**2. Prevention, interception and early detection**

*Disease Burden and Risk:* Approximately 20 million Americans are currently infected with HPV, and another 6 million new infections occur annually. In about 90 percent of these cases, the infection is cleared by the immune system within two years [1, 2]. However, a relatively small subset of infections persists, sometimes resulting in viral oncoprotein-mediated perturbation of cell-cycle controls leading to cervical intraepithelial neoplasia (CIN). Approximately 5 percent of Pap specimens are classified as pre-cancerous (CIN 1, 2 or 3) while 0.3 - 0.5 percent are typically diagnosed as carcinoma *in situ* [3].

Occurring in men and women, HPV infection is most commonly transmitted by sexual contact. According to the National Cancer Institute (NCI), a woman's risk of acquiring HPV and subsequently developing cervical cancer is increased when the age of sexual debut occurs at a younger age and when the number of lifetime sexual partners is higher [4]. In addition, it has been shown that prevalence rates of infection are consistently higher by 70 percent in sexually experienced, low-income populations of racial and ethnic diversity compared to the general population [5-7]. Also, the risk of HPV infection progressing to cancer depends on lifestyle. For example [8], at a woman's first Pap test the risk that HPV16 infection is more likely to progress to carcinoma *in situ* is 70 percent higher in women who currently smoke (Relative Risk for current smokers is 1.9) [8-11].

*Public Education*: Media coverage over the last decade has increased general awareness of HPV infection. However, knowledge regarding how the virus is transmitted and the fact that HPV infection may cause cervical cancer is less well-known, particularly among vulnerable populations [12]. In one survey, when high school students were asked to name a few common sexually transmitted infections (STIs) only 17 percent of students mentioned HPV [13]. A general awareness about HPV infection leading to cervical cancer can be increased through education, but more needs to be done to influence the pre-conceived attitudes about prevention through vaccination and sexual behaviors [14]. For example, HPV infection, like most other STIs, are spread *via* bodily fluids that can be obstructed by condom use [15]. Consequently, this knowledge might heighten the risk-perception (a person's subjective appraisal of danger) of not using a condom during vaginal intercourse encounters and reduce sexually risky behaviors to some appreciable degree.

UNESCO (United Nations Educational, Scientific, and Cultural Organization) has demon‐ strated the effectiveness of sex education in the global fight against HIV/AIDs. Although HPV does not currently compare to HIV/AIDs in terms of mortality and global magnitude, the need for HPV awareness has grown tremendously and it is speculated that such a plan may prove useful here as well. An increase in consciousness may decrease sexual risk behaviors if populations at high-risk for contracting HPV were actively targeted for education [16, 17]. However, it should be considered that gender inequalities experienced by women in locations like Sub-Saharan Africa, as reported by the Global Health Corps and UNESCO, may negatively impact these initiatives. Furthermore, the cost of such programs compared to other interven‐ tional methods has not been determined. But in general, education can be used as a powerful tool in preventing HPV-mediated diseases such as cervical cancer. Ideally, these programs would emphasize risk-perception in both men and women leading to lifestyle modifications, and a further reduction in the incidence of HPV-mediated carcinoma might be realized.

cervical cancer a much more achievable goal than in the past. Several areas of cervical cancer research continue to address the challenges posed by the need for appropriate therapeutic alternatives, and progress is occurring at each level of clinical management ranging from detection to the development of small molecule antiviral leads. Because the field is evolving rapidly in all directions and related disciplines, it is helpful to summarize the status of our growth, and to recognize those pioneering efforts that may ultimately contribute to achieving our goal of eliminating cervical cancer. This review seeks to survey the current understanding of cervical cancer etiology and treatment and to review areas requiring additional progress.

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

*Disease Burden and Risk:* Approximately 20 million Americans are currently infected with HPV, and another 6 million new infections occur annually. In about 90 percent of these cases, the infection is cleared by the immune system within two years [1, 2]. However, a relatively small subset of infections persists, sometimes resulting in viral oncoprotein-mediated perturbation of cell-cycle controls leading to cervical intraepithelial neoplasia (CIN). Approximately 5 percent of Pap specimens are classified as pre-cancerous (CIN 1, 2 or 3) while 0.3 - 0.5 percent

Occurring in men and women, HPV infection is most commonly transmitted by sexual contact. According to the National Cancer Institute (NCI), a woman's risk of acquiring HPV and subsequently developing cervical cancer is increased when the age of sexual debut occurs at a younger age and when the number of lifetime sexual partners is higher [4]. In addition, it has been shown that prevalence rates of infection are consistently higher by 70 percent in sexually experienced, low-income populations of racial and ethnic diversity compared to the general population [5-7]. Also, the risk of HPV infection progressing to cancer depends on lifestyle. For example [8], at a woman's first Pap test the risk that HPV16 infection is more likely to progress to carcinoma *in situ* is 70 percent higher in women who currently smoke

*Public Education*: Media coverage over the last decade has increased general awareness of HPV infection. However, knowledge regarding how the virus is transmitted and the fact that HPV infection may cause cervical cancer is less well-known, particularly among vulnerable populations [12]. In one survey, when high school students were asked to name a few common sexually transmitted infections (STIs) only 17 percent of students mentioned HPV [13]. A general awareness about HPV infection leading to cervical cancer can be increased through education, but more needs to be done to influence the pre-conceived attitudes about prevention through vaccination and sexual behaviors [14]. For example, HPV infection, like most other STIs, are spread *via* bodily fluids that can be obstructed by condom use [15]. Consequently, this knowledge might heighten the risk-perception (a person's subjective appraisal of danger) of not using a condom during vaginal intercourse encounters and reduce sexually risky

**2. Prevention, interception and early detection**

are typically diagnosed as carcinoma *in situ* [3].

(Relative Risk for current smokers is 1.9) [8-11].

behaviors to some appreciable degree.

*Prophylactic Immune Strategies:* Presently, the most effective protective factor against the most prevalent and high-risk types of HPV infection is prophylactic vaccination [18]. It has been seven years since the introduction of the first HPV vaccine [19]. Since then, two vaccines, Gardasil and Cervarix, have been made available to the public to protect against the more common HPV strains [20]. The vaccines induce the production of neutralizing antibodies against HPV L1 capsid virus-like proteins (VLPs), which do not contain virus genetic material. The quadrivalent vaccine, Gardasil, protects against low- and high-risk HPV (LR- and HR-HPV, respectively) types 6, 11, 16, and 18 following full vaccination of all three doses at 0, 1, 2 and 6 months. Alternatively, the bivalent vaccine, Cervarix, prevents infection by HR-HPV types 16 and 18. Both vaccines have been documented to possess compelling prophy‐ lactic efficacy in preventing cervical, genital, and anal diseases. This protection is expected to persist for 7 years, or at least during the years of high infection risk for most individuals [20-22].

HPV infections of types other than the four mentioned above are not reliably prevented by vaccination. Also, studies are warranted regarding the vaccine's long-term effects and how they might impact the occurrence of infections by other HPV types. It has already been noted that quadrivalent and bivalent vaccines may exhibit cross-protection against HPV 31 and other types by 75 to 80 percent [23]. However, concerns are emerging that relate to HPV typereplacement. Type-replacement is an increased prevalence of other HPV strains that are not included in the vaccines, while vaccine-type HPV prevalence is decreased. It was recently reported that vaccine-type HPV has been reduced in vaccinated and nonvaccinated women, while nonvaccine-type HPV has slightly increased overall [24]. Researchers do not expect typereplacement to occur frequently. However, studies are becoming more attentive to changes in the prevalence of various HPV types, which are expected to surface first within the sexually experienced population. Such discoveries could encourage the research community to continue seeking multivalent solutions to as many HPV types as possible without eliciting additional harmful results. To date, clinical trials have revealed that the most common adverse response to both vaccines are injection site reactions, which occur more frequently in vaccine groups rather than in participants given placebos [25].

Though most industrialized countries, like Great Britain, have already implemented struc‐ tured HPV immunization programs, well-functioning programs geared towards adults and young adolescents have yet to be seen in many developing countries. However, there are globally funded systems with strong infrastructure that support the immunization of infants in developing countries [26]. This anomaly is due to several challenges, which include the cost of the vaccines, though the biological, economical, and psychological disease burdens of HPV have also been considered [25, 26]. Therefore, it is no surprise that less developed countries have not made HPV vaccination programs a priority while other issues compete for the same limited governmental resources. The most apparent considerations regarding vaccine distri‐ bution in these countries relate to healthcare infrastructure, which can directly affect a country's ability to establish and maintain immunization programs that target the vaccine's intended population – adolescents. Other factors of significant importance comprise how to best promote these programs in a way that does not aggravate ethnic/cultural sensitivities and attitudes about vaccination against an STI [27, 28]. This would further include easing parental concerns about what an STI vaccine might imply if perceived as socially acceptable within the targeted age groups. Perhaps if immunization programs were set up in an educational setting, a stronger risk-perception might be instilled, which would encourage the formation of better habits of awareness. In years to come, these types of educational agendas might also improve adherence to the 3-dose regimen over the six-month vaccination period and increase compli‐ ance with screening routines throughout a woman's lifetime [29].

neutralized by an earlier developed immunity prior to the individual presenting as HIV+

is based on the premise that the protection conferred against the viral types represented in the vaccine is expected to last for the same time period as in others who are not infected with HIV. However, what remains unclear is whether the vaccine will prove effective for an individual already infected with HIV, or in any immunocompromised state [37]. The current under‐ standing is that humoral immune responses remain relatively intact following HIV infection. However, in such a state of immune weakness, it is unknown whether protection against HPV can be sustained. Overall, individuals who have been vaccinated prior to becoming immune compromised are expected to benefit from vaccination by maintaining immune competency, because new HPV infections from these specific types and the risk of lesions reactivating from an ongoing, latent HPV infection would be reduced. Of course, they, like other individuals, will only be protected from the vaccine-type strains, and it is imperative to note that questions regarding long-term safety in such vulnerable groups remain unanswered [30, 31, 33, 40, 41].

Modern Molecular and Clinical Approaches to Eradicate HPV-Mediated Cervical Cancer

To this end, it is important that innovative therapeutic approaches to improve immunological surveillance and clearance of HPV continue to develop. It is well-documented that cellular immune components contribute directly to natural clearance of the virus in most people. For instance, CD4+ and CD8+ cytotoxic T cells (CTLs) are thought to target HPV 16 early and late proteins, and active HPV-specific CTLs have been identified in patients with existing infections [42]. Furthermore, researchers have found that in response to a vaccine containing E6 and E7 oncoproteins, CD4+ and CD8+ CTLs were stimulated, thus inducing the regression of HPVmediated vulvar intraepithelial neoplasia (VIN) in 50 percent of subjects [43]. A variety of other immunotherapy investigations are underway and will be discussed in more detail later in the

*Physical Barriers:* As noted above, the use of condoms can reduce the rate of HPV infection. One study showed that when condoms are used during all vaginal intercourse encounters, HPV transmission/infection is reduced by 70 percent. Even if a condom is used greater than half of the time, the risk of infection is still reduced by 50 percent [44]. Another study similarly reports that condoms benefit users by promoting viral clearance and possible regression of CIN [4]. The use of barrier protectors such as microbicidal and spermicidal gels can also reduce the risk of HPV infection [45, 46]. The recent utilization of pseudoviruses has proven helpful in better understanding HPV invasion into keratinocytes through cell surface proteins. In these pseudovirus studies, carrageenan exhibited a microbicidal function by blocking virus particle attachment to heparin sulphate proteoglycans on cell basement membranes. Beyond its function on tissue surfaces, carrageenan also exhibited post-attachment inhibitory actions. Carrageenan has been used as a thickening agent in sex lubricants and Pap smear gels, and has shown microbicidal function against a host of STI-causing microbes including HPV [46, 47]. Originally derived from red algae, it is structurally similar to heparin but several times more potent. Therefore, it is able to bind virus more effectively than host cells and thereby acts

*Interception of HPV-Mediated Carcinogenesis:* During the decades that frequently occur between HPV infection and cancer development, there exists a window of opportunity to intercept the process. Any interventions that increase viral clearance, for example, would fit into this

review.

as a decoy receptor [45, 46].

. This

291

http://dx.doi.org/10.5772/55810

One clear limitation of the HPV vaccines is their lack of efficacy for those who have already been exposed to the virus types included in the vaccines. Of course, this exposure is directly correlated to increasing age and sexual experience [30, 31]. For those who fall into this group, including older women regardless of vaccination status, it is important that screenings continue as outlined by the American Cancer Society (ACS) guidelines for Early Detection of Cervical Neoplasia and Cancer [5]. Therefore, integrated approaches of prevention and detection are required if efforts against HPV-mediated cervical cancer are to be maximized. Another important consideration for vaccination is the immune status of potential vaccine recipients; the immune system must be intact [32]. The immune system's ability to clear antigens depends largely on its strength and competence. Thus, immunocompromised women are especially in peril of HR-HPV infection progressing to cancer [33].

The most common scenarios for compromised immunity are seen in HIV+ individuals and organ transplant beneficiaries. Those infected with HIV have a greater chance of HPV coinfection and progression to invasive cervical cancer as compared to those without HIV [34, 35]. The disparity observed here is most likely due to the immune system's inability to effectively clear virus among this subset due to decreased immune reactivity to HPV antigen [36, 37]. It was also found that HPV infection is prevalent among those receiving organ transplants [38], and that the infection increasingly persisted throughout immunosuppressive therapy to moderate graft rejection [39]. Despite these challenges, researchers agree that previous prophylactic HPV vaccination is still beneficial for organ transplant recipients as well as the HPV/HIV co-infected population who receive HPV vaccination before becoming HIV+ . In these cases, any future challenges of HPV infection following vaccination would be neutralized by an earlier developed immunity prior to the individual presenting as HIV+ . This is based on the premise that the protection conferred against the viral types represented in the vaccine is expected to last for the same time period as in others who are not infected with HIV. However, what remains unclear is whether the vaccine will prove effective for an individual already infected with HIV, or in any immunocompromised state [37]. The current under‐ standing is that humoral immune responses remain relatively intact following HIV infection. However, in such a state of immune weakness, it is unknown whether protection against HPV can be sustained. Overall, individuals who have been vaccinated prior to becoming immune compromised are expected to benefit from vaccination by maintaining immune competency, because new HPV infections from these specific types and the risk of lesions reactivating from an ongoing, latent HPV infection would be reduced. Of course, they, like other individuals, will only be protected from the vaccine-type strains, and it is imperative to note that questions regarding long-term safety in such vulnerable groups remain unanswered [30, 31, 33, 40, 41].

Though most industrialized countries, like Great Britain, have already implemented struc‐ tured HPV immunization programs, well-functioning programs geared towards adults and young adolescents have yet to be seen in many developing countries. However, there are globally funded systems with strong infrastructure that support the immunization of infants in developing countries [26]. This anomaly is due to several challenges, which include the cost of the vaccines, though the biological, economical, and psychological disease burdens of HPV have also been considered [25, 26]. Therefore, it is no surprise that less developed countries have not made HPV vaccination programs a priority while other issues compete for the same limited governmental resources. The most apparent considerations regarding vaccine distri‐ bution in these countries relate to healthcare infrastructure, which can directly affect a country's ability to establish and maintain immunization programs that target the vaccine's intended population – adolescents. Other factors of significant importance comprise how to best promote these programs in a way that does not aggravate ethnic/cultural sensitivities and attitudes about vaccination against an STI [27, 28]. This would further include easing parental concerns about what an STI vaccine might imply if perceived as socially acceptable within the targeted age groups. Perhaps if immunization programs were set up in an educational setting, a stronger risk-perception might be instilled, which would encourage the formation of better habits of awareness. In years to come, these types of educational agendas might also improve adherence to the 3-dose regimen over the six-month vaccination period and increase compli‐

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

One clear limitation of the HPV vaccines is their lack of efficacy for those who have already been exposed to the virus types included in the vaccines. Of course, this exposure is directly correlated to increasing age and sexual experience [30, 31]. For those who fall into this group, including older women regardless of vaccination status, it is important that screenings continue as outlined by the American Cancer Society (ACS) guidelines for Early Detection of Cervical Neoplasia and Cancer [5]. Therefore, integrated approaches of prevention and detection are required if efforts against HPV-mediated cervical cancer are to be maximized. Another important consideration for vaccination is the immune status of potential vaccine recipients; the immune system must be intact [32]. The immune system's ability to clear antigens depends largely on its strength and competence. Thus, immunocompromised women

The most common scenarios for compromised immunity are seen in HIV+ individuals and organ transplant beneficiaries. Those infected with HIV have a greater chance of HPV coinfection and progression to invasive cervical cancer as compared to those without HIV [34, 35]. The disparity observed here is most likely due to the immune system's inability to effectively clear virus among this subset due to decreased immune reactivity to HPV antigen [36, 37]. It was also found that HPV infection is prevalent among those receiving organ transplants [38], and that the infection increasingly persisted throughout immunosuppressive therapy to moderate graft rejection [39]. Despite these challenges, researchers agree that previous prophylactic HPV vaccination is still beneficial for organ transplant recipients as well as the HPV/HIV co-infected population who receive HPV vaccination before becoming HIV+

In these cases, any future challenges of HPV infection following vaccination would be

.

ance with screening routines throughout a woman's lifetime [29].

are especially in peril of HR-HPV infection progressing to cancer [33].

To this end, it is important that innovative therapeutic approaches to improve immunological surveillance and clearance of HPV continue to develop. It is well-documented that cellular immune components contribute directly to natural clearance of the virus in most people. For instance, CD4+ and CD8+ cytotoxic T cells (CTLs) are thought to target HPV 16 early and late proteins, and active HPV-specific CTLs have been identified in patients with existing infections [42]. Furthermore, researchers have found that in response to a vaccine containing E6 and E7 oncoproteins, CD4+ and CD8+ CTLs were stimulated, thus inducing the regression of HPVmediated vulvar intraepithelial neoplasia (VIN) in 50 percent of subjects [43]. A variety of other immunotherapy investigations are underway and will be discussed in more detail later in the review.

*Physical Barriers:* As noted above, the use of condoms can reduce the rate of HPV infection. One study showed that when condoms are used during all vaginal intercourse encounters, HPV transmission/infection is reduced by 70 percent. Even if a condom is used greater than half of the time, the risk of infection is still reduced by 50 percent [44]. Another study similarly reports that condoms benefit users by promoting viral clearance and possible regression of CIN [4]. The use of barrier protectors such as microbicidal and spermicidal gels can also reduce the risk of HPV infection [45, 46]. The recent utilization of pseudoviruses has proven helpful in better understanding HPV invasion into keratinocytes through cell surface proteins. In these pseudovirus studies, carrageenan exhibited a microbicidal function by blocking virus particle attachment to heparin sulphate proteoglycans on cell basement membranes. Beyond its function on tissue surfaces, carrageenan also exhibited post-attachment inhibitory actions. Carrageenan has been used as a thickening agent in sex lubricants and Pap smear gels, and has shown microbicidal function against a host of STI-causing microbes including HPV [46, 47]. Originally derived from red algae, it is structurally similar to heparin but several times more potent. Therefore, it is able to bind virus more effectively than host cells and thereby acts as a decoy receptor [45, 46].

*Interception of HPV-Mediated Carcinogenesis:* During the decades that frequently occur between HPV infection and cancer development, there exists a window of opportunity to intercept the process. Any interventions that increase viral clearance, for example, would fit into this category. Another possible approach would be to target the process by which viral DNA integrates into the host chromosome, a relatively rare event that greatly increases the incidence of cancer. One study, for example, has postulated that chronic inflammation and the subse‐ quent generation of reactive oxygen/nitrogen species (ROS/RNS) are harbingers of DNA damage, causing high HPV integration rates. Furthermore, smoking, cervical trauma induced by high parity, co-infection with other STDs, and long-term use of oral contraceptives have all been linked to cellular oxidative stress [48]. Thus, breaks in the DNA induced by this oxidative stress increase the probability of viral integration [49]. Interestingly, HR-HPV types integrate more frequently than do LR-HPV. The difference in integration occurrence suggests a distinc‐ tion in the molecular variation and/or susceptibility between high- and low-grade lesions. Therefore, progression to cervical, and some anogenital, cancers is dependent on the presence of HR-HPV integration into the host genome. Furthermore, scientists are finding certain patterns in HPV integration events. In particular, the E2 ORF region of the viral genome is strongly preferred over other sites of integration, and integration at this site, with its accom‐ panying loss of functional E2 protein, is linked to an increase in E6 and E7 oncogene expression [50, 51]. Consequentially, integration leads to uncontrolled expression of the oncogenes and ultimately to cellular transformation.

an integral component of preventing invasive disease at all stages [5]. Because cervical cancer detection programs are the most expensive preventive measure in developed countries, enhancements to existing screening techniques, or the development of completely innovative

Modern Molecular and Clinical Approaches to Eradicate HPV-Mediated Cervical Cancer

http://dx.doi.org/10.5772/55810

293

The most utilized and successful of screening methods in lowering cervical cancer incidence rates (by 70 percent) is exfoliative cervicovaginal cytology, or the Pap test. The Pap test satisfies the aforementioned objectives for reducing the occurrence of squamous cervical carcinoma through appropriate screening [5]. Pap tests are recommended for all sexually active women and/or women ages 21 and older. Now, a modified liquid-based version of the Pap smear is available. In a liquid-based Pap test such as Cytoscreen or Thinprep, the cells are first filtered and fixed in preservative. Then the specimen is smeared on a glass slide, which is slightly in contrast to the conventional method of directly smearing a sample onto a microscope slide. Other tests such as visual inspection with acetic acid (VIA) are useful in resource-limited settings. Further modifications of VIA include magnified visual inspection with acetic acid (VIAM) and visual inspection with Lugol's iodine solution (VILI) [57]. Colposcopy, though considered more diagnostic, also allows a magnified visualization of abnormal cervical cells [56]. Other cervical cancer screening tests may also be applied: pelvic examination – involving internal palpation of the reproductive organs; automated cervical screening techniques – supplemental imaging that reduces false positives from the cytological tests; computer imaging; polar probe – measuring the differences in electrical stimulation between normal and abnormal cervical tissue; laser-induced fluorescence – measuring spectroscopic differences in florescence between normal and diseased cervix; speculoscopy – cervical inspection using acetic acid with chemiluminescent light; and cervicography – photo development while using

Complementing the Pap test is the detection of HPV DNA. The direct testing for HPV DNA is becomingstandardinmanycervical cancer screeningregimens,as its combinedusewithliquidbasedcytologyhasgeneratedresultswithevenbettersensitivity(upto100percent)forpredicting high-grade cervical dysplasia [58]. HPV DNA is usually obtained from cervical scrapes and/or biopsy specimens, and recent clinical studies continue to assert the unique value of HPV-DNA testing over cytology [59-61]. Nevertheless, only time will tell the extent to which the Pap test will be replaced by the more economically appealing HPV-DNA test. To date, the FDA has approved five HPV-DNA tests: the Hybrid Capture 2 HPV DNA test, the Cervista HPV HR test, the Cervista HPV16/18 test, the Cobas 4800 HPV test, and the Aptima HPV assay. Other commonly used assays not approved by the FDA include PCR and Southern Blot hybridiza‐ tion, the latter being the laboratory gold standard. Some other recent innovative HPV detec‐ tion methods are complete HPV genotyping, HPV mRNA detection, HPV load quantitation, identifying HPV integration, p16 ELISA, methylation profiles, and the E6 Strip test [62, 63].

Cervical cancer incidence and mortality seem to be on a downward swing in the U.S., primarily due to cytological gynecologic screening through the Pap test. Nevertheless, the global burden of HPV infection remains. Because no single detection method is optimal for every situation, it is essential that novel techniques to identify cervical cancer and HPV infection be continu‐ ously developed. Ideally, these new procedures/assays would allow clinicians to easily

and economical methods would be beneficial.

acetic acid [56].

The role of chronic inflammation and its link to radical species production in cancer patho‐ genesis is widely recognized. If increased levels of oxidative stress and ROS do indeed increase the frequency of integration and cancer, one would predict that antioxidant mechanisms that counteract the generation of radical species could therefore exert chemopreventative and chemotherapeutic effects; such mechanisms have indeed been described [52, 53]. In contrast, other groups are studying ways to therapeutically harness the power of oxidative stress for actions against cancer cells. For example, the antimalarial drug, artemisin, was found to induce apoptosis in cervical cancer cells. The mechanism of action involves artemisin interacting with reduced iron to generate oxidative stress through ROS, as well as the destabilization of mitochondrial oxidative mechanisms [45, 54]. These discoveries merit further research that continues to seek ways of preventing HPV-mediated oncogenesis.

*Screening for HPV and Cervical Cancer:* The ultimate goal of cervical cancer screening, as outlined by the American Cancer Society (ACS) guidelines for the prevention and early detection of cervical cancer, is to prevent morbidity and mortality by determining appropriate treatment plans. In detecting the presence of HPV in the cervix, screening methods should serve to distinguish transient from persistent infections, and to effectively diagnose disease while minimizing or avoiding unnecessary complications induced by these techniques. Because 50 percent of women diagnosed with cervical cancer in the U.S. have never been screened, the importance of diligent watchfulness cannot be over-stated. Moreover, when HIV+ women comply with regular detection methods and schedules, their otherwise 10-fold higher risk of progression to invasive cervical carcinoma is diminished [30, 31, 55]. Earlier detection corre‐ sponds to better prognosis [56]. Thus, it is ideal for precancerous women who are at signifi‐ cantly higher risk for invasive cervical carcinoma to be promptly identified and to undergo intervention. Generally, it is recommended that cervical cancer screening start no earlier than age 21 for both non-vaccinated and vaccinated women, implying that cervical monitoring is an integral component of preventing invasive disease at all stages [5]. Because cervical cancer detection programs are the most expensive preventive measure in developed countries, enhancements to existing screening techniques, or the development of completely innovative and economical methods would be beneficial.

category. Another possible approach would be to target the process by which viral DNA integrates into the host chromosome, a relatively rare event that greatly increases the incidence of cancer. One study, for example, has postulated that chronic inflammation and the subse‐ quent generation of reactive oxygen/nitrogen species (ROS/RNS) are harbingers of DNA damage, causing high HPV integration rates. Furthermore, smoking, cervical trauma induced by high parity, co-infection with other STDs, and long-term use of oral contraceptives have all been linked to cellular oxidative stress [48]. Thus, breaks in the DNA induced by this oxidative stress increase the probability of viral integration [49]. Interestingly, HR-HPV types integrate more frequently than do LR-HPV. The difference in integration occurrence suggests a distinc‐ tion in the molecular variation and/or susceptibility between high- and low-grade lesions. Therefore, progression to cervical, and some anogenital, cancers is dependent on the presence of HR-HPV integration into the host genome. Furthermore, scientists are finding certain patterns in HPV integration events. In particular, the E2 ORF region of the viral genome is strongly preferred over other sites of integration, and integration at this site, with its accom‐ panying loss of functional E2 protein, is linked to an increase in E6 and E7 oncogene expression [50, 51]. Consequentially, integration leads to uncontrolled expression of the oncogenes and

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

The role of chronic inflammation and its link to radical species production in cancer patho‐ genesis is widely recognized. If increased levels of oxidative stress and ROS do indeed increase the frequency of integration and cancer, one would predict that antioxidant mechanisms that counteract the generation of radical species could therefore exert chemopreventative and chemotherapeutic effects; such mechanisms have indeed been described [52, 53]. In contrast, other groups are studying ways to therapeutically harness the power of oxidative stress for actions against cancer cells. For example, the antimalarial drug, artemisin, was found to induce apoptosis in cervical cancer cells. The mechanism of action involves artemisin interacting with reduced iron to generate oxidative stress through ROS, as well as the destabilization of mitochondrial oxidative mechanisms [45, 54]. These discoveries merit further research that

*Screening for HPV and Cervical Cancer:* The ultimate goal of cervical cancer screening, as outlined by the American Cancer Society (ACS) guidelines for the prevention and early detection of cervical cancer, is to prevent morbidity and mortality by determining appropriate treatment plans. In detecting the presence of HPV in the cervix, screening methods should serve to distinguish transient from persistent infections, and to effectively diagnose disease while minimizing or avoiding unnecessary complications induced by these techniques. Because 50 percent of women diagnosed with cervical cancer in the U.S. have never been screened, the importance of diligent watchfulness cannot be over-stated. Moreover, when HIV+ women comply with regular detection methods and schedules, their otherwise 10-fold higher risk of progression to invasive cervical carcinoma is diminished [30, 31, 55]. Earlier detection corre‐ sponds to better prognosis [56]. Thus, it is ideal for precancerous women who are at signifi‐ cantly higher risk for invasive cervical carcinoma to be promptly identified and to undergo intervention. Generally, it is recommended that cervical cancer screening start no earlier than age 21 for both non-vaccinated and vaccinated women, implying that cervical monitoring is

continues to seek ways of preventing HPV-mediated oncogenesis.

ultimately to cellular transformation.

The most utilized and successful of screening methods in lowering cervical cancer incidence rates (by 70 percent) is exfoliative cervicovaginal cytology, or the Pap test. The Pap test satisfies the aforementioned objectives for reducing the occurrence of squamous cervical carcinoma through appropriate screening [5]. Pap tests are recommended for all sexually active women and/or women ages 21 and older. Now, a modified liquid-based version of the Pap smear is available. In a liquid-based Pap test such as Cytoscreen or Thinprep, the cells are first filtered and fixed in preservative. Then the specimen is smeared on a glass slide, which is slightly in contrast to the conventional method of directly smearing a sample onto a microscope slide. Other tests such as visual inspection with acetic acid (VIA) are useful in resource-limited settings. Further modifications of VIA include magnified visual inspection with acetic acid (VIAM) and visual inspection with Lugol's iodine solution (VILI) [57]. Colposcopy, though considered more diagnostic, also allows a magnified visualization of abnormal cervical cells [56]. Other cervical cancer screening tests may also be applied: pelvic examination – involving internal palpation of the reproductive organs; automated cervical screening techniques – supplemental imaging that reduces false positives from the cytological tests; computer imaging; polar probe – measuring the differences in electrical stimulation between normal and abnormal cervical tissue; laser-induced fluorescence – measuring spectroscopic differences in florescence between normal and diseased cervix; speculoscopy – cervical inspection using acetic acid with chemiluminescent light; and cervicography – photo development while using acetic acid [56].

Complementing the Pap test is the detection of HPV DNA. The direct testing for HPV DNA is becomingstandardinmanycervical cancer screeningregimens,as its combinedusewithliquidbasedcytologyhasgeneratedresultswithevenbettersensitivity(upto100percent)forpredicting high-grade cervical dysplasia [58]. HPV DNA is usually obtained from cervical scrapes and/or biopsy specimens, and recent clinical studies continue to assert the unique value of HPV-DNA testing over cytology [59-61]. Nevertheless, only time will tell the extent to which the Pap test will be replaced by the more economically appealing HPV-DNA test. To date, the FDA has approved five HPV-DNA tests: the Hybrid Capture 2 HPV DNA test, the Cervista HPV HR test, the Cervista HPV16/18 test, the Cobas 4800 HPV test, and the Aptima HPV assay. Other commonly used assays not approved by the FDA include PCR and Southern Blot hybridiza‐ tion, the latter being the laboratory gold standard. Some other recent innovative HPV detec‐ tion methods are complete HPV genotyping, HPV mRNA detection, HPV load quantitation, identifying HPV integration, p16 ELISA, methylation profiles, and the E6 Strip test [62, 63].

Cervical cancer incidence and mortality seem to be on a downward swing in the U.S., primarily due to cytological gynecologic screening through the Pap test. Nevertheless, the global burden of HPV infection remains. Because no single detection method is optimal for every situation, it is essential that novel techniques to identify cervical cancer and HPV infection be continu‐ ously developed. Ideally, these new procedures/assays would allow clinicians to easily distinguish between low-risk and high-risk HPV status without causing undue concern in patients with transient infection. However, the cost of cervical cancer screening programs, even in developed countries, may hamper the implementation of these new advances.

as they fit the scope of this review. Therefore, the sections below are not intended to represent

Modern Molecular and Clinical Approaches to Eradicate HPV-Mediated Cervical Cancer

http://dx.doi.org/10.5772/55810

295

*Imaging:* One of the most important aspects of cervical cancer treatment is identifying and evaluating abnormal tissue morphology using radiological technologies such as magnetic resonance imaging (MRI), x-ray computed tomography (CT scan), and positron emission tomography (PET). Because the effectiveness of these devices depends on clinical expertise and equipment, diagnostic imaging possesses several inherent discrepancies [64]. However, many researchers have begun studies that will help to improve these methods and/or to enable clinicians to draw better conclusions. Advancements in the functionality of diagnostic imaging are making it easier than ever to assess and exploit tumor parameters such as cellularity, blood flow, and glucose metabolism. Recent studies are showing that the glucose analogue, fluorinelabeled fluoro-2-deoxy-D-glucose (FDG), is particularly useful in gauging tumor metabolic activity. When combined with PET (FDG-PET), FDG is considered to have high sensitivity in detecting primary cervical tumors [69]. Additionally, combining PET and CT is becoming more acceptable as a way to eliminate the guesswork involved in evaluating metastasis to lymph nodes [70]. However, MRI is the preferred imaging method in managing cervical cancer due to the high quality of anatomic resolution it provides in the pelvis; this enhances its ability to evaluate primary tumor volume [67, 71]. New developments in MRI technology such as diffusion-weighted MRI (DWI) compare normal and abnormal tissues based on the Brownian motion of water molecules, the movement of which impacts cellular membrane integrity. However, these features may not be as distinct or reliable in excessively necrotic tumors. Another derivative of MRI is dynamic contrast-enhanced MRI (DCE-MRI), which provides an unprecedented appraisal of tumor vasculature through contrast distribution over time. Therefore, DCE-MRI may prove to be distinctly helpful in ascertaining a tumor's unique

*Surgery:* Surgery is the advised treatment for cervical carcinoma at stages I and II. The preferred procedure, radical hysterectomy (RH), has a 75 to 80 percent cure rate according to the NCI and is the gold standard of treatment. RH is the complete removal of the uterus, cervix, and upper portion of the vagina, and involves measuring metastasis to the parametrial and pelvic lymph nodes [73, 74]. Other procedures, such as total and subtotal hysterectomies, do not require the removal of the vagina and cervix, respectively. At the early phase of stage I, less invasive techniques labeled *excisional* therapies selectively remove pathologic tissue [75, 76]. Large loop loop excision of the transformation zone (LLETZ) and cold knife conization procedures are classified as excisional therapies and are used without great risk of the cancer recurring. Conical biopsies, or the removal and microscopic examination of presumably abnormal tissue, may suffice in some situations. *Ablative* therapies such as laser and cryosur‐ gery are utilized in expunging carcinomas *in situ* of lesser risk. This distinction between therapies is supported by studies showing that lesions from more progressed carcinomas return at a higher rate when treated with ablative techniques as compared to excisional ones [76]. In some instances, neither excision nor ablative therapies are suitable for the grade of disease, and hysterectomy is recommended. Indeed, patients not interested in fertility loss or those with lymph and vascular space involvement (LVSI) should elect for RH (RH). Nonethe‐

a comprehensive discussion of these modalities.

response to therapies [67, 72].
