurte,* 

Lung cancer continues to be the most common neoplasia and represents the leading cause of cancer-related death in the world. Nonetheless, contrary to expected projections, the decrease in incidence expected by decrease in tobacco exposure has been partially halted due to an increasing amount of lung cancer cases in nonsmokers, particularly in female patients. This led to the development of new hypotheses in terms of lung cancer etiology, including the involvement of oncogenic viruses such as the human papillomavirus (HPV). HPV role in the pathophysiology of lung cancer, including adenocarcinoma and squamous cell carcinoma, is currently under research. Exposure to HPV, and the resulting infection, can occur in several possible ways, including sexual transmission and airborne fomites. Main pathogenic occurrences include alterations in inhibition of p53 and retinoblastoma. This chapter presents the current evidence as to the role of HPV in the development of lung cancer, methods to establish HPV infection, and also explores the role of predisposing factors, as well as immunological and inflammatory factors in nonsmokers. Additionally, the role of other molecular factors, such as EGFR, interleukins 6 and 10, and others, is discussed. Finally, future perspectives in this new paradigm of lung cancer in nonsmokers are

*Leonardo Rojas, Zyanya Lucia Zatarain-Barrón,* 

**Keywords:** HPV, lung cancer, inflamation, immunogenicity, viral DNA

Lung cancer is the most common cancer in the world. In 2012, 1.8 million new cases were diagnosed and 1.6 million people died as a consequence of this disease [1]. It is one of the top 10 leading causes of death worldwide [2]. About 90% of lung cancer cases in men and 75% in women in the United States and Europe are caused by tobacco smoke [3, 4]. An important proportion of lung cancer cases presents in nonsmokers, as shown in several reports. In the Caucasian population, the rate of nonsmall cell lung cancer (NSCLC) in non-smokers is 10% for men and 20% for women, while for Asian populations the rate reached 30–40% [5, 6]. In the United States, the overall lung cancer incidence rates and mortality have been declining for the past two decades, and the reduction in both of these parameters has been more prominent in men than in women, a trend that likely reflects the decrease in smoking rates in the

### **Chapter 4**

*Current Perspectives in Human Papillomavirus*

Nelson M, Boslego J, Sattler C, Barr E, Koutsky LA. Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. New England Journal of Medicine.

[39] Lynge E, Rygaard C, Baillet MV, Dugue PA, Sander BB, Bonde J, Rebolj M. Cervical cancer screening at crossroads. APMIS. 2014;**122**:667-673

[40] Skorstengaard M, Thamsborg LH, Lynge E. Burden of HPV-caused cancers in Denmark and the potential effect of HPV-vaccination. Vaccine.

[41] Bolhassani A. Future prospects in HPV prevention and treatment.

Infections: Diagnosis, Prevention, and Treatment. Bentham Science Publishers; 2018. p. 220-226. DOI: 10.2174/97816810861701180101

In: Azam Bolhassani. HPV

2007;**356**:1928-1943

2017;**35**:5939-5945

**46**

## Human Papillomavirus Infection and Lung Cancer

*Andrés F. Cardona, Alejandro Ruiz-Patiño, Luisa Ricaurte, Leonardo Rojas, Zyanya Lucia Zatarain-Barrón, Oscar Arrieta and Rafael Rosell*

### **Abstract**

Lung cancer continues to be the most common neoplasia and represents the leading cause of cancer-related death in the world. Nonetheless, contrary to expected projections, the decrease in incidence expected by decrease in tobacco exposure has been partially halted due to an increasing amount of lung cancer cases in nonsmokers, particularly in female patients. This led to the development of new hypotheses in terms of lung cancer etiology, including the involvement of oncogenic viruses such as the human papillomavirus (HPV). HPV role in the pathophysiology of lung cancer, including adenocarcinoma and squamous cell carcinoma, is currently under research. Exposure to HPV, and the resulting infection, can occur in several possible ways, including sexual transmission and airborne fomites. Main pathogenic occurrences include alterations in inhibition of p53 and retinoblastoma. This chapter presents the current evidence as to the role of HPV in the development of lung cancer, methods to establish HPV infection, and also explores the role of predisposing factors, as well as immunological and inflammatory factors in nonsmokers. Additionally, the role of other molecular factors, such as EGFR, interleukins 6 and 10, and others, is discussed. Finally, future perspectives in this new paradigm of lung cancer in nonsmokers are broadly reviewed.

**Keywords:** HPV, lung cancer, inflamation, immunogenicity, viral DNA

### **1. Introduction**

Lung cancer is the most common cancer in the world. In 2012, 1.8 million new cases were diagnosed and 1.6 million people died as a consequence of this disease [1]. It is one of the top 10 leading causes of death worldwide [2]. About 90% of lung cancer cases in men and 75% in women in the United States and Europe are caused by tobacco smoke [3, 4]. An important proportion of lung cancer cases presents in nonsmokers, as shown in several reports. In the Caucasian population, the rate of nonsmall cell lung cancer (NSCLC) in non-smokers is 10% for men and 20% for women, while for Asian populations the rate reached 30–40% [5, 6]. In the United States, the overall lung cancer incidence rates and mortality have been declining for the past two decades, and the reduction in both of these parameters has been more prominent in men than in women, a trend that likely reflects the decrease in smoking rates in the

male population [7]. Interestingly, in developed countries, lung cancer incidence has been gradually increasing for non-smokers [8–10]. In Asian countries, the situation is similar; lung cancer incidence and mortality have been increasing despite the implementation of successful anti-smoking campaigns [11, 12].

Among the histologic subtypes of NSCLC, squamous cell lung cancer (SCC) is more common in men (44% cases in men vs. 25% in women) and adenocarcinoma (ADC) is more common in women (28% cases in men and 42% in women). SCC and small cell lung cancers (SCLC) are more closely associated with smoking, in contrast to ADC that is most commonly found in non-smokers [13]. In fact, the calculated histological distribution of lung cancer among smokers and non-smokers in 17 different studies has shown that 53% of the cases in smokers and 19% in non-smokers are SCC while 62% in non-smokers and 18% in smokers are ADC [14]. Furthermore, ADC in non-smokers appears to have a less complex histology with a higher presence of targetable driver mutations, particularly *EGFR*, *Her2* as well as *ALK* and *ROS* translocations [15, 16].

The differences in epidemiology, genetic profile, and survival outcomes of lung cancer in non-smokers have made it clear that this malignancy is a separate entity from lung cancer in smokers [17]. Over the last decades, the investigation of the preventable risk factors associated with lung cancer in non-smokers has gained much attention. Of interest, human papillomavirus (HPV) has been reported in


#### **Figure 1.**

*Forest plot of random effects model stratified by study design. Individual study OR and grouped ORs sub (reproduced with authorization from Ref. [24].*

**49**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

infection and lung cancer.

respiratory diseases [15].

association [41].

**smokers**

non-smoker population [25, 26].

lung cancer tissues from Western and Eastern countries, being types 18 and 16 the most common pathogenic species [18–21]. Likewise, recent studies have demonstrated a significantly increased risk of acquiring lung cancer in non-smokers who are exposed to HPV infection (OR 5.32; 95% CI 1.75–16.17) [22, 23]. **Figure 1** depicts a meta-analysis of recent studies evaluating the association between HPV

**2. Immune and inflammation markers in lung cancer among never** 

Multiple studies have shown an increased incidence of non-smoker lung cancer in females [3, 9, 10, 27–30]. After analyzing a cohort of 975 patients in Singapore, Yano et al. identified that non-smoker lung cancer patients presented at a younger age and with an earlier stage at diagnosis than their smoker counterparts [10]. The most important risk factors for lung cancer in non-smokers are second-hand smoke, indoor air pollution, occupational exposures, genetic susceptibility, family history, estrogen levels, HPV infection, and pre-existing

Respiratory diseases elicit a deleterious chronic inflammatory response in lung tissue, which in turn causes an increased rate of cell division leading to an augmented risk of DNA damage [31]. Additionally, inflammation stimulates antiapoptotic signal activation and angiogenesis further promoting tumorigenesis [32, 33]. Previous studies have also suggested an important role for lung diseases in the development of lung cancer, even when not associated with tobacco use. In 2012, Brenner et al. carried out an extensive analysis from 17 studies and identified that a history of emphysema, pneumonia, and tuberculosis elevated the risk for lung cancer development among non-smokers [34]. Likewise, other case-control studies across various populations have concluded that a history of respiratory diseases, including chronic obstructive pulmonary disease (COPD), asthma, pneumonia, and tuberculosis, would increase the risk of developing lung cancer [35–40]. A systematic review demonstrated that pre-existing tuberculosis increased lung cancer risk in non-smokers (RR 1.78, 95% CI 1.42–2.23); interestingly, the increased risk was only associated with ADC histology, while SCC and SCLC showed no

Due to the association between respiratory diseases and lung cancer among non-smokers, inflammatory pathways and markers have been the focus of much interest in recent years and hence have been widely studied. A case-control study nested within three prospective cohort studies carried out in Australia and Sweden identified a higher lung cancer risk for participants who had elevated concentrations of interleukin (IL)-6 and IL-8. These associations were stronger for former smokers (smoking cessation at least 10 years before study was performed) than for current smokers, both for IL-6 and IL-8; however, these associations were not observed in never-smoker patients [42]. Other inflammatory markers have been found to be significantly associated with lung cancer risk. In a previous case-control study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening trial (PLCO), 11 markers of importance were identified. Interestingly, nine of these markers were significantly associated with lung cancer among non-smokers, which

Importantly, the public health impact of these recent findings has been recently explored, since vaccination against HPV could represent an efficacious measure to prevent lung cancer. Additionally, the timely detection of HPV in the respiratory tract could warrant a method for early diagnosis of lung cancer, particularly in the

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Current Perspectives in Human Papillomavirus*

*ALK* and *ROS* translocations [15, 16].

mentation of successful anti-smoking campaigns [11, 12].

male population [7]. Interestingly, in developed countries, lung cancer incidence has been gradually increasing for non-smokers [8–10]. In Asian countries, the situation is similar; lung cancer incidence and mortality have been increasing despite the imple-

Among the histologic subtypes of NSCLC, squamous cell lung cancer (SCC) is more common in men (44% cases in men vs. 25% in women) and adenocarcinoma (ADC) is more common in women (28% cases in men and 42% in women). SCC and small cell lung cancers (SCLC) are more closely associated with smoking, in contrast to ADC that is most commonly found in non-smokers [13]. In fact, the calculated histological distribution of lung cancer among smokers and non-smokers in 17 different studies has shown that 53% of the cases in smokers and 19% in non-smokers are SCC while 62% in non-smokers and 18% in smokers are ADC [14]. Furthermore, ADC in non-smokers appears to have a less complex histology with a higher presence of targetable driver mutations, particularly *EGFR*, *Her2* as well as

The differences in epidemiology, genetic profile, and survival outcomes of lung cancer in non-smokers have made it clear that this malignancy is a separate entity from lung cancer in smokers [17]. Over the last decades, the investigation of the preventable risk factors associated with lung cancer in non-smokers has gained much attention. Of interest, human papillomavirus (HPV) has been reported in

*Forest plot of random effects model stratified by study design. Individual study OR and grouped ORs sub* 

**48**

**Figure 1.**

*(reproduced with authorization from Ref. [24].*

lung cancer tissues from Western and Eastern countries, being types 18 and 16 the most common pathogenic species [18–21]. Likewise, recent studies have demonstrated a significantly increased risk of acquiring lung cancer in non-smokers who are exposed to HPV infection (OR 5.32; 95% CI 1.75–16.17) [22, 23]. **Figure 1** depicts a meta-analysis of recent studies evaluating the association between HPV infection and lung cancer.

Importantly, the public health impact of these recent findings has been recently explored, since vaccination against HPV could represent an efficacious measure to prevent lung cancer. Additionally, the timely detection of HPV in the respiratory tract could warrant a method for early diagnosis of lung cancer, particularly in the non-smoker population [25, 26].

### **2. Immune and inflammation markers in lung cancer among never smokers**

Multiple studies have shown an increased incidence of non-smoker lung cancer in females [3, 9, 10, 27–30]. After analyzing a cohort of 975 patients in Singapore, Yano et al. identified that non-smoker lung cancer patients presented at a younger age and with an earlier stage at diagnosis than their smoker counterparts [10]. The most important risk factors for lung cancer in non-smokers are second-hand smoke, indoor air pollution, occupational exposures, genetic susceptibility, family history, estrogen levels, HPV infection, and pre-existing respiratory diseases [15].

Respiratory diseases elicit a deleterious chronic inflammatory response in lung tissue, which in turn causes an increased rate of cell division leading to an augmented risk of DNA damage [31]. Additionally, inflammation stimulates antiapoptotic signal activation and angiogenesis further promoting tumorigenesis [32, 33]. Previous studies have also suggested an important role for lung diseases in the development of lung cancer, even when not associated with tobacco use. In 2012, Brenner et al. carried out an extensive analysis from 17 studies and identified that a history of emphysema, pneumonia, and tuberculosis elevated the risk for lung cancer development among non-smokers [34]. Likewise, other case-control studies across various populations have concluded that a history of respiratory diseases, including chronic obstructive pulmonary disease (COPD), asthma, pneumonia, and tuberculosis, would increase the risk of developing lung cancer [35–40]. A systematic review demonstrated that pre-existing tuberculosis increased lung cancer risk in non-smokers (RR 1.78, 95% CI 1.42–2.23); interestingly, the increased risk was only associated with ADC histology, while SCC and SCLC showed no association [41].

Due to the association between respiratory diseases and lung cancer among non-smokers, inflammatory pathways and markers have been the focus of much interest in recent years and hence have been widely studied. A case-control study nested within three prospective cohort studies carried out in Australia and Sweden identified a higher lung cancer risk for participants who had elevated concentrations of interleukin (IL)-6 and IL-8. These associations were stronger for former smokers (smoking cessation at least 10 years before study was performed) than for current smokers, both for IL-6 and IL-8; however, these associations were not observed in never-smoker patients [42]. Other inflammatory markers have been found to be significantly associated with lung cancer risk. In a previous case-control study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening trial (PLCO), 11 markers of importance were identified. Interestingly, nine of these markers were significantly associated with lung cancer among non-smokers, which

included the epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5) and IL-7, and also associated with lung cancer overall, and others not associated with lung cancer overall, which included human granulocyte chemotactic protein-2 (GCP2/CXCL6), granulocyte colony stimulating factor (G-CSF), IL-6, macrophage inflammatory protein 1B, 2 and 4 (MIP-1B/CCL4, MCP-2/CCL8, MCP-4/CCL13), and stromal cell derived factor-1 (SDF-1 A-B/CXCL12) [43]. Two years later, the PLCO trial was nested to its replication in a case-control study. Both of the nested case-control trials demonstrated that circulating levels of C-reactive protein (CRP), serum amyloid A (SAA), soluble tumor necrosis factor receptor 2 (sTNFRII), and monokine induced by gamma interferon (CXCL9/MIG) were associated with lung cancer risk [43, 44]. These associations were limited to smokers, and the study was considered to be underpowered to evaluate associations among non-smokers [44]. Finally, a nested case-control study carried out within the Shanghai Women's Health Study evaluated 61 inflammatory markers among non-smoker Chinese women. Nine markers were statistically significantly associated with lung cancer: soluble IL-6 receptor (sIL6R) and chemokine ligand 2/monocyte chemotactic protein 2 (CCL2/MCP-1) were associated with an increased risk of lung cancer, while IL-21, chemokine (C-X3-C motif) ligand 1/fractalkine (CX3CL1/fractalkine), soluble vascular endothelial growth factor receptor 2 (sVEGFR2) and sTNFRII and CRP were associated with a decreased risk. Interestingly sIL-6R was associated with an increased lung cancer risk even 7.5 years prior to diagnosis. The results of this study further support our current knowledge in terms of the role of inflammation and immune response on the development of lung cancer among the female nonsmoker population.

### **3. HPV transmission in lung cancer**

Since the respiratory tract is composed of two different epitheliums, it contains various squamous columnar junctions (SCJ). In the bronchi, the SCJ may occur naturally or most commonly as squamous metaplasia (SQM) secondary to cigarette smoking [45]. The SQM process initiates with the activation and posterior hyperproliferation of the SQM quiescent basal cells present in the pseudostratified epithelium. As a consequence, the epithelial cells begin to show a squamous cell differentiation, given by the expression of squamous epithelial cytokeratins and cell adhesion molecule SQM1. Finally, cells express involucrin, a protein which indicates that the cells are in a terminal differentiation stage [46–52]. The biochemical and metabolic changes that SQM induces make the bronchial epithelium susceptible to HPV infection. Hence, the multiple foci of SQM in the bronchi are analogous to the transformation zone of the uterine cervix, as both work as the point of entry for HPV [53, 54].

To date, there are three hypotheses regarding HPV infection and transmission into the pulmonary tissue [24, 55]. The first one states that the HPV infection occurs in the reproductive system (male or female) and then hematogenously transmitted to the lung tissue. A study reports that approximately 80% of female HPV-positive lung cancer patients also have cervical intraepithelial neoplasia [56]. Peripheral blood cells (B cells, dendritic cells, NK cells, and neutrophils) on healthy men have been shown to be infected by high- and low-risk HPV [57]. Furthermore, Bodaghi et al. identified HPV types 16 and 18 on peripheral blood from healthy transfusion donors [58]. Since the lung is a highly vascularized tissue, this makes it susceptible to capture the virus and eventually develop the tumor. More evidence to support this hypothesis is the high prevalence of HPV DNA in peripheral blood samples

**51**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Possible transmission pathways of HPV infection.*

**Figure 2.**

These hypotheses are summarized in **Figure 2**.

seen in NSCLC patients [25]. These findings have led some authors to suggest that these peripheral blood cells can be a viral reservoir for the infection of other organs and even contribute to viral spread in a sexual contact-independent manner [55]. Another hypothesis refers to oral-genital HPV infection that causes transmission through the throat into the lung. HPV infection may be propagated either through oral-oral contact or genital to oral contact. A survey that followed 222 men and their female patterns showed an infection rate in men of 7.2% with the majority of their female partners having either cervical or oral HPV infection. And finally, a third hypothesis is that HPV may be transmitted as an airborne disease. Carpagnano et al. reported the presence of HPV DNA in exhaled breath condensate of lung cancer patients, hence proposing that HPV transmission can occur through inhalation [59].

**4. Possible molecular mechanisms of HPV-induced lung carcinogenesis**

HPV initially interacts with cellular receptors and infects the basal lamina, transferring its viral genomes to the nucleus [60]. These events are followed by an initial phase of genome amplification and afterwards by a steady maintenance of the viral episome at low copy number [61, 62], particularly around 200 copies per cell, based on the study of episomal cell lines derived from cervical lesions [63]. The replication process requires the hijacking of the of the Retinoblastoma family of proteins (RB, p108 and p130), which regulate cell proliferation, as well as the inhibition of p53, which disrupts apoptosis [64]. The carcinogenic potential comes from the presence of the genes E6 and E7 in the HPV genome, especially in serotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. The E6 protein is composed of roughly 150 amino acids and has the ability to interact with many targets due to its structure containing two zinc fingers built by two pairs of CXX motifs [65]. The main target of this protein is p53. After binding with E3 ubiquitin ligase UBE3A/E6AP (E6-associated protein), the E6/ E6AP complex marks p53 for proteasome-dependent degradation. E6 is also involved in other steps of oncogenesis including guarantying survival of the infected cells and offering evasion of the infected cells from the immune system. This protein has in turn the ability to stimulate interleukin-10 (IL10) expression, a cytokine responsible for immunomodulation and anti-inflammatory effects. Among its effects, IL10 has the ability to induce autocrine and paracrine immunologic tolerance due to activation of T helper 2 and T regulatory lymphocytes, inhibition of pro-inflammatory cytokines, and downregulation of the MHC classes I and II, arresting DC maturation

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Current Perspectives in Human Papillomavirus*

smoker population.

HPV [53, 54].

**3. HPV transmission in lung cancer**

included the epithelial neutrophil-activating peptide 78 (ENA-78/CXCL5) and IL-7, and also associated with lung cancer overall, and others not associated with lung cancer overall, which included human granulocyte chemotactic protein-2 (GCP2/CXCL6), granulocyte colony stimulating factor (G-CSF), IL-6, macrophage inflammatory protein 1B, 2 and 4 (MIP-1B/CCL4, MCP-2/CCL8, MCP-4/CCL13), and stromal cell derived factor-1 (SDF-1 A-B/CXCL12) [43]. Two years later, the PLCO trial was nested to its replication in a case-control study. Both of the nested case-control trials demonstrated that circulating levels of C-reactive protein (CRP), serum amyloid A (SAA), soluble tumor necrosis factor receptor 2 (sTNFRII), and monokine induced by gamma interferon (CXCL9/MIG) were associated with lung cancer risk [43, 44]. These associations were limited to smokers, and the study was considered to be underpowered to evaluate associations among non-smokers [44]. Finally, a nested case-control study carried out within the Shanghai Women's Health Study evaluated 61 inflammatory markers among non-smoker Chinese women. Nine markers were statistically significantly associated with lung cancer: soluble IL-6 receptor (sIL6R) and chemokine ligand 2/monocyte chemotactic protein 2 (CCL2/MCP-1) were associated with an increased risk of lung cancer, while IL-21, chemokine (C-X3-C motif) ligand 1/fractalkine (CX3CL1/fractalkine), soluble vascular endothelial growth factor receptor 2 (sVEGFR2) and sTNFRII and CRP were associated with a decreased risk. Interestingly sIL-6R was associated with an increased lung cancer risk even 7.5 years prior to diagnosis. The results of this study further support our current knowledge in terms of the role of inflammation and immune response on the development of lung cancer among the female non-

Since the respiratory tract is composed of two different epitheliums, it contains various squamous columnar junctions (SCJ). In the bronchi, the SCJ may occur naturally or most commonly as squamous metaplasia (SQM) secondary to cigarette smoking [45]. The SQM process initiates with the activation and posterior hyperproliferation of the SQM quiescent basal cells present in the pseudostratified epithelium. As a consequence, the epithelial cells begin to show a squamous cell differentiation, given by the expression of squamous epithelial cytokeratins and cell adhesion molecule SQM1. Finally, cells express involucrin, a protein which indicates that the cells are in a terminal differentiation stage [46–52]. The biochemical and metabolic changes that SQM induces make the bronchial epithelium susceptible to HPV infection. Hence, the multiple foci of SQM in the bronchi are analogous to the transformation zone of the uterine cervix, as both work as the point of entry for

To date, there are three hypotheses regarding HPV infection and transmission into the pulmonary tissue [24, 55]. The first one states that the HPV infection occurs in the reproductive system (male or female) and then hematogenously transmitted to the lung tissue. A study reports that approximately 80% of female HPV-positive lung cancer patients also have cervical intraepithelial neoplasia [56]. Peripheral blood cells (B cells, dendritic cells, NK cells, and neutrophils) on healthy men have been shown to be infected by high- and low-risk HPV [57]. Furthermore, Bodaghi et al. identified HPV types 16 and 18 on peripheral blood from healthy transfusion donors [58]. Since the lung is a highly vascularized tissue, this makes it susceptible to capture the virus and eventually develop the tumor. More evidence to support this hypothesis is the high prevalence of HPV DNA in peripheral blood samples

**50**

**Figure 2.** *Possible transmission pathways of HPV infection.*

seen in NSCLC patients [25]. These findings have led some authors to suggest that these peripheral blood cells can be a viral reservoir for the infection of other organs and even contribute to viral spread in a sexual contact-independent manner [55]. Another hypothesis refers to oral-genital HPV infection that causes transmission through the throat into the lung. HPV infection may be propagated either through oral-oral contact or genital to oral contact. A survey that followed 222 men and their female patterns showed an infection rate in men of 7.2% with the majority of their female partners having either cervical or oral HPV infection. And finally, a third hypothesis is that HPV may be transmitted as an airborne disease. Carpagnano et al. reported the presence of HPV DNA in exhaled breath condensate of lung cancer patients, hence proposing that HPV transmission can occur through inhalation [59]. These hypotheses are summarized in **Figure 2**.

### **4. Possible molecular mechanisms of HPV-induced lung carcinogenesis**

HPV initially interacts with cellular receptors and infects the basal lamina, transferring its viral genomes to the nucleus [60]. These events are followed by an initial phase of genome amplification and afterwards by a steady maintenance of the viral episome at low copy number [61, 62], particularly around 200 copies per cell, based on the study of episomal cell lines derived from cervical lesions [63]. The replication process requires the hijacking of the of the Retinoblastoma family of proteins (RB, p108 and p130), which regulate cell proliferation, as well as the inhibition of p53, which disrupts apoptosis [64]. The carcinogenic potential comes from the presence of the genes E6 and E7 in the HPV genome, especially in serotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. The E6 protein is composed of roughly 150 amino acids and has the ability to interact with many targets due to its structure containing two zinc fingers built by two pairs of CXX motifs [65]. The main target of this protein is p53. After binding with E3 ubiquitin ligase UBE3A/E6AP (E6-associated protein), the E6/ E6AP complex marks p53 for proteasome-dependent degradation. E6 is also involved in other steps of oncogenesis including guarantying survival of the infected cells and offering evasion of the infected cells from the immune system. This protein has in turn the ability to stimulate interleukin-10 (IL10) expression, a cytokine responsible for immunomodulation and anti-inflammatory effects. Among its effects, IL10 has the ability to induce autocrine and paracrine immunologic tolerance due to activation of T helper 2 and T regulatory lymphocytes, inhibition of pro-inflammatory cytokines, and downregulation of the MHC classes I and II, arresting DC maturation

and downregulating intercellular adhesion molecules and co-stimulating mediators. Additionally, IL10 has been shown to promote early E6 and E7 expression, consolidating a vicious cycle [66]. Other interleukins such as IL6 have been shown to be both a stimulator and an inhibitor of cell proliferation, depending on the cell line exposed. Through a complex process in which E6 causes STAT3 activation and IL6 expression, the cancer-associated fibroblast cells, present in the tumor microenvironment, suffer from IL6-induced and p16-mediated senescence. This phenomenon of adjacent cell dysfunction has been shown to promote neoplastic growth due to paracrine stimulation, chronic inflammation, and loss of oncolytic countermeasures [67]. Furthermore, IL6 has the ability to induce antiapoptotic Mcl-1 expression in HPVinfected lung cancer cells [68]. Mcl-1, member of the BCL-2 gene family is responsible, together with the Bcl-2 protein, for the apoptotic response of the mitochondria. Mcl-1 inhibits apoptosis by capturing BH3 and thus inhibiting Bax/Bcl-xl translocation, crucial steps in propagating an apoptotic signal in the cell [69]. Similarly, Bcl-2 offers a comparable Bax translocation inhibition, thus offering a strong antiapoptotic signal [70]. All in all, E6-activated and Il6-mediated BCL2 family modulation seems to be responsible for contributing to apoptosis inhibition and immortalization of HPV-infected cells. Other mechanisms of E6-induced immortalization include the expression of cIAP2, which is considered to be a very potent antiapoptotic factor in these cells by being the upstream inhibitor of caspase 3 activity. This hypothesis was validated in a study of induction of apoptosis in HeLa cells transfected with E6 and E7 proteins by knockdown of this molecule. E6 in turn causes the binding of p52 to NF-κB leading to an upregulation in the expression of cIAP [71, 72]. Additionally, this molecule lays as a downstream step in EGFR signaling, and its expression has also been strongly correlated with the presence of *EGFR* mutations [73]. One possible hypothesis to explain this phenomenon relates to the E6 inhibition of p53. Inactivation of this protein in turn leads to the loss of function in the MMR pathway, especially in MLH1 and MSH2, thus causing an increase in reactive oxygen species, which also strongly correlates with exon 19 *EGFR* mutations in lung cancer [74]. Finally, matrix metalloproteinases (MMP) seem to be also influenced by E6 interactions. These enzymes are responsible for degradation of the extracellular matrix, process required for cell migration and development of metastasis. MT1-MMP, MMP-2, and MMP-9 are the main members of this family that were upregulated by the expression of this oncogene [75], possibly by the induction of microRNAs [76].

E7, on the other hand, is responsible for binding and degrading pRb, p105, p107, and p130, especially in the upper epithelial layers. Additionally, E7 causes genome instability by deregulating the centrosome cycle [60]. Additionally, occurs the induction of the aryl hydrocarbon receptor signaling, a transcription factor involved in proliferation, differentiation, and apoptosis. Members of this family and inhibitors of cyclin-dependent kinases p16 and p21 have been proven to bind to E7 increasing pRb phosphorylation and promoting furthermore cell cycle deregulation [77]. Other activities can also be impaired by E7, such as epigenetic cell function. This molecule has the capacity to displace histone deacetylases, specially HDAC 1, 4, and 7 blocking their binding sites to the HIF-1α promoter regions and leading to an upregulation of its expression. This, in turn, is considered a key step in angiogenesis and thus strongly contributes to tumor growth [78].

### **5. HPV gene expression and detection in lung tissue**

The relation between HPV and lung cancer was postulated since the decade of the 1970s. In 1975, Roglic et al., followed by Rubel and Reynolds in 1979, observed

**53**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

development of lung cancer.

main goal.

sequencing [83, 84].

rized in **Table 1**.

**5.1 HPV detection and genotyping**

risk serotypes but also low risk serotypes.

koilocytosis, a classical pattern in HPV infection, in sputum samples from benign bronchial lesions [79, 80]. Simultaneously, Syrjanen described that the epithelial changes seen in bronchial carcinoma closely resembled HPV-induced genital lesions [81]. Afterwards, several epidemiological data, particularly in non-smoking lung cancer patients, firmly established the relationship between HPV infection and

Although several studies showed the presence of oncogenic HPV in lung cancer tissue, demonstrating a causal role is necessary. Taking into account that integration of HPV DNA within the host is the critical point for oncogenic transformation, demonstration of the HPV DNA presence in lung cancer DNA cells is the

Cheng et al. demonstrated that HPV DNA was integrated into lung cancer DNA cells but not in the adjacent non-tumor cells and also demonstrated that HPV (+) lung cancer patients were predominantly non-smoking females, suggesting a role of HPV infection in the development of lung cancer in non-smokers (OR 10.12, CI 95%: 3.88–26.38 for non-smoking females), and it was one of the first proofs of concept of this causative role [82]. In this study, HPV DNA of high-risk serotypes 16 and 18 was determined by nested PCR and *in situ* hybridization (ISH). Taking this under consideration, PCR is an ideal method for determining HPV-Host DNA integration. The presence of HPV infection can be assessed through different methods, including the use of lung tissue samples (Frozen, Fresh or Formalin fixed and paraffin embedded tissue) but also serological samples (using techniques such as Bead-based multiplex serology method or Multiplex liquid bead microarray antibody assay) [24]. However, one must consider that serologic analysis is usually limited by the low amount of HPV circulating in the bloodstream and also by the low sensitivity and specificity of serological detection techniques; therefore, lung tissue is usually better accepted. In a previous meta-analysis, Xiong et al. [24] evaluated the association between HPV and lung cancer in over 6000 lung cancer patients and 24,000 HPV-exposed individuals. The results showed an association between lung cancer and HPV (OR 3.64; 95% CI: 2.60–5.08), with most studies (75% [28/37]) using polymerase chain reaction (PCR) analysis in lung cancer tissue for HPV detection. The sensitivity and false-positive rate of PCR is higher than with other methods, including *in situ* hybridization, Southern blot, dot blot, and

In brief, DNA is extracted from lung cancer tissue and then different PCR methods can be used. One of the most common PCR methods used for HPV detection and genotyping is INNO-LiPA Genotyping Extra assay (Innogenetics N.V., Ghent, Belgium) [84]. This assay can detect 18 high-risk types using a reverse hybridization line probe (16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73, 82), 7 low-risk types (6, 11, 40, 43, 44, 54, 70), and some additional types (69, 71, 74). The assay also includes negative and positive controls (HPV6), as well as an internal control (HLA-DPB1 gene), to confirm DNA quality and the absence of PCR inhibitors. The results of trials conducted using each technique are summa-

In conclusion, the association between HPV infection and lung cancer should demonstrate the integration between HPV DNA and lung tumor cells DNA. The method usually performed for this assessment is PCR, and different techniques for genotyping have been used, mainly methodologies that include detection of high-

### *Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Current Perspectives in Human Papillomavirus*

and downregulating intercellular adhesion molecules and co-stimulating mediators. Additionally, IL10 has been shown to promote early E6 and E7 expression, consolidating a vicious cycle [66]. Other interleukins such as IL6 have been shown to be both a stimulator and an inhibitor of cell proliferation, depending on the cell line exposed. Through a complex process in which E6 causes STAT3 activation and IL6 expression, the cancer-associated fibroblast cells, present in the tumor microenvironment, suffer from IL6-induced and p16-mediated senescence. This phenomenon of adjacent cell dysfunction has been shown to promote neoplastic growth due to paracrine stimulation, chronic inflammation, and loss of oncolytic countermeasures [67]. Furthermore, IL6 has the ability to induce antiapoptotic Mcl-1 expression in HPVinfected lung cancer cells [68]. Mcl-1, member of the BCL-2 gene family is responsible, together with the Bcl-2 protein, for the apoptotic response of the mitochondria. Mcl-1 inhibits apoptosis by capturing BH3 and thus inhibiting Bax/Bcl-xl translocation, crucial steps in propagating an apoptotic signal in the cell [69]. Similarly, Bcl-2 offers a comparable Bax translocation inhibition, thus offering a strong antiapoptotic signal [70]. All in all, E6-activated and Il6-mediated BCL2 family modulation seems to be responsible for contributing to apoptosis inhibition and immortalization of HPV-infected cells. Other mechanisms of E6-induced immortalization include the expression of cIAP2, which is considered to be a very potent antiapoptotic factor in these cells by being the upstream inhibitor of caspase 3 activity. This hypothesis was validated in a study of induction of apoptosis in HeLa cells transfected with E6 and E7 proteins by knockdown of this molecule. E6 in turn causes the binding of p52 to NF-κB leading to an upregulation in the expression of cIAP [71, 72]. Additionally, this molecule lays as a downstream step in EGFR signaling, and its expression has also been strongly correlated with the presence of *EGFR* mutations [73]. One possible hypothesis to explain this phenomenon relates to the E6 inhibition of p53. Inactivation of this protein in turn leads to the loss of function in the MMR pathway, especially in MLH1 and MSH2, thus causing an increase in reactive oxygen species, which also strongly correlates with exon 19 *EGFR* mutations in lung cancer [74]. Finally, matrix metalloproteinases (MMP) seem to be also influenced by E6 interactions. These enzymes are responsible for degradation of the extracellular matrix, process required for cell migration and development of metastasis. MT1-MMP, MMP-2, and MMP-9 are the main members of this family that were upregulated by the expression of this oncogene [75], possibly by the induction of microRNAs [76]. E7, on the other hand, is responsible for binding and degrading pRb, p105, p107, and p130, especially in the upper epithelial layers. Additionally, E7 causes genome instability by deregulating the centrosome cycle [60]. Additionally, occurs the induction of the aryl hydrocarbon receptor signaling, a transcription factor involved in proliferation, differentiation, and apoptosis. Members of this family and inhibitors of cyclin-dependent kinases p16 and p21 have been proven to bind to E7 increasing pRb phosphorylation and promoting furthermore cell cycle deregulation [77]. Other activities can also be impaired by E7, such as epigenetic cell function. This molecule has the capacity to displace histone deacetylases, specially HDAC 1, 4, and 7 blocking their binding sites to the HIF-1α promoter regions and leading to an upregulation of its expression. This, in turn, is considered a key step in

angiogenesis and thus strongly contributes to tumor growth [78].

The relation between HPV and lung cancer was postulated since the decade of the 1970s. In 1975, Roglic et al., followed by Rubel and Reynolds in 1979, observed

**5. HPV gene expression and detection in lung tissue**

**52**

koilocytosis, a classical pattern in HPV infection, in sputum samples from benign bronchial lesions [79, 80]. Simultaneously, Syrjanen described that the epithelial changes seen in bronchial carcinoma closely resembled HPV-induced genital lesions [81]. Afterwards, several epidemiological data, particularly in non-smoking lung cancer patients, firmly established the relationship between HPV infection and development of lung cancer.

Although several studies showed the presence of oncogenic HPV in lung cancer tissue, demonstrating a causal role is necessary. Taking into account that integration of HPV DNA within the host is the critical point for oncogenic transformation, demonstration of the HPV DNA presence in lung cancer DNA cells is the main goal.

Cheng et al. demonstrated that HPV DNA was integrated into lung cancer DNA cells but not in the adjacent non-tumor cells and also demonstrated that HPV (+) lung cancer patients were predominantly non-smoking females, suggesting a role of HPV infection in the development of lung cancer in non-smokers (OR 10.12, CI 95%: 3.88–26.38 for non-smoking females), and it was one of the first proofs of concept of this causative role [82]. In this study, HPV DNA of high-risk serotypes 16 and 18 was determined by nested PCR and *in situ* hybridization (ISH). Taking this under consideration, PCR is an ideal method for determining HPV-Host DNA integration. The presence of HPV infection can be assessed through different methods, including the use of lung tissue samples (Frozen, Fresh or Formalin fixed and paraffin embedded tissue) but also serological samples (using techniques such as Bead-based multiplex serology method or Multiplex liquid bead microarray antibody assay) [24]. However, one must consider that serologic analysis is usually limited by the low amount of HPV circulating in the bloodstream and also by the low sensitivity and specificity of serological detection techniques; therefore, lung tissue is usually better accepted. In a previous meta-analysis, Xiong et al. [24] evaluated the association between HPV and lung cancer in over 6000 lung cancer patients and 24,000 HPV-exposed individuals. The results showed an association between lung cancer and HPV (OR 3.64; 95% CI: 2.60–5.08), with most studies (75% [28/37]) using polymerase chain reaction (PCR) analysis in lung cancer tissue for HPV detection. The sensitivity and false-positive rate of PCR is higher than with other methods, including *in situ* hybridization, Southern blot, dot blot, and sequencing [83, 84].

### **5.1 HPV detection and genotyping**

In brief, DNA is extracted from lung cancer tissue and then different PCR methods can be used. One of the most common PCR methods used for HPV detection and genotyping is INNO-LiPA Genotyping Extra assay (Innogenetics N.V., Ghent, Belgium) [84]. This assay can detect 18 high-risk types using a reverse hybridization line probe (16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73, 82), 7 low-risk types (6, 11, 40, 43, 44, 54, 70), and some additional types (69, 71, 74). The assay also includes negative and positive controls (HPV6), as well as an internal control (HLA-DPB1 gene), to confirm DNA quality and the absence of PCR inhibitors. The results of trials conducted using each technique are summarized in **Table 1**.

In conclusion, the association between HPV infection and lung cancer should demonstrate the integration between HPV DNA and lung tumor cells DNA. The method usually performed for this assessment is PCR, and different techniques for genotyping have been used, mainly methodologies that include detection of highrisk serotypes but also low risk serotypes.


**55**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

Yu, 2013 China PCR,

7 European countries

Anantharaman,

Sarchianaki, 2014

Anantharaman,

Colombara, 2015

Colombara, 2016

**Table 1.**

2014

2014

**Author, year Country Method HPV** 

Greece PCR,

Yu, 2015 China PCR L1, 16,

Gupta, 2016 India PCR 16, 18,

Robinson, 2016 USA Microarray,

Xiong, 2016 China PCR,

10 European countries

reverse blot hybridization, SB

genotyping

oncovirus panel, genotyping PCR

reverse blot hybridization

USA LBMA 6, 11, 16,

China LBMA 6, 11, 16,

BMSM 6, 11, 16,

Sagerup, 2014 Norway PCR 15 types Tissue 13/334 0/13

Fan, 2016 China ICC 16 Pe 42/95 1/55

Lu, 2016 China PCR 16, 18 Tissue 33/72 2/54

Simen, 2010 Finland ELISA 16, 18 Serum 67/311 220/930

BMSM 6, 11, 16,

18, 31

18, 31, 33, 52, 58

18, 31, 33, 52, 58

**types**

18, 31

18

31, 33, 45

**Sample type**

37 types Tissue 19/

FNAC, tissue

25 types Tissue 75/170 21/91

Blood 791/

Tissue 100/

28 types Tissue 15/57 1/10

21 types Tissue 7/83 6/83

Blood 604/

1449

Serum 4/200 15/200

Serum 8/183 8/2

**Cases (n/N)**

1634

100

180

5/73 0/75

**Controls (n/N)**

991/2729

0/16

8/110

601/1599

**6. HPV in lung cancer clinical information and perspective**

*Summary of reported trials conducted using each HPV detection and genotyping technique.*

There are at least three postulated ways for HPV virus to reach the tracheobronchial tract and cause epithelial transformation and malignancy: (1) cervical to lung transmission, (2) from an infected reproductive system to the mouth, throat, and finally lungs, and (3) airborne transmission. All of these have been supported by solid epidemiological data [56, 59, 85, 86]. Once HPV reaches the tracheobronchial epithelium, several molecular and cytological changes can occur as consequence of proteins E6 and E7 from HPV. These oncogene proteins can regulate expression of


### *Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Current Perspectives in Human Papillomavirus*

Béjui-Thivolet, 1990

**Author, year Country Method HPV** 

Fong, 1995 Australia PCR 6, 11, 16,

Yang, 1998 China PCR 6/11, 16,

Ciotti, 2006 Italy PCR,

Giuliani, 2007 Italy PCR,

Nadji, 2007 Iran PCR,

Wang, 2008 China PCR, ISH,

Joh, 2010 USA PCR,

Carpagnano, 2011

Jain, 2005 India PCR 16, 18 Tissue

sequencing

reverse blot hybridization, sequencing

sequencing

IHC

sequencing

sequencing, INFINITI HPV-QUAD assay

Krikelis, 2010 Greece PCR 16 Tissue,

Italy PCR,

Gatta, 2012 Italy PCR 16, 18,

France ISH 6, 11, 16,

**types**

18

18, 31, 33, 52b, 58

31/33

16, 18, 31

Li, 1995 China PCR, DB 16, 18 Tissue 16/50 0/22

Niyaz, 2000 China PCR 16, 18 Tissue 44/110 1/40 Cheng, 2001 China PCR, ISH 16, 18 Tissue 77/141 16/60 Chiou, 2003 China PCR 16, 18 Blood 71/149 22/174 Cheng, 2004 China PCR, ISH 6, 11 Tissue 40/141 1/60

Fei, 2006 China ISH 16, 18 Tissue 23/73 2/34

Buyru, 2008 Turkey PCR, SB 16, 18 Blood 1/65 0/87

Yu, 2009 China PCR 25 types Tissue 43/109 16/71 Xu, 2009 China ISH 16/18 Tissue 32/44 0/15

Wang, 2010 China PCR 16, 18 Tissue 18/45 0/16

Galvan, 2012 Italy, UK PCR,DB 35 types Tissue 0/100 0/100

16, 18, 30, 31, 33, 45, 35/68, 39/56, 58/52, 59/51, 6/11

33, 35, 52, 58

**Sample type**

(case) Blood (control) **Cases (n/N)**

Tissue 6/33 0/10

Tissue 2/104 0/104

Tissue 13/50 3/30

Tissue 8/38 0/38

— Tissue 10/78 0/78

— Tissue 33/129 8/89

16, 18 Tissue 138/313 4/96

— Tissue 5/30 0/21

BW

Tissue, BW, EBC 2/40 0/40

36/58 11/16

12/89 0/68

Tissue 2/50 1/23

**Controls (n/N)**

**54**

**Table 1.**

*Summary of reported trials conducted using each HPV detection and genotyping technique.*

### **6. HPV in lung cancer clinical information and perspective**

There are at least three postulated ways for HPV virus to reach the tracheobronchial tract and cause epithelial transformation and malignancy: (1) cervical to lung transmission, (2) from an infected reproductive system to the mouth, throat, and finally lungs, and (3) airborne transmission. All of these have been supported by solid epidemiological data [56, 59, 85, 86]. Once HPV reaches the tracheobronchial epithelium, several molecular and cytological changes can occur as consequence of proteins E6 and E7 from HPV. These oncogene proteins can regulate expression of

several target genes and proteins, which derives in promoted lung cell proliferation, angiogenesis, and cell immortalization. Among the genes and proteins affected are p53, pRb, HIF-1α, VEGF, IL-6, IL-10, Mcl-1, Bcl-2, cIAP-2, EGFR, FHIT, hTERT, HER-2, ALK, ROS1, and AhR [55, 87–90]. Evidence which supports the association between HPV infection and lung cancer continues to grow, but debate will likely continue due to heterogeneous methodologies for HPV detection in lung tissue. At least eight systematic reviews and meta-analysis have consistently found that HPV infection is a risk factor for lung cancer [22–24, 84, 91–94]. One of them included longitudinal studies: a nested case-control and a cohort study with high causality [24]. According to subgroup analyses from several trials, the HPV infection constitutes a risk for lung cancer, especially in non-smokers, similar to the findings in head and neck cancers, females and Asian race [22, 24, 82]. Additionally considering HPV affinity to squamous cells, HPV infection constitutes a risk for squamous cell lung cancer; however, other histologic subtypes including ADC and SCLC have also been related to HPV infection [23, 24, 94].

HPV serotypes 16 and 18, known as high-risk serotypes, are mainly associated with lung cancer risk, though low-risk serotypes are believed to cause benign, non-malignant, lesions [23, 24, 95]. However, this relationship has not been fully studied, and other serotypes including HPV 11 and HPV 31 have a less clear role in terms of lung cancer association. Currently available vaccines against HPV could theoretically prevent lung cancer development; however, this important issue has seldom been explored and more research is needed to draw robust conclusions. HPV status modifies treatment modalities and prognosis in head and neck cancers. Further research is necessary to determine whether lung cancer treatment should change according to HPV infection status. HPV coinfection in lung cancer favors the inclusion of E5 oncoprotein, which alters the mitogenic signaling downstream of Ras, EGFR, and PKC, as well as the constitutive activation of AP-1, which through c-jun may result in cell survival [96]. In the same way, E6 HPV protein blocks p53 activation causing an inhibition of p21 action, upregulating the expression of EGFR and inhibiting apoptosis by activating cIAP28. Additionally, the inclusion of the E7 protein leads to the downregulation of p16INK4 by hypermethylation and migration of tumor-infiltrating lymphocytes (TILs) [97]. Recently, Cheng et al. have found that HPV infection increases tumor activity via hypermethylation of the XRCC3 and XRCC5, an event that generates induced DNA [98]. In parallel, Zhang et al. proposed that inflammation related to HPV lung cancer is induced by increasing levels of HIF, VEGF and [90]. We previously reported a high HPV positivity rate in Hispanic patients suffering lung ADC; in addition, we described that presence of viral DNA leads to a better prognosis in *EGFR* and *KRAS*—mutated lung ADC and increases the expression of PDL1 [99]. Based on this information, HPV infection could modify host immune response and subsequently predict response to immunotherapy, which is currently a treatment modality in certain subgroups of lung cancer patients. Similarly, HPV infection appears to be associated with lung cancer in non-smokers, as are *EGFR* mutations; therefore, it is possible that a synergistic approach could be reached when treating the infection in *EGFR*-mutated lung cancer patients who receive targeted agents. In this regard, a previous study by Li et al. demonstrated that the presence of HPV DNA was significantly associated with *EGFR* mutations in advanced lung ADC. Interestingly, patients with both HPV infections and *EGFR* mutations have a reduced risk of progression compared to those without HPV infection or *EGFR* mutation (adjusted HR = 0.640; 95% confidence interval: 0.488–0.840; P = 0.001), suggesting a prognostic role for HPV status in this patient subgroup [73]. Another likely suitable target for therapy is MEK, a mitogenic signaling pathway protein

**57**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

activated as a result of *KRAS* mutations in HPV, and some anti-MEK therapies have been tested in lung cancer [100]. Pathophysiology of infection and main molecular

If lung cancer patients with HPV infection need to de-escalate treatment, as in head and neck cancer patients, they requires further investigation. Some arguments are in favor of de-escalation considering, for example, the fact that lung cancer patients with HPV infection seem to have a better prognosis. Wang et al. described ADC with HPV 16/18 infections as having significantly higher survival rates compared to those that are HPV16/18 negative [101]. In a similar way, Hsu et al. reported survival benefits for stage I NSCLC patients who expressed the HPV16/18

In conclusion, HPV infection constitutes a risk factor for lung cancer develop-

Future work in this field will likely include the validation of a screening test for HPV infection in lung cancer patients and also a strategy to follow HPV-infected individuals who might be at a higher risk of developing lung cancer. Additionally, the potential efficacy of anti-HPV vaccination for reducing the incidence of lung

ment, especially in patients infected with high-risk serotypes 16 and 18, nonsmokers and females. HPV vaccination could have a potential role in the prevention of development of HPV-associated lung cancer. Furthermore, HPV status could modify some lung cancer treatment decisions; however, more information is

pathways compromised are presented in **Figure 3**.

*Pathophysiology of HPV infection and development of lung cancer.*

E6 oncoprotein [102].

needed to draw definitive conclusions.

cancer must be adequately explored.

**8. Future perspectives**

**7. Conclusion**

**Figure 3.**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

*Current Perspectives in Human Papillomavirus*

also been related to HPV infection [23, 24, 94].

several target genes and proteins, which derives in promoted lung cell proliferation, angiogenesis, and cell immortalization. Among the genes and proteins affected are p53, pRb, HIF-1α, VEGF, IL-6, IL-10, Mcl-1, Bcl-2, cIAP-2, EGFR, FHIT, hTERT, HER-2, ALK, ROS1, and AhR [55, 87–90]. Evidence which supports the association between HPV infection and lung cancer continues to grow, but debate will likely continue due to heterogeneous methodologies for HPV detection in lung tissue. At least eight systematic reviews and meta-analysis have consistently found that HPV infection is a risk factor for lung cancer [22–24, 84, 91–94]. One of them included longitudinal studies: a nested case-control and a cohort study with high causality [24]. According to subgroup analyses from several trials, the HPV infection constitutes a risk for lung cancer, especially in non-smokers, similar to the findings in head and neck cancers, females and Asian race [22, 24, 82]. Additionally considering HPV affinity to squamous cells, HPV infection constitutes a risk for squamous cell lung cancer; however, other histologic subtypes including ADC and SCLC have

HPV serotypes 16 and 18, known as high-risk serotypes, are mainly associated with lung cancer risk, though low-risk serotypes are believed to cause benign, non-malignant, lesions [23, 24, 95]. However, this relationship has not been fully studied, and other serotypes including HPV 11 and HPV 31 have a less clear role in terms of lung cancer association. Currently available vaccines against HPV could theoretically prevent lung cancer development; however, this important issue has seldom been explored and more research is needed to draw robust conclusions. HPV status modifies treatment modalities and prognosis in head and neck cancers. Further research is necessary to determine whether lung cancer treatment should change according to HPV infection status. HPV coinfection in lung cancer favors the inclusion of E5 oncoprotein, which alters the mitogenic signaling downstream of Ras, EGFR, and PKC, as well as the constitutive activation of AP-1, which through c-jun may result in cell survival [96]. In the same way, E6 HPV protein blocks p53 activation causing an inhibition of p21 action, upregulating the expression of EGFR and inhibiting apoptosis by activating cIAP28. Additionally, the inclusion of the E7 protein leads to the downregulation of p16INK4 by hypermethylation and migration of tumor-infiltrating lymphocytes (TILs) [97]. Recently, Cheng et al. have found that HPV infection increases tumor activity via hypermethylation of the XRCC3 and XRCC5, an event that generates induced DNA [98]. In parallel, Zhang et al. proposed that inflammation related to HPV lung cancer is induced by increasing levels of HIF, VEGF and [90]. We previously reported a high HPV positivity rate in Hispanic patients suffering lung ADC; in addition, we described that presence of viral DNA leads to a better prognosis in *EGFR* and *KRAS*—mutated lung ADC and increases the expression of PDL1 [99]. Based on this information, HPV infection could modify host immune response and subsequently predict response to immunotherapy, which is currently a treatment modality in certain subgroups of lung cancer patients. Similarly, HPV infection appears to be associated with lung cancer in non-smokers, as are *EGFR* mutations; therefore, it is possible that a synergistic approach could be reached when treating the infection in *EGFR*-mutated lung cancer patients who receive targeted agents. In this regard, a previous study by Li et al. demonstrated that the presence of HPV DNA was significantly associated with *EGFR* mutations in advanced lung ADC. Interestingly, patients with both HPV infections and *EGFR* mutations have a reduced risk of progression compared to those without HPV infection or *EGFR* mutation (adjusted HR = 0.640; 95% confidence interval: 0.488–0.840; P = 0.001), suggesting a prognostic role for HPV status in this patient subgroup [73]. Another likely suitable target for therapy is MEK, a mitogenic signaling pathway protein

**56**

**Figure 3.** *Pathophysiology of HPV infection and development of lung cancer.*

activated as a result of *KRAS* mutations in HPV, and some anti-MEK therapies have been tested in lung cancer [100]. Pathophysiology of infection and main molecular pathways compromised are presented in **Figure 3**.

If lung cancer patients with HPV infection need to de-escalate treatment, as in head and neck cancer patients, they requires further investigation. Some arguments are in favor of de-escalation considering, for example, the fact that lung cancer patients with HPV infection seem to have a better prognosis. Wang et al. described ADC with HPV 16/18 infections as having significantly higher survival rates compared to those that are HPV16/18 negative [101]. In a similar way, Hsu et al. reported survival benefits for stage I NSCLC patients who expressed the HPV16/18 E6 oncoprotein [102].

### **7. Conclusion**

In conclusion, HPV infection constitutes a risk factor for lung cancer development, especially in patients infected with high-risk serotypes 16 and 18, nonsmokers and females. HPV vaccination could have a potential role in the prevention of development of HPV-associated lung cancer. Furthermore, HPV status could modify some lung cancer treatment decisions; however, more information is needed to draw definitive conclusions.

### **8. Future perspectives**

Future work in this field will likely include the validation of a screening test for HPV infection in lung cancer patients and also a strategy to follow HPV-infected individuals who might be at a higher risk of developing lung cancer. Additionally, the potential efficacy of anti-HPV vaccination for reducing the incidence of lung cancer must be adequately explored.

### **Author details**

Andrés F. Cardona1,2,3\*, Alejandro Ruiz-Patiño1 , Luisa Ricaurte1 , Leonardo Rojas1,4, Zyanya Lucia Zatarain-Barrón5 , Oscar Arrieta<sup>5</sup> and Rafael Rosell6

1 Clinical and Translational Oncology Group, Thoracic Oncology Unit, Institute of Oncology, Clínica del Country, Bogotá, Colombia

2 Foundation for Clinical and Applied Cancer Research—FICMAC, Bogotá, Colombia

3 Molecular Oncology and Biology Systems Research Group (Fox-G), Bogotá, Colombia

4 Clinical Oncology Department, Clínica Colsanitas, Bogotá, Colombia

5 Thoracic Oncology Unit and Laboratory of Personalized Medicine, Instituto Nacional de Cancerología (INCan), México City, Mexico

6 Cancer Biology and Precision Medicine Program, Catalan Institute of Oncology, Barcelona, Spain

\*Address all correspondence to: a\_cardonaz@yahoo.com

© 2018 The Author(s). Licensee IntechOpen. 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.

**59**

*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

> Journal of the American Society of Clinical Oncology. 2007;**25**:561-570. DOI: 10.1200/JCO.2006.06.8015

[9] Wakelee HA, Chang ET, Gomez SL, Keegan TH, Feskanich D, Clarke CA, et al. Lung cancer incidence in never smokers. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 2007;**25**:472-478.

[10] Yano T, Miura N, Takenaka T, Haro A, Okazaki H, Ohba T, et al. Neversmoking nonsmall cell lung cancer as a separate entity: Clinicopathologic features and survival. Cancer. 2008;**113**:1012-1018. DOI: 10.1002/

DOI: 10.1200/JCO.2006.07.2983

[11] Gupta P, Haldar D, Naru J, Dey P, Aggarwal AN, Minz RW, et al. Prevalence of human

papillomavirus, Epstein-Barr virus, and cytomegalovirus in fine needle aspirates from lung carcinoma: A casecontrol study with review of literature. Diagnostic Cytopathology. 2016;**44**: 987-993. DOI: 10.1002/dc.23613

[12] Maehara Y. Primary lung cancer in never smokers. International Journal of Clinical Oncology. 2011;**16**:285-286. DOI: 10.1007/s10147-010-0159-1

[13] Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. Lyon: International Agency for Research on

[14] Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers—A different disease. Nature Reviews. Cancer. 2007;**7**:778-790. DOI: 10.1038/nrc2190

[15] Okazaki I, Ishikawa S, Sohara Y. Genes associated with succeptibility to lung adenocarcinoma among never

cncr.23679

Cancer; 2015

[1] World Health Organization, International Agency for Research on Cancer. GLOBOCAN 2012. Cancer fact sheets [WWW Document]. Population Fact Sheets. 2016. Available from: http:// globocan.iarc.fr/Pages/fact\_sheets\_ cancer.aspx [Accessed: 06-02-2018]

[2] Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the global burden of disease study 2010. Lancet (London, England). 2012;**380**:2095-2128. DOI: 10.1016/

[3] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA: A Cancer Journal for Clinicians. 2002;**55**:74-108. DOI: 10.3322/

[4] Tyczynski JE, Bray F, Parkin DM. Lung cancer in Europe in 2000: Epidemiology, prevention, and early detection. The Lancet Oncology.

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[6] Scagliotti GV, Longo M, Novello S. Nonsmall cell lung cancer in never smokers. Current Opinion in Oncology.

2009;**21**:99-104. DOI: 10.1097/ CCO.0b013e328321049e

[7] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA: A Cancer Journal for Clinicians. 2017;**67**:7-30.

[8] Subramanian J, Govindan R. Lung cancer in never smokers: A review. Journal of Clinical Oncology: Official

DOI: 10.3322/caac.21387

S0140-6736(12)61728-0

canjclin.55.2.74

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*Human Papillomavirus Infection and Lung Cancer DOI: http://dx.doi.org/10.5772/intechopen.80706*

### **References**

*Current Perspectives in Human Papillomavirus*

**58**

**Author details**

Colombia

Colombia

Barcelona, Spain

Andrés F. Cardona1,2,3\*, Alejandro Ruiz-Patiño1

Oncology, Clínica del Country, Bogotá, Colombia

Zyanya Lucia Zatarain-Barrón5

provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. 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,

, Oscar Arrieta<sup>5</sup>

2 Foundation for Clinical and Applied Cancer Research—FICMAC, Bogotá,

3 Molecular Oncology and Biology Systems Research Group (Fox-G), Bogotá,

5 Thoracic Oncology Unit and Laboratory of Personalized Medicine, Instituto

6 Cancer Biology and Precision Medicine Program, Catalan Institute of Oncology,

4 Clinical Oncology Department, Clínica Colsanitas, Bogotá, Colombia

Nacional de Cancerología (INCan), México City, Mexico

\*Address all correspondence to: a\_cardonaz@yahoo.com

1 Clinical and Translational Oncology Group, Thoracic Oncology Unit, Institute of

, Luisa Ricaurte1

and Rafael Rosell6

, Leonardo Rojas1,4,

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Section 3

Molecular Pathogenesis of

Human Papillomavirus

### Section 3
