**2. The diagnosis of HPV infection to screening cervical cancer**

Carcinoma of the uterine cervix is the second most common cancer among women worldwide, with very high mortality rates in developing countries. It was observed more than 20 years ago that some types of HPV were more frequent in malignant than in benign lesions, and infection with high-risk types of HPV is now considered the major risk factor for the devel‐ opment of cancer of the uterine cervix [1]. Oncogenic HPV types 16 and 18 and history of other concurrent sexually transmitted diseases were found to be significantly associated with progression to cervical cancer [5].

Studies have demonstrated a strong association between lifetime number of sexual part‐ ners and genital HPV acquisition. The acquisition of new sexual partners continues through‐ out all age groups. In addition, studies have shown consistently that the risk of cervical cancer can be predicted as much by a woman's own sexual behaviour as by the sexual behaviour of her husband/partner. The presence of HPV DNA in the penis and urethra of her sexual partner(s) is directly related to her HPV carrier status and therefore her risk of developing cervical cancer [13-15].

Of the genital HPVs, which are sexually transmitted, 15 are categorized as high risk and are considered the causative agents of most cervical cancers, with over 99% of cervical lesions containing viral sequences [1]. The remaining viral types are rarely found in malignancies. High-risk HPVs (HR-HPV) are also associated with many vulvar, anal and penile carcinomas and contribute to oral cancers [17], 2006). Additionally, these cancers, in contrast to cervical cancer, appear to be preferentially associated with HPV16 [11,14]. For instance, in the subset of penile cancer attributed to HPV infection, HPV 16 was found in 60,23% of cases [18]. A vaccine has recently been introduced that can prevent the initial infection by two of these highrisk types, HPV 16 and 18, which are responsible for about 70% of cervical cancers [19].

The process by which HPV facilitates tumour initiation and fosters tumour progression is an exceptional model to understand the development of many other human cancers and also allows identification of additional signalling pathways targeted in malignant progression [19]. The association between HPV and human cancer was first proposed more than three decades ago by Harald zur Hausen, and he was honored with one of two 2008 Nobel Prizes in Medicine for his isolation and characterization of HPV 16 in 1983 and later HPV 18 in cervical cancer [20]. The award recognized not only the importance of his discovery in the eventual documentation of the etiology of HPV in cervical and a number of other cancers, but also the importance of the application of his discovery to the clinical use of HPV testing and in implementation of the HPV vaccine.

result of lack of screening programs and poor access to medical care [1]. The prevalence of HPV and the distribution of its types probably plays an important role as well. On the other hand, the relationship between others cancer types and HPV-associated is just emerging [10]. The variability in HPV-attributable proportions for non cervical cancers, in part, arises from differences in HPV detection methods across studies as well as from true geographic differ‐ ences in HPV-attributable proportions [11]. Despite this variability, 90%–93% of anal cancers, 12%–63% of oropharyngeal cancers, 36%–46.9% of penile cancers, 40%–64% of vaginal cancers,

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

and 40%–51% of vulvar cancers are potentially attributable to HPV infection [11-15].

**2. The diagnosis of HPV infection to screening cervical cancer**

and molecular tools to detect HPV-DNA/RNA.

progression to cervical cancer [5].

cervical cancer [13-15].

Abnormal cervical epithelial cells can be detected microscopically following Papanicolaou (Pap) staining of conventional cervical smears or of the more homogeneous cell suspension from liquid cytology medium. This forms the basis of cervical screening programmes for detection of women at risk of disease progression, and also for incident infections [9,16]. Molecular detection of HPV provides a different approach to screening and patient manage‐ ment. In this chapter was described the diagnosis of HPV infection to screening cervical cancer

Carcinoma of the uterine cervix is the second most common cancer among women worldwide, with very high mortality rates in developing countries. It was observed more than 20 years ago that some types of HPV were more frequent in malignant than in benign lesions, and infection with high-risk types of HPV is now considered the major risk factor for the devel‐ opment of cancer of the uterine cervix [1]. Oncogenic HPV types 16 and 18 and history of other concurrent sexually transmitted diseases were found to be significantly associated with

Studies have demonstrated a strong association between lifetime number of sexual part‐ ners and genital HPV acquisition. The acquisition of new sexual partners continues through‐ out all age groups. In addition, studies have shown consistently that the risk of cervical cancer can be predicted as much by a woman's own sexual behaviour as by the sexual behaviour of her husband/partner. The presence of HPV DNA in the penis and urethra of her sexual partner(s) is directly related to her HPV carrier status and therefore her risk of developing

Of the genital HPVs, which are sexually transmitted, 15 are categorized as high risk and are considered the causative agents of most cervical cancers, with over 99% of cervical lesions containing viral sequences [1]. The remaining viral types are rarely found in malignancies. High-risk HPVs (HR-HPV) are also associated with many vulvar, anal and penile carcinomas and contribute to oral cancers [17], 2006). Additionally, these cancers, in contrast to cervical cancer, appear to be preferentially associated with HPV16 [11,14]. For instance, in the subset of penile cancer attributed to HPV infection, HPV 16 was found in 60,23% of cases [18]. A vaccine has recently been introduced that can prevent the initial infection by two of these highrisk types, HPV 16 and 18, which are responsible for about 70% of cervical cancers [19].

Additional, many studies have demonstrated the direct role of HPV infection in the develop‐ ment of several human cancers [12-15,21]. HPV 16 and HPV 18 are the most frequently found HPV types in cervical cancers worldwide, being detected in approximately 50 and 20% of the cases, respectively [2-22]. For this reason, the majority of the biological studies were focused on these two HPV types.

The viral genomes are replicated in synchrony with cellular DNA replication. After cell division, one daughter cell migrates away from the basal layer and undergoes differentiation. Differentiation of HPV-positive cells induces the productive phase of the viral life cycle, which requires cellular DNA synthesis machinery. The expression of E6 and E7 deregulates cell cycle control, pushing differentiating cells into S phase, allowing viral genome amplification in cells that normally would have exited the cell cycle. The late-phase L1 and L2 proteins encapsidate newly synthesized viral genomes and virions are shed from the uppermost layers of the epithelium [19].

The induction of hyperproliferation by the E7 oncoprotein triggers apoptosis, which is blocked by the actions of the E6 oncoprotein. The cooperative actions of E6 and E7 efficiently immor‐ talize cells and this process is augmented by the actions of the E5 protein. The ability of E6 and E7 to target crucial regulators of proliferation, apoptosis, immortalization and genomic stability collectively promotes the emergence of a clonal population of cells with a growth advantage and an increased propensity for transformation and malignant progression [19].

The best-characterized HPV 16 E6 activity is its ability to induce degradation of the tumor suppressor protein p53 via the ubiquitin pathway. This cellular protein is a transcription factor that can trigger cell cycle arrest or apoptosis in response to a large variety of cellular stresses, such as hypoxia or DNA damages. Overall, the role of p53 is to ensure the integrity of the cellular genome, preventing cell division after DNA damage or delaying it until the damage has been repaired. The induction of p53 degradation appears to be an exclusive feature of E6 proteins from the HR-HPV types [23].

Codon 72 polymorphism on the 4th exon of TP53 is involved in multiple steps of carcinogenesis and may also account for genetic differences in susceptibility to cancer [24-26]. This most common polymorphism results in a non-conservative change at codon 72 of an arginine to a proline within a proline - rich region of p53 which is known to be important for the growth suppression and apoptotic functions [25]. It has been demonstrated that the TP53 polymor‐ phism varies according to ethnic and geographical distribution, like most human genetic polymorphisms [24].

acceptable method for managing women over the age of 20 years with ASCUS, but HPV-DNA testing is the preferred approach. Several population-based studies have established that tests for HR-HPV DNA have higher clinical sensitivity than cytology in detecting cervical intrae‐ pithelial neoplasia (CIN) of grades 2 and above (CIN2+), and that combined HPV and cytology testing shows the highest negative predictive values (NPV) for CIN2+ [31-69]. Thus, the HPV DNA test should be used in conjunction with Pap's smear test wherever feasible and affordable or potentially as a stand-alone test [32], because both combined has been shown to greatly

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31

In general, the prevalence of HPV is higher in young women compared to women over 30 years [34-35]. Most HPV infections are transient [9,36] and natural history studies have shown that HPV DNA is detectable in cells from the cervix for less than a year in most infected women. Therefore, the presence or absence of HPV DNA at a single time point is a poor indicator of lifetime exposure. To mitigate this problem, serological tools to detect HPV antibodies have

However, the serological assays have only limited accuracy and HPV cannot be grown in conventional cell cultures. As infection with HPV is followed by a humoral immune response against the major capsid protein [37], with antibodies remaining detectable for many years, serology is not suitable for distinguishing present and past infections. Consequently, accurate

The Hybrid Capture Assay I (HC1) was first introduced by Digene in 1995 [39]. HC1 was a relatively fast, liquid hybridization assay designed to detect 14 HPV types divided into highrisk types (HPV 16, 18, 31, 33, 35, 45, 51, 52 and 56) and low-risk types (HPV 6, 11, 42, 43, and 44). Initially, this was to be used to augment the sensitivity of conventional Pap testing and to

The second generation of Hybrid Capture Asssay, the hybrid capture-II (HC2) DNA test - Digene (Now Qiagen, Valencia, Calif Gaithersburg, MD, USA), which uses a micro titer plate instead of tubes and has been approved by the US Food and Drug Administration (FDA) for DNA- HPV identification [40], as an adjunct to cervical screening in the US women in aged 30 years and over. HC2 is a semiquantitative measure of viral load relative to 1pg/ml and uses RNA-labeled probes for targeting DNA sequences from 13 high-risk HPV (16, 18, 31, 33, 35,

Although its use has become the standard in many countries, it has several limitations including the inability to identify specific types and the lack of internal control for the amount of input DNA 7,40-42]. In addition, the reliance on sample volume as a proxy for cellularity may give rise to false negatives in samples with few infected cells. The lack of a negative control within the test also prevents detection of false negatives due to procedural or reagent problems. Another source of concern is the fact that the HR-HPV probe set is not all-inclusive. Women with unusual types may have a true-negative HC2 test yet still harbor a virus capable of causing

Studies comparing HC2 and Polymarese Chain Reaction (PCR) results have also shown that the hybrid capture probes are not entirely type specific [43]. The detection limit of approxi‐

provide a meaningful negative predictive value for assessing cervical dysplasia [20].

39, 45, 51, 52, 56, 58, 59, and 68) 5 low-risk types (6, 11, 42, 43 and 44) [7,40-41].

diagnosis of HPV infection relies on the detection of viral nucleic acid [9,38].

improve the ability to detect pre-cancerous states [33].

been developed.

cancer [43].

A meta-analysis of such studies revealed that the arginine homozygous genotype is associated with an increased risk of invasive cervical cancers, but not with squamous intraepithelial lesions supporting the hypothesis that the p53 codon 72 polymorphism may have a principal role in progression to HPV-related cancer, rather than in initiation of the neoplasia [27].

For prevention and early detection of cervical cancer, it is important to detect not only cervical intraepithelial changes, but also to identify the presence of HR-HPV and its type as well. If the results of HR-HPV test are positive, the possibility of cervical intraepithelial neoplasia (CIN) can be prognosticated even if there are no cytologic changes in the cervix. The possibility for regression of CIN 2 cervical changes caused by HPV type 16 is lower compared with cervical changes caused by other HR-HPV types. The risk of mild cervical changes leading to severe cervical changes (CIN 3) is higher when detecting HR-HPV types, especially HPV type 16, compared with lower-risk HPV types. HR-HPV-positive women, even without cytologic changes, have a 210-fold higher risk of developing CIN 3 in 6 years as compared with HR-HPV-negative women [28].

Starting in the late 1960s significant advances were made in understanding the cellular changes leading to invasive cervical cancer, but it was not until 1976 that Meisels and Fortin first established HPV as the etiologic agent in an abnormal cervical cytologic finding (koilocytotic atypia) [20]. The advent of screening to identify and treat cervical cancer precursor lesions, CIN, has led to a substantial reduction in the incidence of cervical cancer in those countries where routine screening is in place. Conversely, most cervical cancer-related mortality occurs in countries where there is no routine cervical screening, although Pap's smear is a cost effective screening method in low resource settings [10].

Cervical screening is considered to have been the most effective cancer-screening test ever introduced and in developed countries with organized programmes. A successful screening programme however is dependent on understanding and acceptance of the need for a particular test, the need for further investigation of abnormalities and particularly, the need for quality assurance of all parts of the system [29].

In the evaluation by IARC in 2005 was concluded that there is sufficient evidence that screening women ages 35 to 64 for cervical cancer precursors by conventional cytology every 3 to 5 years within high-quality programs reduces incidence of invasive cervical cancer by at least 80% among those screened [6]. Despite the large amount of data available on the value of HPV-DNA testing for the detection of cervical cancer precursors, both in the primary cervical screening and in the management of 'borderline' or Atypical Squamous Cells of Undetermined Significance (ASCUS) cytology, HPV-DNA tests have not always and correctly been translated into clinical practice by clinicians and within national cervical screening programs [30-31].

Although Pap's smears have reduced the incidence of cervical cancer by over 80% in the United States, cervical cancer is the second leading cause of cancer deaths in women worldwide, and effective implementation of the HPV vaccine and continued screening should dramatically reduce the incidence of these cancers [19]. Cervical cytologic testing or colposcopy is an acceptable method for managing women over the age of 20 years with ASCUS, but HPV-DNA testing is the preferred approach. Several population-based studies have established that tests for HR-HPV DNA have higher clinical sensitivity than cytology in detecting cervical intrae‐ pithelial neoplasia (CIN) of grades 2 and above (CIN2+), and that combined HPV and cytology testing shows the highest negative predictive values (NPV) for CIN2+ [31-69]. Thus, the HPV DNA test should be used in conjunction with Pap's smear test wherever feasible and affordable or potentially as a stand-alone test [32], because both combined has been shown to greatly improve the ability to detect pre-cancerous states [33].

phism varies according to ethnic and geographical distribution, like most human genetic

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

A meta-analysis of such studies revealed that the arginine homozygous genotype is associated with an increased risk of invasive cervical cancers, but not with squamous intraepithelial lesions supporting the hypothesis that the p53 codon 72 polymorphism may have a principal role in progression to HPV-related cancer, rather than in initiation of the neoplasia [27].

For prevention and early detection of cervical cancer, it is important to detect not only cervical intraepithelial changes, but also to identify the presence of HR-HPV and its type as well. If the results of HR-HPV test are positive, the possibility of cervical intraepithelial neoplasia (CIN) can be prognosticated even if there are no cytologic changes in the cervix. The possibility for regression of CIN 2 cervical changes caused by HPV type 16 is lower compared with cervical changes caused by other HR-HPV types. The risk of mild cervical changes leading to severe cervical changes (CIN 3) is higher when detecting HR-HPV types, especially HPV type 16, compared with lower-risk HPV types. HR-HPV-positive women, even without cytologic changes, have a 210-fold higher risk of developing CIN 3 in 6 years as compared with HR-

Starting in the late 1960s significant advances were made in understanding the cellular changes leading to invasive cervical cancer, but it was not until 1976 that Meisels and Fortin first established HPV as the etiologic agent in an abnormal cervical cytologic finding (koilocytotic atypia) [20]. The advent of screening to identify and treat cervical cancer precursor lesions, CIN, has led to a substantial reduction in the incidence of cervical cancer in those countries where routine screening is in place. Conversely, most cervical cancer-related mortality occurs in countries where there is no routine cervical screening, although Pap's smear is a cost

Cervical screening is considered to have been the most effective cancer-screening test ever introduced and in developed countries with organized programmes. A successful screening programme however is dependent on understanding and acceptance of the need for a particular test, the need for further investigation of abnormalities and particularly, the need

In the evaluation by IARC in 2005 was concluded that there is sufficient evidence that screening women ages 35 to 64 for cervical cancer precursors by conventional cytology every 3 to 5 years within high-quality programs reduces incidence of invasive cervical cancer by at least 80% among those screened [6]. Despite the large amount of data available on the value of HPV-DNA testing for the detection of cervical cancer precursors, both in the primary cervical screening and in the management of 'borderline' or Atypical Squamous Cells of Undetermined Significance (ASCUS) cytology, HPV-DNA tests have not always and correctly been translated into clinical practice by clinicians and within national cervical screening programs [30-31]. Although Pap's smears have reduced the incidence of cervical cancer by over 80% in the United States, cervical cancer is the second leading cause of cancer deaths in women worldwide, and effective implementation of the HPV vaccine and continued screening should dramatically reduce the incidence of these cancers [19]. Cervical cytologic testing or colposcopy is an

polymorphisms [24].

HPV-negative women [28].

effective screening method in low resource settings [10].

for quality assurance of all parts of the system [29].

In general, the prevalence of HPV is higher in young women compared to women over 30 years [34-35]. Most HPV infections are transient [9,36] and natural history studies have shown that HPV DNA is detectable in cells from the cervix for less than a year in most infected women. Therefore, the presence or absence of HPV DNA at a single time point is a poor indicator of lifetime exposure. To mitigate this problem, serological tools to detect HPV antibodies have been developed.

However, the serological assays have only limited accuracy and HPV cannot be grown in conventional cell cultures. As infection with HPV is followed by a humoral immune response against the major capsid protein [37], with antibodies remaining detectable for many years, serology is not suitable for distinguishing present and past infections. Consequently, accurate diagnosis of HPV infection relies on the detection of viral nucleic acid [9,38].

The Hybrid Capture Assay I (HC1) was first introduced by Digene in 1995 [39]. HC1 was a relatively fast, liquid hybridization assay designed to detect 14 HPV types divided into highrisk types (HPV 16, 18, 31, 33, 35, 45, 51, 52 and 56) and low-risk types (HPV 6, 11, 42, 43, and 44). Initially, this was to be used to augment the sensitivity of conventional Pap testing and to provide a meaningful negative predictive value for assessing cervical dysplasia [20].

The second generation of Hybrid Capture Asssay, the hybrid capture-II (HC2) DNA test - Digene (Now Qiagen, Valencia, Calif Gaithersburg, MD, USA), which uses a micro titer plate instead of tubes and has been approved by the US Food and Drug Administration (FDA) for DNA- HPV identification [40], as an adjunct to cervical screening in the US women in aged 30 years and over. HC2 is a semiquantitative measure of viral load relative to 1pg/ml and uses RNA-labeled probes for targeting DNA sequences from 13 high-risk HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) 5 low-risk types (6, 11, 42, 43 and 44) [7,40-41].

Although its use has become the standard in many countries, it has several limitations including the inability to identify specific types and the lack of internal control for the amount of input DNA 7,40-42]. In addition, the reliance on sample volume as a proxy for cellularity may give rise to false negatives in samples with few infected cells. The lack of a negative control within the test also prevents detection of false negatives due to procedural or reagent problems. Another source of concern is the fact that the HR-HPV probe set is not all-inclusive. Women with unusual types may have a true-negative HC2 test yet still harbor a virus capable of causing cancer [43].

Studies comparing HC2 and Polymarese Chain Reaction (PCR) results have also shown that the hybrid capture probes are not entirely type specific [43]. The detection limit of approxi‐ mately 5000 genome is the power of HC2 and it makes it less sensitive than PCR and crossreactivity of the two probe cocktails can reduce the clinical relevance of a positive result [20,40].

is transcribed from HPV DNA that exists either as circular, extrachromosomal episomes or as DNA that is integrated into the chromosome. As pre-malignant lesions progress, the abun‐ dance of mRNAs that encode the oncogenes, such as E6 and E7 (E6–7), may increase and the mRNAs encoding non-oncogenic HPV proteins, such as E2 and L1, may decrease [49]. The incidence of HPV integration may reduce further transcripts encoding E2 and other down‐ stream genes, such as L1, because integration usually occurs at the E2 loci [50]. The E2 gene productisanimportantdown-regulatorofoncogenicE6–7expression[19].Thus,lowerE2levels may correlate with disease progression. Therefore, it may be useful to measure the ratio of E6– 7overE2transcriptsincervicalspecimensandcomparethisratiowiththeseverityofdisease[51]. In the study [48] was compared the performance of a prototype version of the Hybrid Capture 3 (HC3) human papillomavirus (HPV) DNA assay to the current generation Hybrid Capture 2 (HC2) assay, both of which target 13 oncogenic HPV types, for the detection of cervical intraepi‐ thelial neoplasia grade 3 and cancer (CIN3+) with cervicovaginal lavage specimens collected at enrollmentintoa 10-year cohort studyatKaiserPermanente.The authors suggestthatHC3may be a slightly more sensitive, equally specific test for the detection of CIN3+ over the duration of typical screening intervals compared to its predecessor, HC2. The increased sensitivity of HC3 compared to HC2 appears to be the result of increased detection of CIN3+ in women who were 30 years of age or older and were cytologically negative. They emphasize that further valida‐

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33

tion studies of HC3 are needed with more clinically relevant cervical specimens.

cervical samples in screening [20].

widespread adoption of this approach [43].

Diagnosis of HPV infection relies on the detection of the viral DNA in clinical samples; thus accurate detection and genotyping of HPV are of critical importance for determining the prevalence of HPVs in a given population and for determining the risks associated with infections of a particular type [52]. The first evaluation of the use of HPV testing in a potential clinical application was published in 1989 by Tidy et al. on the detection of HPV 16 by PCR both in normal and in dyskaryotic smears from 21 women. The results was sufficiently compelling to predict that HPV testing might eventually supplement cytologic analysis of

PCR is a highly sensitive technique and allows testing on samples with less tissue or fewer cells [7,46]. Multiplex HPV genotyping methods based on hybridization to fluorescently labeled beads have also been reported [53]. These methods are capable of detecting very small copy numbers of virus, and extremely high sensitivities have been reported. However, because HC2 has been established as the criterion standard for clinical treatment algorithms, such high positive rates are actually a drawback rather than an advantage for clinical use. The relative complexity of PCR-based methods, concerns about contamination of the laboratory by PCR products, and patent protection on some aspects of the technology have further hindered

The sensitivity and specificity of PCR based methods vary, depending mainly on the primer set, the size of the PCR product, the reaction conditions and efficacy of the DNA polymerase used in the reaction, the spectrum of HPV types amplified, the ability to detect multiple types and the availability of a type-specific assay. PCR can theoretically produce 109 copies from a single double stranded DNA molecule after 30 cycles of amplification. Therefore, care must be taken to avoid false-positive results derived from cross-contaminated specimens or

Despite the HC2 present some limitations [33], this assay has shown high sensitivity for the detection of CIN of grade 2 and worse (CIN2+) and it was recently recommended to be used as a benchmark for performance assessment of new candidate HPV tests for primary cervical cancer screening in women of 30 years and older. The emergence of competing platforms like the Linear Array (Roche) and INNO-LiPA HPV (Innogenetics) has led to the development of some new elements by Qiagen that are designed to counteract perceived advantages of alternative tests [43]. This others platforms are commonly used in the HPV typing assays and follow up of persistent infections to monitor the presence of specific HPV genotypes [44].

Has been demonstrated that HC2 has proven its effectiveness in large clinical trials and everyday practice, but the search continues for markers with superior specificity for highgrade disease without excessive corresponding loss of sensitivity. A number of new assays have been developed for molecular and immunostaining platforms with the intention of meeting this need [43].

As a screening test, cervical cytology for CIN has reduced the incidence of mortality worldwide [45], however this methodology has a limited sensitivity. So, as described by [46] a shift from conventional cytology to a molecular approach integrated into cervical cancer screening is the most likely solution to the goals of improved screening in both the developed and developing world. Therefore, molecular tests have become available for clinical and research purposes in response to the need for identifying infection during earlier stages and improving patient follow-up [9,40].

A modified, experimental Hybrid Capture assay named Hybrid Capture 3 uses RNA probes, as in Hybrid Capture 2, but in combination with biotinylated capture oligonucleotides that are directed to unique sequence regions within the desired target to increase test specificity [47]. The assay has been developed further to reduce cross-reactivity while maintaining sensitivity and for use either on DNA or RNA as targets. A recent comparison study concluded that, at the optimal cut-off points, Hybrid Capture 2 and 3 had similar screening performance characteristics for high-grade lesions diagnosed at the enrolment visit [48].

Hybrid Capture 3 (HC3) is being evaluated as the next generation of hybrid capture clinical assays that target 13 oncogenic HPV types for the detection of cervical precancerous cervical intraepithelial neoplasia grade 3 (CIN3). A primary technical distinction between HC3 and HC2 is that HC3 employs a biotinylated DNA oligonucleotide specific for selected HPV DNA sequences for the capture of the DNA-RNA complexes on streptavidin-coated wells, whereas HC2 uses wells coated with polyclonal antibody against DNA-RNA complexes for hybrid capture. The use of capture oligonucleotide instead of an immobilized antibody also dimin‐ ishes the possibility of nonspecific RNA-DNA hybrids, present as the result of improperly alkali-denatured specimens, from binding to the microplate well and consequently may reduce false positivity for HC3 compared to HC2 [48].

This ratio assaymay reflectthenaturalpatternof HPVmRNAexpressionthat occursduring the progression of disease in cervical epithelium. After initial infection, polycistronic, pre-mRNA

is transcribed from HPV DNA that exists either as circular, extrachromosomal episomes or as DNA that is integrated into the chromosome. As pre-malignant lesions progress, the abun‐ dance of mRNAs that encode the oncogenes, such as E6 and E7 (E6–7), may increase and the mRNAs encoding non-oncogenic HPV proteins, such as E2 and L1, may decrease [49]. The incidence of HPV integration may reduce further transcripts encoding E2 and other down‐ stream genes, such as L1, because integration usually occurs at the E2 loci [50]. The E2 gene productisanimportantdown-regulatorofoncogenicE6–7expression[19].Thus,lowerE2levels may correlate with disease progression. Therefore, it may be useful to measure the ratio of E6– 7overE2transcriptsincervicalspecimensandcomparethisratiowiththeseverityofdisease[51].

mately 5000 genome is the power of HC2 and it makes it less sensitive than PCR and crossreactivity of the two probe cocktails can reduce the clinical relevance of a positive result [20,40]. Despite the HC2 present some limitations [33], this assay has shown high sensitivity for the detection of CIN of grade 2 and worse (CIN2+) and it was recently recommended to be used as a benchmark for performance assessment of new candidate HPV tests for primary cervical cancer screening in women of 30 years and older. The emergence of competing platforms like the Linear Array (Roche) and INNO-LiPA HPV (Innogenetics) has led to the development of some new elements by Qiagen that are designed to counteract perceived advantages of alternative tests [43]. This others platforms are commonly used in the HPV typing assays and follow up of persistent infections to monitor the presence of specific HPV genotypes [44].

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

Has been demonstrated that HC2 has proven its effectiveness in large clinical trials and everyday practice, but the search continues for markers with superior specificity for highgrade disease without excessive corresponding loss of sensitivity. A number of new assays have been developed for molecular and immunostaining platforms with the intention of

As a screening test, cervical cytology for CIN has reduced the incidence of mortality worldwide [45], however this methodology has a limited sensitivity. So, as described by [46] a shift from conventional cytology to a molecular approach integrated into cervical cancer screening is the most likely solution to the goals of improved screening in both the developed and developing world. Therefore, molecular tests have become available for clinical and research purposes in response to the need for identifying infection during earlier stages and improving patient

A modified, experimental Hybrid Capture assay named Hybrid Capture 3 uses RNA probes, as in Hybrid Capture 2, but in combination with biotinylated capture oligonucleotides that are directed to unique sequence regions within the desired target to increase test specificity [47]. The assay has been developed further to reduce cross-reactivity while maintaining sensitivity and for use either on DNA or RNA as targets. A recent comparison study concluded that, at the optimal cut-off points, Hybrid Capture 2 and 3 had similar screening performance

Hybrid Capture 3 (HC3) is being evaluated as the next generation of hybrid capture clinical assays that target 13 oncogenic HPV types for the detection of cervical precancerous cervical intraepithelial neoplasia grade 3 (CIN3). A primary technical distinction between HC3 and HC2 is that HC3 employs a biotinylated DNA oligonucleotide specific for selected HPV DNA sequences for the capture of the DNA-RNA complexes on streptavidin-coated wells, whereas HC2 uses wells coated with polyclonal antibody against DNA-RNA complexes for hybrid capture. The use of capture oligonucleotide instead of an immobilized antibody also dimin‐ ishes the possibility of nonspecific RNA-DNA hybrids, present as the result of improperly alkali-denatured specimens, from binding to the microplate well and consequently may reduce

This ratio assaymay reflectthenaturalpatternof HPVmRNAexpressionthat occursduring the progression of disease in cervical epithelium. After initial infection, polycistronic, pre-mRNA

characteristics for high-grade lesions diagnosed at the enrolment visit [48].

false positivity for HC3 compared to HC2 [48].

meeting this need [43].

follow-up [9,40].

In the study [48] was compared the performance of a prototype version of the Hybrid Capture 3 (HC3) human papillomavirus (HPV) DNA assay to the current generation Hybrid Capture 2 (HC2) assay, both of which target 13 oncogenic HPV types, for the detection of cervical intraepi‐ thelial neoplasia grade 3 and cancer (CIN3+) with cervicovaginal lavage specimens collected at enrollmentintoa 10-year cohort studyatKaiserPermanente.The authors suggestthatHC3may be a slightly more sensitive, equally specific test for the detection of CIN3+ over the duration of typical screening intervals compared to its predecessor, HC2. The increased sensitivity of HC3 compared to HC2 appears to be the result of increased detection of CIN3+ in women who were 30 years of age or older and were cytologically negative. They emphasize that further valida‐ tion studies of HC3 are needed with more clinically relevant cervical specimens.

Diagnosis of HPV infection relies on the detection of the viral DNA in clinical samples; thus accurate detection and genotyping of HPV are of critical importance for determining the prevalence of HPVs in a given population and for determining the risks associated with infections of a particular type [52]. The first evaluation of the use of HPV testing in a potential clinical application was published in 1989 by Tidy et al. on the detection of HPV 16 by PCR both in normal and in dyskaryotic smears from 21 women. The results was sufficiently compelling to predict that HPV testing might eventually supplement cytologic analysis of cervical samples in screening [20].

PCR is a highly sensitive technique and allows testing on samples with less tissue or fewer cells [7,46]. Multiplex HPV genotyping methods based on hybridization to fluorescently labeled beads have also been reported [53]. These methods are capable of detecting very small copy numbers of virus, and extremely high sensitivities have been reported. However, because HC2 has been established as the criterion standard for clinical treatment algorithms, such high positive rates are actually a drawback rather than an advantage for clinical use. The relative complexity of PCR-based methods, concerns about contamination of the laboratory by PCR products, and patent protection on some aspects of the technology have further hindered widespread adoption of this approach [43].

The sensitivity and specificity of PCR based methods vary, depending mainly on the primer set, the size of the PCR product, the reaction conditions and efficacy of the DNA polymerase used in the reaction, the spectrum of HPV types amplified, the ability to detect multiple types and the availability of a type-specific assay. PCR can theoretically produce 109 copies from a single double stranded DNA molecule after 30 cycles of amplification. Therefore, care must be taken to avoid false-positive results derived from cross-contaminated specimens or reagents. Several procedures are available to avoid the potential problems of using PCR protocols for HPV DNA detection [30].

PGMY09/11 PCR, 22 HPV types with GP5+/6+ PCR 15 HPV types with YBT L1/GP-1 PCR, and 18 HPV types with GP5+/6+ PCR. These multiplex HPV genotyping (MPG) methods have been compared with other well established HPV detection methods such as HC2, restriction fragment length polymorphism (RFLP) and DNA chip technology for the evaluation of their performance. However, none of these well established assays are perfect and suitable for the "gold standard". Sequencing gives the most conclusive genotype information, although it is the most labor intensive. However, sequencing is the most desirable way to validate HPV

> Sandwich capture molecular

PCR/reverse

PCR/reverse

hybdridization line blot

hybdridization line blot

**detected**

Molecular Tools for Detection Human Papillomavirus

http://dx.doi.org/10.5772/55710

35

Invader chemistry DNA, *L1*, *E6, E7* Hologic (WI, USA)

Real-time PCR DNA, *L1* Roche (Rotkreuz,

Real-time PCR DNA, *L1* Abbott (IL, USA)

PCR/microarray DNA, *L1* Genomica

PCR/microarray DNA, *L1* Greiner BioOne

DNA, *L1* Qiagen (MD,

DNA, *L1* Innogenetics

DNA, *L1* Innogenetics

USA)

Switzerland)

(Madrid, Spain)

(Gent, Belgium)

(Gent, Belgium)

(Frickenhaunsen, Germany)

**Producer**

**Assay name HPV genotypes detected Detection technology DNA and HPV gene**

genotyping methods [62].

High-risk

8 Low –risk 6, 11, 42, 43, 44

16,18,31,33,35,39,45,51,52,56,58,59,6

16, 18 and bulk (31, 33, 35, 39, 45, 51,

16, 18 and bulk (31, 33, 35, 39, 45, 51,

43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84,

45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 71, 71, 72, 73, 81, 82,

6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68,

44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68,

Cervista 16, 18 and bulk (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68)

52, 56, 58, 59, 66, 68)

52, 56, 58, 59, 66, 68)

Clinical Array 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42,

Linear Array 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42,

69, 70, 71, 73, 74, 82.

PapilloCheck 6,11, 16, 18, 31, 33, 35, 39, 40, 42, 43,

85, 89.

83, 84.

70, 73, 82

**Table 1.** Commercial HPV-DNA assays

Hybrid capture 2 (HC2)

Cobas HPV test

Abbott´s Real Time High Risk

HPV

INNO-LiPA Genotyping Extra

\* Modified from [62]

The limitations associated with PCR-based HPV-DNA detection are related to primer selec‐ tion and optimal protocol standardization [46,54]. The PCR assay most likely to be amendable to broad screening and/or surveillance applications are based on consensus primer amplifica‐ tion of a broad spectrum of genotypes which are subsequently differentiated by type-specific oligonucleotide probe hybridization. The most commonly used consensus PCR target is the highly conserved L1 open reading frame (ORF), of genital HPV genomes [52]. Among these are the singlepair of consensusprimersGP5/6 [55] andits extendedversionGP5+/6+ [35,56] andthe MY09/11 degenerate primers [57] and its modified version, PGMY09/11 [30,58] and SPF 10 [59]. In addition, none of these assays can be automated or deployed on a high-throughput plat‐ form,features that are essentialfor anassayintendedforusewitha largevolumeofpatients [60].

The viral nucleic acid must be preserved to avoid false-negative results caused by degradation by endogenous endonucleases. This is especially important when analyzing HPV-RNA transcripts. To assess the integrity of genomic DNA in the specimen and its suitability for molecular analysis, adequate controls, such as β-globin gene amplification or spiking of the samplewithknownpositivematerial, are crucial.Several commerciallyavailable samplingkits, originallyintendedforcytology(e.g.PreservCyt,CytycCorp.)adequatelypreservenucleicacids for molecular diagnosis even after prolonged storage at ambient temperatures [61].

The commercial HPV assays are based on L1 or E1 PCR for high-risk HPV DNA detection and genotyping are now available from different companies: AmplicorR and Linear Array (Roche Molecular Systems, CA, USA), INNO-LiPAR (Inngenetics, Ghent, Belgium), PapiloCheck (Greiner Bio-One GmbH, Germany), Multiplex HPV Genotyping Kit (Multimetrix GmmbH, Heildelberg, Germany) [Table 01]. After amplifying parts of the L1 region, such assays hybridize the resulting PCR product to a detection and visualization unit (microarray or a reverse line blot) [62].

HPV-DNA assays can be performed using the same specimen as used for cytological exami‐ nation, which is an important logistic aspect of routine clinical testing. However, a cervical scrape is only a small sample of the cervical epithelium and sampling errors may influence cytology grading. Only a portion of the cervical cell suspension is used for DNA isolation with only a fraction of the isolated DNA being used for specific DNA detection. Therefore, if a specimen only contains a limited number of HPV-DNA copies, sampling errors may produce inconsistencies even in a sensitive assay. Furthermore, the outcome of a HPV-DNA assay can vary depending on the menstrual cycle [9].

Luminex (xMAP) suspension array technology is based on polystyrene beads that are inter‐ nally dyed with various ratios of two spectrally distinct fluorophores. Different molecules such as individual oligonucleotide probes can be coupled to different bead sets with specific absorption spectra. These sets are combined to a suspension array and allow up to 100 different probes to be measured simultaneously in a single reaction (multiplexing) [62].

This technology can potentially be fully automated, dramatically decreasing the personal cost component of the assay; this assay has been used for the genotyping of 45 HPV types with PGMY09/11 PCR, 22 HPV types with GP5+/6+ PCR 15 HPV types with YBT L1/GP-1 PCR, and 18 HPV types with GP5+/6+ PCR. These multiplex HPV genotyping (MPG) methods have been compared with other well established HPV detection methods such as HC2, restriction fragment length polymorphism (RFLP) and DNA chip technology for the evaluation of their performance. However, none of these well established assays are perfect and suitable for the "gold standard". Sequencing gives the most conclusive genotype information, although it is the most labor intensive. However, sequencing is the most desirable way to validate HPV genotyping methods [62].


**Table 1.** Commercial HPV-DNA assays

reagents. Several procedures are available to avoid the potential problems of using PCR

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

The limitations associated with PCR-based HPV-DNA detection are related to primer selec‐ tion and optimal protocol standardization [46,54]. The PCR assay most likely to be amendable to broad screening and/or surveillance applications are based on consensus primer amplifica‐ tion of a broad spectrum of genotypes which are subsequently differentiated by type-specific oligonucleotide probe hybridization. The most commonly used consensus PCR target is the highly conserved L1 open reading frame (ORF), of genital HPV genomes [52]. Among these are the singlepair of consensusprimersGP5/6 [55] andits extendedversionGP5+/6+ [35,56] andthe MY09/11 degenerate primers [57] and its modified version, PGMY09/11 [30,58] and SPF 10 [59]. In addition, none of these assays can be automated or deployed on a high-throughput plat‐ form,features that are essentialfor anassayintendedforusewitha largevolumeofpatients [60]. The viral nucleic acid must be preserved to avoid false-negative results caused by degradation by endogenous endonucleases. This is especially important when analyzing HPV-RNA transcripts. To assess the integrity of genomic DNA in the specimen and its suitability for molecular analysis, adequate controls, such as β-globin gene amplification or spiking of the samplewithknownpositivematerial, are crucial.Several commerciallyavailable samplingkits, originallyintendedforcytology(e.g.PreservCyt,CytycCorp.)adequatelypreservenucleicacids

for molecular diagnosis even after prolonged storage at ambient temperatures [61].

The commercial HPV assays are based on L1 or E1 PCR for high-risk HPV DNA detection and genotyping are now available from different companies: AmplicorR and Linear Array (Roche Molecular Systems, CA, USA), INNO-LiPAR (Inngenetics, Ghent, Belgium), PapiloCheck (Greiner Bio-One GmbH, Germany), Multiplex HPV Genotyping Kit (Multimetrix GmmbH, Heildelberg, Germany) [Table 01]. After amplifying parts of the L1 region, such assays hybridize the resulting PCR product to a detection and visualization unit (microarray or a

HPV-DNA assays can be performed using the same specimen as used for cytological exami‐ nation, which is an important logistic aspect of routine clinical testing. However, a cervical scrape is only a small sample of the cervical epithelium and sampling errors may influence cytology grading. Only a portion of the cervical cell suspension is used for DNA isolation with only a fraction of the isolated DNA being used for specific DNA detection. Therefore, if a specimen only contains a limited number of HPV-DNA copies, sampling errors may produce inconsistencies even in a sensitive assay. Furthermore, the outcome of a HPV-DNA assay can

Luminex (xMAP) suspension array technology is based on polystyrene beads that are inter‐ nally dyed with various ratios of two spectrally distinct fluorophores. Different molecules such as individual oligonucleotide probes can be coupled to different bead sets with specific absorption spectra. These sets are combined to a suspension array and allow up to 100 different

This technology can potentially be fully automated, dramatically decreasing the personal cost component of the assay; this assay has been used for the genotyping of 45 HPV types with

probes to be measured simultaneously in a single reaction (multiplexing) [62].

protocols for HPV DNA detection [30].

reverse line blot) [62].

vary depending on the menstrual cycle [9].
