**2. HPV epidemiology: The changes coming**

Although the risk of cervical cancer was long known to be associated with risky sexual behavior in both the woman and her partner, it was not until around 3 decades ago that HPV was identified the likely culprit by Dr. Zur Hausen, whose work in cervical cancer won him the Nobel Prize. The development of superior molecular techniques advanced the field rapidly cementing the association between HPV and cervical cancer. This finding quickly led scientists to examine HPV's association with other anogenital cancers in women including vulvar, vaginal and anal cancers and in men, penile and anal cancers [2]. Most recently, oropharyngeal cancers have joined the list of HPV-associated cancers. Although HPV was found to be associated with these other anogenital cancers, most of the work continues to focus on cervical cancers since it remains 20 times more common than any of the other HPV-associated cancers. This phenomenon is likely due to the vulnerability of the cervix to HPV. Although the mechanisms are not well understood, the transformation zone (TZ) is likely one of the most vulnerable epithelia to HPV's carcinogenic powers [3]. The TZ change of columnar epithelium to stratified epithelium Mullerian urogenital area in the ectocervix. In the puberty columnar epithelium differentiates into stratified squamous epithelium. Metaplastic tissue is more vulnerable to HPV because its thickness to the basal layer is smaller and the virus requires cells in differentiation state for their survival, which is accelerated in metaplastic tissues [4]. It is likely that this metaplastic tissue supports viral persistence which is essential for cancer development. Interestingly metaplastic tissue is also found in the anal and tonsillar tissue.

According to a recent meta-analysis that included data from more than 1 million women in 59 countries, the prevalence of genital HPV infection among those with normal cytology ranges from 1.6% to 41.9% [5]. The estimated average global prevalence of HPV in this particular study was 11.7%. This study, along with others [6], reported an interesting trend in the female agespecific distribution of HPV whereby there is a first peak at younger ages (< 25 years) in all regions; and in the Americas, Africa and Europe, a clear second peak among individuals 45 years or older. The first peak, which comes shortly after sexual debut for most women, is generally attributed to higher levels of sexual activity with multiple partners and low viral immunity. After the first peak, a consistent age-related decline in HPV prevalence has been documented in numerous epidemiological studies. Although the reason for the smaller second peak at middle age still remains unclear, possible explanations include immunosenescence, hormonal changes prior to menopause, changes in male/female sexual behavior, cohort effects, or perhaps higher rates of HPV persistence at older ages [7]. However, new sexual exposures and latent reactivation are often invoked as explanation for the second peak of HPV prevalence observed in older women, particularly in Latin America [6]. Studies in humans are unable to directly demonstrate establishment of latency and induction of reactivation from the latent state. However, animal models of papillomavirus infection have provided sufficient experi‐ mental evidence for papillomavirus latency [8]. HPV infects basal epithelial cells at sites of microtrauma, likely a normal consequence of sexual intercourse. Infected basal cells induced to differentiate to fill the wound will result in active papillomavirus infection. A few infected basal stem cells will retain HPV, but do not differentiate, and these infected cells are unlikely to be sampled using standard exfoliative techniques employed in most epidemiologic studies, which sample only the surface epithelium. Thus, HPV in a basal stem cell may remain undetectable until triggered to differentiate by undetermined stimuli such as wound repair and hormonal regulation [9]. With high cellular turnover in the cervical epithelium, one may expect relatively constant detection of HPV in this model. However, it is likely that a reacti‐ vated infection subsequent to latently infected stem cell differentiation is immediately recognized and brought under control by a functional memory immune response resulting in very short duration of active infection. This model would be consistent with the increase in new HPV DNA detection immediately following memory T cell depletion in acute HIV and in sexually abstinent women with chronic HIV infection [10, 11]. The relatively lower rates of recurrent HPV DNA detection in healthy populations suggests that the normal immune response is able to retain recurrence, but that background reactivation is common and likely associated with short duration of detectable viral DNA [12].

The availability of sensitive diagnostic assays for HPV, reproducible and cost effectively will be critical in the monitoring of current vaccines and the design and implementation of vaccination programs. The HPV vaccine offers a unique opportunity to fight against cancer

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

Although the risk of cervical cancer was long known to be associated with risky sexual behavior in both the woman and her partner, it was not until around 3 decades ago that HPV was identified the likely culprit by Dr. Zur Hausen, whose work in cervical cancer won him the Nobel Prize. The development of superior molecular techniques advanced the field rapidly cementing the association between HPV and cervical cancer. This finding quickly led scientists to examine HPV's association with other anogenital cancers in women including vulvar, vaginal and anal cancers and in men, penile and anal cancers [2]. Most recently, oropharyngeal cancers have joined the list of HPV-associated cancers. Although HPV was found to be associated with these other anogenital cancers, most of the work continues to focus on cervical cancers since it remains 20 times more common than any of the other HPV-associated cancers. This phenomenon is likely due to the vulnerability of the cervix to HPV. Although the mechanisms are not well understood, the transformation zone (TZ) is likely one of the most vulnerable epithelia to HPV's carcinogenic powers [3]. The TZ change of columnar epithelium to stratified epithelium Mullerian urogenital area in the ectocervix. In the puberty columnar epithelium differentiates into stratified squamous epithelium. Metaplastic tissue is more vulnerable to HPV because its thickness to the basal layer is smaller and the virus requires cells in differentiation state for their survival, which is accelerated in metaplastic tissues [4]. It is likely that this metaplastic tissue supports viral persistence which is essential for cancer development. Interestingly metaplastic tissue is also found in the anal and tonsillar tissue. According to a recent meta-analysis that included data from more than 1 million women in 59 countries, the prevalence of genital HPV infection among those with normal cytology ranges from 1.6% to 41.9% [5]. The estimated average global prevalence of HPV in this particular study was 11.7%. This study, along with others [6], reported an interesting trend in the female agespecific distribution of HPV whereby there is a first peak at younger ages (< 25 years) in all regions; and in the Americas, Africa and Europe, a clear second peak among individuals 45 years or older. The first peak, which comes shortly after sexual debut for most women, is generally attributed to higher levels of sexual activity with multiple partners and low viral immunity. After the first peak, a consistent age-related decline in HPV prevalence has been documented in numerous epidemiological studies. Although the reason for the smaller second peak at middle age still remains unclear, possible explanations include immunosenescence, hormonal changes prior to menopause, changes in male/female sexual behavior, cohort effects, or perhaps higher rates of HPV persistence at older ages [7]. However, new sexual exposures and latent reactivation are often invoked as explanation for the second peak of HPV prevalence observed in older women, particularly in Latin America [6]. Studies in humans are unable to directly demonstrate establishment of latency and induction of reactivation from the latent

and diseases associated with HPV infection.

**2. HPV epidemiology: The changes coming**

Among sexually active males, genital HPV infection is also very common; however, prevalence varies widely depending on geographic region, risk group, anatomical site, sampling method, and HPV testing methodology [13, 14]. In a recent systematic review, Smith et al [15] estimated HPV prevalence to be between 1% and 84% in low-risk sexually active men, and between 2% and 93% in high risk men. HPV infection declines substantially after about 30 years of age, The prevalence of HPV infection in males generally remains constant or declines only slightly with age after peak prevalence. One possible explanation for this is that men experience a higher rate of reinfection compared to women. Anal cancer is very rare in the general population, but much more common in well defined, high-risk populations, including women with a previous cervical precancer, men having sex with men (MSM), and immune-compromised individuals. Infection with carcinogenic human papillomavirus (HPV) has been increasingly recognised to cause anal cancer and Machalek et al [16] recorded a prevalence of high-risk anal HPV in HIVpositive MSM of 73.5%.

In 1995, the International Agency for Research on Cancer (IARC) first classified HPV types 16 and 18 as carcinogenic to humans, but based on more recent evidence, the list of carcinogenic HPV types has been expanded to include a total of 13 mucosotropic anogenital HPV types as being definite or probable carcinogens (grade 1 or 2a) based on their frequent association with invasive cervical cancer (ICC) and cervical intraepithelial neoplasia (CIN) [17]. The oncogenic types (mostly HPV 16) are also causally implicated in other cancers, including penile, anal, vulvar and vaginal cancers [18, 19]. The remaining genital types (e.g., HPV types 6, 11, 42, 43, 44 and some rarer types) are considered to be of low or no oncogenic risk [20]. However, these types may cause subclinical and clinically visible benign lesions known as flat and acuminate condylomata, respectively.

In descending order, the most common HPV types implicated in cervical cancer globally are: 16, 18, 58, 33, 45, 31, 52, 35, 59, 39, 51 and 56 [21]. HPV types 16 and 18 are the most dominant types implicated in cervical cancer in all continents, being responsible for ~ 70% of ICC cases globally. In many studies, estimating the fraction of cervical cancer cases attributable to the different HPV types is difficult due to the high prevalence of multiple type infections. For example, a recent meta-analysis estimated the prevalence of co-infection (≥ 2 HPV types) in tumor specimens at 15.7% [21]. Other recent meta-analyses and cross sectional studies evaluating the worldwide distribution of HPV infections consistently reveal the same HPV prevalence patterns [5, 22]. This widespread circulation of HR-HPV types strengthens the potential for a phenomenon known as type-replacement, i.e., an increase in other non-vaccine genotypes following HPV vaccination. However, based on evidence that HPVs evolve very slowly and that HPV types do not normally compete with one another during natural infection [23-25], it is still unlikely that some other HPV type(s) will evolve to fill the niche currently occupied by vaccine target types. Furthermore, phase III trials evaluating both bivalent and quadrivalent vaccines indicate partial cross-type protection (cross-immunity) against many phylogenetically related HPV types [26-28], suggesting that the benefit from vaccination may be even greater than expected.

undetected. These observations and the fact that antibodies to HPV are less common in men than in women suggest that most infections in men are relatively superficial and clearance is likely directed by local innate immune responses, but not by memory immune responses [36]. Since men do not appear to develop adequate HPV specific immune responses, they are not protected from re-infection. This is another argument for HPV vaccination in men. It may be that sexually active males would benefit from vaccination. Benefits would include prevention of penile, anal and oropharyngeal cancers and prevention of infections would, in theory, also

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 61

After breast and colorectal cancer, cervical cancer is the 3rd leading cancer site worldwide irrespective of gender and second among women. In 2008, there were an estimated 530,000 cases and 270,000 deaths attributed to cervical cancer, with 86% of cases and 88% of deaths occurring in developing countries [38]. In these developing countries, the age-standardized incidence rate (ASIR) and age-standardized mortality rate (ASMR) were 18 and 10 per 100,000 women, respectively; whereas in more developed countries, the ASIR and ASMR were 9 and 3 per 100,000 women, respectively. Globally, incidence of ICC ranges from < 3 to > 50 cases per 100,000 women for low- and high-burden countries, respectively. These differences between countries are believed to reflect protection from screening, and variance in exposure to HPV and other cofactors like smoking and oral contraceptive use, and other sexually transmitted

The global burden of other HPV related cancers is also substantial. Worldwide, approximately 97,215 cases of non-cervical cancers for which HPV infection may be an etiologic factor are diagnosed annually; roughly 50,780 in men (520 penile, 26,775 oropharyngeal and 13,485 anal cancers) and 46,435 in women (25,600 vaginal/vulvar, 6048 oropharyngeal and 14,787 anal cancers) [39]. However, it is important to recognize that not all of these cases are attributable to HPV and that these estimates represent the upper limit for the annual burden of cancers caused by HPV. only a quarter of oropharyngeal cancers are attributable to HPV; meanwhile approximately 90% of anal cancers, and 40% of penile, vaginal or vulvar cancers are attribut‐ able to the virus. Although there is some evidence implicating HPV with several other cancers (e.g., lung, colon, ovary, breast, prostate, urinary bladder, esophageal and nasal/sinonasal cancers), current molecular and epidemiological data are sparse and do not yet support a causal role for HPV in the etiology of these cancers [30, 40-42]. The role of HPV in the causation

Globally, HPV accounts for roughly 5.2% of the total cancer burden — the highest among all infectious agents. However, as may be expected, the distribution varies considerably according to country development status, where HPV accounts for approximately 7.7% and 2.2% of all

HPV cannot be cultured in conventional cell cultures. Other classical direct virological diagnostic techniques, such as immunohistochemistry, lack the sensitivity as well as specificity

benefit women [37].

infections such as human immunodeficiency virus [38].

of esophageal squamous cell carcinoma is unclear.

cancer cases in developing and developed countries, respectively [42].

**3. Screening methods for detect HPV infection**

Infection of the oral cavity with high risk HPV is also now recognized as an important cause of oral and oropharyngeal cancers [29]. However, unlike cervical cancer in which 100% of cases are attributable to infection with HPV, only 25–35% of these cancers are attributable to HPV [2, 30]; the major risk factors being alcohol and tobacco use. Among cases of oral/oropharyngeal cancer linked to HPV infection, HPV 16 is by far the most common type detected in tumor specimens [31, 32]. Based on evaluation of risk factor profiles for cancers of the head and neck, comparing HPV 16-positive and HPV 16-negative cases, some researchers have decided that these should actually be considered distinct cancers [33]. In their study, sexual behavior (but not alcohol or tobacco use) was an important predictor of head and neck cancers among HPV 16-positive subjects, meanwhile the opposite was observed for HPV 16-negative subjects.

HPV 16 is the most common in all the HPV associated anogenital and oropharyngeal cancers therefore the HPV vaccines currently on the market are likely to make a significant impact on all these cancers in women and in men. In contrast, the role of HPV in penile cancers is less understood. Trauma and inflammation appear to be important risks suggesting that tissue damage and/or repair processes play a role in cancer development [34]. The natural history of HPV in the penis is also not well described. Although there is a histologic equivalent of the precancerous CIN 3 found in men, referred to as penile intraepithelial neoplasia (PIN), nothing is known about the natural history of these lesions and whether they have the same potential as CIN 3 (~ 12%) to progress to invasive cancer. One intriguing observation is that the penile cancer has a much lower prevalence than cervical cancer and yet the prevalence of HPV is much higher in the genitals of men than what is observed in women. Across all ages, HPV is found 2–4 times more common in the genital area of men than seen in women and the decline observed with age in women is not seen in men with similar prevalence across all ages. In contrast, the natural history of HPV in anogenital area in men behaves similar to HPV in women [35] in that HPV appears to be rapidly cleared so by 6–8 months, most infections are undetected. These observations and the fact that antibodies to HPV are less common in men than in women suggest that most infections in men are relatively superficial and clearance is likely directed by local innate immune responses, but not by memory immune responses [36]. Since men do not appear to develop adequate HPV specific immune responses, they are not protected from re-infection. This is another argument for HPV vaccination in men. It may be that sexually active males would benefit from vaccination. Benefits would include prevention of penile, anal and oropharyngeal cancers and prevention of infections would, in theory, also benefit women [37].

In descending order, the most common HPV types implicated in cervical cancer globally are: 16, 18, 58, 33, 45, 31, 52, 35, 59, 39, 51 and 56 [21]. HPV types 16 and 18 are the most dominant types implicated in cervical cancer in all continents, being responsible for ~ 70% of ICC cases globally. In many studies, estimating the fraction of cervical cancer cases attributable to the different HPV types is difficult due to the high prevalence of multiple type infections. For example, a recent meta-analysis estimated the prevalence of co-infection (≥ 2 HPV types) in tumor specimens at 15.7% [21]. Other recent meta-analyses and cross sectional studies evaluating the worldwide distribution of HPV infections consistently reveal the same HPV prevalence patterns [5, 22]. This widespread circulation of HR-HPV types strengthens the potential for a phenomenon known as type-replacement, i.e., an increase in other non-vaccine genotypes following HPV vaccination. However, based on evidence that HPVs evolve very slowly and that HPV types do not normally compete with one another during natural infection [23-25], it is still unlikely that some other HPV type(s) will evolve to fill the niche currently occupied by vaccine target types. Furthermore, phase III trials evaluating both bivalent and quadrivalent vaccines indicate partial cross-type protection (cross-immunity) against many phylogenetically related HPV types [26-28], suggesting that the benefit from vaccination may

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

Infection of the oral cavity with high risk HPV is also now recognized as an important cause of oral and oropharyngeal cancers [29]. However, unlike cervical cancer in which 100% of cases are attributable to infection with HPV, only 25–35% of these cancers are attributable to HPV [2, 30]; the major risk factors being alcohol and tobacco use. Among cases of oral/oropharyngeal cancer linked to HPV infection, HPV 16 is by far the most common type detected in tumor specimens [31, 32]. Based on evaluation of risk factor profiles for cancers of the head and neck, comparing HPV 16-positive and HPV 16-negative cases, some researchers have decided that these should actually be considered distinct cancers [33]. In their study, sexual behavior (but not alcohol or tobacco use) was an important predictor of head and neck cancers among HPV 16-positive subjects, meanwhile the opposite was observed for HPV 16-negative subjects.

HPV 16 is the most common in all the HPV associated anogenital and oropharyngeal cancers therefore the HPV vaccines currently on the market are likely to make a significant impact on all these cancers in women and in men. In contrast, the role of HPV in penile cancers is less understood. Trauma and inflammation appear to be important risks suggesting that tissue damage and/or repair processes play a role in cancer development [34]. The natural history of HPV in the penis is also not well described. Although there is a histologic equivalent of the precancerous CIN 3 found in men, referred to as penile intraepithelial neoplasia (PIN), nothing is known about the natural history of these lesions and whether they have the same potential as CIN 3 (~ 12%) to progress to invasive cancer. One intriguing observation is that the penile cancer has a much lower prevalence than cervical cancer and yet the prevalence of HPV is much higher in the genitals of men than what is observed in women. Across all ages, HPV is found 2–4 times more common in the genital area of men than seen in women and the decline observed with age in women is not seen in men with similar prevalence across all ages. In contrast, the natural history of HPV in anogenital area in men behaves similar to HPV in women [35] in that HPV appears to be rapidly cleared so by 6–8 months, most infections are

be even greater than expected.

After breast and colorectal cancer, cervical cancer is the 3rd leading cancer site worldwide irrespective of gender and second among women. In 2008, there were an estimated 530,000 cases and 270,000 deaths attributed to cervical cancer, with 86% of cases and 88% of deaths occurring in developing countries [38]. In these developing countries, the age-standardized incidence rate (ASIR) and age-standardized mortality rate (ASMR) were 18 and 10 per 100,000 women, respectively; whereas in more developed countries, the ASIR and ASMR were 9 and 3 per 100,000 women, respectively. Globally, incidence of ICC ranges from < 3 to > 50 cases per 100,000 women for low- and high-burden countries, respectively. These differences between countries are believed to reflect protection from screening, and variance in exposure to HPV and other cofactors like smoking and oral contraceptive use, and other sexually transmitted infections such as human immunodeficiency virus [38].

The global burden of other HPV related cancers is also substantial. Worldwide, approximately 97,215 cases of non-cervical cancers for which HPV infection may be an etiologic factor are diagnosed annually; roughly 50,780 in men (520 penile, 26,775 oropharyngeal and 13,485 anal cancers) and 46,435 in women (25,600 vaginal/vulvar, 6048 oropharyngeal and 14,787 anal cancers) [39]. However, it is important to recognize that not all of these cases are attributable to HPV and that these estimates represent the upper limit for the annual burden of cancers caused by HPV. only a quarter of oropharyngeal cancers are attributable to HPV; meanwhile approximately 90% of anal cancers, and 40% of penile, vaginal or vulvar cancers are attribut‐ able to the virus. Although there is some evidence implicating HPV with several other cancers (e.g., lung, colon, ovary, breast, prostate, urinary bladder, esophageal and nasal/sinonasal cancers), current molecular and epidemiological data are sparse and do not yet support a causal role for HPV in the etiology of these cancers [30, 40-42]. The role of HPV in the causation of esophageal squamous cell carcinoma is unclear.

Globally, HPV accounts for roughly 5.2% of the total cancer burden — the highest among all infectious agents. However, as may be expected, the distribution varies considerably according to country development status, where HPV accounts for approximately 7.7% and 2.2% of all cancer cases in developing and developed countries, respectively [42].
