**9. Progression markers**

#### **9.1. HPV viral load**

HPV viral load is a product of the number of cells infected and number of viruses per infected cell and is therefore influenced by two main factors: (i) the extent of an HPV infection on the cervical surface and (ii) the level of viral production in the area of infection. Viral load has been suggested to be a potential biomarker for cervical intraepithelial neoplasia grade 2 (CIN2) or greater, but currently there is no consistent evidence that a one-time measurement of viral load is a useful marker of prevalent disease or disease progression [101]. A widespread productive infection might be associated with high viral load, while a small incipient CIN3 with low-level virus production might be associated with low viral load. Furthermore, viral load in a cytological sample is subject to sampling variation in which there are varying proportions of lesional cells, normal epithelial cells, inflammatory exudate, and blood. A further complication in using viral load to predict neoplasia of CIN2 or greater is the high prevalence of multiple carcinogenic HPV infections detected in cervical samples. The current paradigm is that cervical lesions clonally expand following infection with one specific genotype (one virus-one lesion concept). On the cervical surface, multiple independent infections or lesions may occur that are caused by different genotypes. Without specific genotyping conducted *in situ*, assigning a causal HPV genotype to a specific lesion can only be based on assumptions [102]. HPV16 is the only genotype for which there is some indication that viral load may predict viral persis‐ tence and progression to precancer [103-105].

transcriptase real time PCR [95] or by nucleic acid sequence-based amplification (NASBA). For the latter, three commercially available assays that detect E6/E7 transcripts are currently available. PreTect HPV-Proofer (HPV-Proofer; NorChip, Klokkarstua, Norway) is an assay based on NASBA technology, which allows qualitative determination of E6/E7 mRNA tran‐ scriptsofthefivemostfrequentlyidentifiedhr-HPVtypesincervicalcancerworldwide:HPV-16, HPV-18, HPV-31, HPV-33 and HPV-45 [96]. The NucliSENS EasyQ HPV V1 assay (NucliS‐ ENS; bioMérieux) was launched in 2007 and is based on the original HPV-Proofer assay, except for the proprietary hardware platform and the software for NASBA measurements and data analysis[97].APTIMAHPVAssay(APTIMA;Gen-Probe,SanDiego,CA,USA)isatranscriptionmediated amplification-based assay, which allows the detection of E6/E7 mRNA transcripts of 14HPVtypes:HPV-16,HPV-18,HPV-31,HPV-33,HPV-35,HPV-39,HPV-45,HPV-51,HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. The assay generates a qualitative result for the presence/absenceof14targetedHPVsanddoesnotallowtheexactdeterminationofHPVtype(s) presentinaclinical specimen[98].APTIMAyieldedsimilar sensitivityforCIN2+comparedwith hc2,Amplicor andLinearArray(95.2vs 99.6%, 98.9%and98.2%,respectively),but a significant‐ ly higher specificity (42.2 vs 28.4%, 21.7% and 32.8%, respectively) [55]. In a comparative evaluation of APTIMA and hc2 on PreservCyt specimens collected from 800 women referred to colposcopy, APTIMA showed comparable sensitivity to hc2 for the detection of CIN2+ (91 vs 95%), as well as CIN3+ (98 vs 99%), but had higher clinical specificity (>55 vs 47% for CIN2+; 53 vs 44% for CIN3+) [99]. APTIMA had the best sensitivity/specificity balance measured by AUC (areaunderROCcurve) comparisontest(significantforCIN2+),andthe colposcopyreferralrate

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

*In situ* hybridization (ISH) is the only molecular method allowing reliable detection and identification of HPVs in topographical relation to their pathological lesions. Unlike other molecular methods, in ISH the whole HPV detection procedure occurs within the nuclei of infected cells and not on solid supports or in solutions. The result of the hybridization reaction is evaluated microscopically and the appearance of an appropriate precipitate within the nuclei of epithelial cells is indicative of the presence of HPVs in the specimen being tested. In addition, the physical state of the virus can be evaluated by the presence of punctuate signals for integrated virus and diffuse signals for episomal virus. Although commercially available HPV assays based on ISH have been validated technically, they are insufficiently clinically validat‐ ed. In addition, current ISH-based assays are considered by many experts in the field to be too laborious and to have insufficient clinical sensitivity to be used in routine screening [52].

HPV viral load is a product of the number of cells infected and number of viruses per infected cell and is therefore influenced by two main factors: (i) the extent of an HPV infection on the

(9.2%) comparable to that of liquid citology (8.7%) [100].

**8. In situ hybridization metods**

**9. Progression markers**

**9.1. HPV viral load**

Quantitating HPV viral load seems to be a rational strategy of identifying women at risk for persistent HPV infection and progression to high-grade dysplasia. Accordingly, a high viral load could represent many cells with few virions each or a few cells containing many virions. An inaccurate description of the viral biology and the possible implications for the host could result from this discrepancy. Recently, studies have focused on longitudinal observations of viral load to predict viral clearance or lesion progression [106, 107]. Initial data indicate that repeated measurements can improve prediction of persistence or clearance, but these data are, so far, limited to HPV16 only. Although signal intensities from HC2 or various endpoint PCRbased assays have been proposed and partly used as surrogates for viral load, these approaches have limitations [108, 109]. HC2 only gives aggregate signal strength for a pool of 13 carcino‐ genic types, and commercial genotyping assays, such as the Roche Linear Array (LA) or Innogenetics InnoLiPA (line probe assay), do not formally report quantitative results. Over‐ estimation of the presence of oncogenic HPV may result. Despite these caveats, the develop‐ ment of HPV viral load assays that may reliably be used as an adjunct screening tool to identify women at increased risk of progression to CIN 2+ and cervical cancer remains a promising tool in cervical cancer screening. Recently, Wentzensen et al [110] measuring signal intensities on LA HPV genotyping strips provides quantitative information comparable to viral load measurements based on Q-PCR. This approach offers the potential for viral load assessment for 37 types in parallel, simplifying conducting repeated measurements of viral load in epidemiologic studies and addressing the problems of multiple HPV genotype infections in studies of HPV load.

Screening for HPV integration into the host genome is a subcategory of HPV diagnostics. HPV integration is a key molecular event in the transition from an innocuous HPV infection to one that has oncogenic potential. Human papilloma virus integration results in increased expres‐ sion of the viral E6 and E7 proteins. Increased expression of these proteins ultimately results in the disruption of host cell proteins, p53 and retinoblastoma protein [111]. Tests that detect

the integration of HPV into the host cell and corresponding risk of CIN 2+ or cancer are in development, and may provide a useful way of screening women at risk for cervical cancer. Studies have shown that viral integrants are detected in 100% of HPV-18-positive and 70–80% of HPV-16-positive cases of cervical carcinoma [112, 113]. A smaller subset of HSIL (15%) and 0% of LSIL contain transcriptionally active viral integrants [111].

chromosome band 3q26), which seems to play a key role in progression from low-grade dysplasia to cancer [120]. Many studies indicate that TERC identification may become a useful screening tool for cervical cancer. A prospective study by Andersson et al. [121] found a correlation between increasing TERC detection in cytology specimens and higher grade of dysplasia. They showed that progression to cervical cancer is never seen without TERC amplification and that, conversely, specimens without extra copies of TERC were likely to

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 71

Other biomarkers under early evaluation for cervical cancer screening include CDC6 and MCM5. These proteins are present in normal cells only during the activation of the cell cycle and help form prereplicative DNA complexes during the G1 phase. They are absent from the cell during quiescence and differentiation. Dysplastic cells have unregulated cell cycles and, as a result, CDC6 and MCM5 reflect cell proliferation [122]. Studies indicate that CDC6 may be a biomarker of high-grade and invasive lesions of the cervix, with limited use in low-grade dysplasia. MCM5 seems to be a biomarker that is expressed independent of high-risk HPV infection, and may in the future serve as a useful marker for both HPV dependent and HPV-

General markers of cell proliferation like MIB-1, MCMs or ProEx C, and surrogate markers of high risk HPV infection like p16 INK4A have shown promising results. Other potential candidates need to be tested until we find an ideal combination. Following the example of the p16 INK4A /MIB-1 dual staining combination and the MCM2/TOP2A combination (ProEx C), combinations are often superior to any single marker and should be tested. A critical deter‐ minant for the success of future investigations will be the standardization of sample prepara‐ tion and interpretation. Furthermore, before reaching the point of routine application, this ideal 'biomolecular' or 'immune-enhanced Papanicolaou test' needs to be evaluated in large prospective clinical trials with appropriate colposcopic, histological, and clinical endpoints as

In order to facilitate the acceptance of novel HPV assays, mainly for cervical screening purposes, several recommendations have recently been published [70, 124, 125]. Meijer *et al.* proposed that before a new HPV assay can be used for cervical screening purposes, it should demonstrate at least similar if not better clinical characteristics (sensitivity, specificity, reproducibility, and so on) for the detection of CIN2+ as hc2 [125]. Other experts believe that large-scale clinical trials, with an assessment of prospective disease outcomes, are required to validate any proposed HPV screening test and that cross-sectional comparisons of new HPV assay to HC2 using several hundred specimens are not an acceptable form of assay validation [70]. Stoler *et al.* recently proposed that any novel HPV assay aiming to be used for cervical screening should have a clinical sensitivity of 92% ± 3% for CIN3+ to render a high NPV or the capacity to predict the future detection of a CIN3+ outcome that might occur during a recommended screening interval [124]. The HPV assay aiming to be used for cervical screening should also have a clinical specificity of at least 85% to achieve an adequate PPV for CIN3. The common idea behind all proposed recommendations is that a clinically useful HPV assay

undergo spontaneous regression of HPV infection.

independent cervical dysplasia [122].

well as adequate follow-up [123].

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

Detection of p16(INK4a) correlates tightly with viral integration. In a normal cell, p16 blocks cyclindependent kinases (CDK) 4/6. Increased expression of the E6 and E7 oncogenes disrupt cell–cycle regulation, resulting in cell–cycle progression. In the normal cell, cell–cycle pro‐ gression is activated by CDK 4/6 and in part regulated by p16. Because in HPV-transformed cells, cell–cycle activation is caused by E7 and not by CDK 4/6, p16 has no effect on the cell– cycle activation. Increased expression of p16 in cells driven by viral oncogene-mediated cellcycle dysregulation can be detected through cellular immunostaining [114].

Because the correlation between HPV mRNA and high-grade dysplasia is a biologically plausible biomarker of risk, HPV mRNA detection may improve the specificity in the evalu‐ ation of women with ASCUS and LSIL Pap smears [115]. Many women have lesions that will not progress to CIN3 or invasive cancer, and these women currently present a treatment dilemma. No reliable methods can identify those lesions that are likely to regress. As a result, these women are monitored with serial colposcopic examinations at great expense to patients and the healthcare community. Detection and quantification of mRNA transcripts in these women may further refine current broad-spectrum, high-risk HPV DNA typing by allowing clinicians to know whether or not the virus is actively replicating E6 and E7 oncogenes. Messenger RNA transcript assays show great promise for being able to stratify the risk of progression to high-grade dysplasia in women with abnormal cytology.

The E6 strip test is also a biomarker that indicates viral integration. Schweizer et al. [116] evaluated the correlation of the HPV E6 test (Arbor Vita Corporation, Fremont, CA), which takes an hour to carry out and detects the HPV-E6 oncoprotein of HPV types 16,18 and 45, with detection of oncogenic HPV DNA in cytologic samples.

#### **9.2. Screening methods identifying epigenetic changes**

Many genes are currently being evaluated as potential methylation biomarkers for cervical cancer, but assay reliability for these methylation markers is highly variable. Within the human genome, methylation of cytosines in the CpG dinucleotides (also known as CpG sites) clustered into islands associated with transcriptional promoters is an important cellular mechanism to regulate gene expression. Methylation of HPV DNA by infected cells may alter the expression patterns of viral genes that are relevant for infection and transformation [117]. Increased methylation of CpG sites within the HPV16 genome before diagnosis and at the time of diagnosis was associated with cervical precancer [118]. Some promising candidate genes include DAPK1, CADM1, and RARB [119].

Another area of biomarker research is in the use of telomerase RNA component (TERC) identification by fluorescence in-situ hybridisation. Most cervical cancers have an extra copy of the long arm of chromosome 3, and consequently show amplification of TERC (present on chromosome band 3q26), which seems to play a key role in progression from low-grade dysplasia to cancer [120]. Many studies indicate that TERC identification may become a useful screening tool for cervical cancer. A prospective study by Andersson et al. [121] found a correlation between increasing TERC detection in cytology specimens and higher grade of dysplasia. They showed that progression to cervical cancer is never seen without TERC amplification and that, conversely, specimens without extra copies of TERC were likely to undergo spontaneous regression of HPV infection.

Other biomarkers under early evaluation for cervical cancer screening include CDC6 and MCM5. These proteins are present in normal cells only during the activation of the cell cycle and help form prereplicative DNA complexes during the G1 phase. They are absent from the cell during quiescence and differentiation. Dysplastic cells have unregulated cell cycles and, as a result, CDC6 and MCM5 reflect cell proliferation [122]. Studies indicate that CDC6 may be a biomarker of high-grade and invasive lesions of the cervix, with limited use in low-grade dysplasia. MCM5 seems to be a biomarker that is expressed independent of high-risk HPV infection, and may in the future serve as a useful marker for both HPV dependent and HPVindependent cervical dysplasia [122].

General markers of cell proliferation like MIB-1, MCMs or ProEx C, and surrogate markers of high risk HPV infection like p16 INK4A have shown promising results. Other potential candidates need to be tested until we find an ideal combination. Following the example of the p16 INK4A /MIB-1 dual staining combination and the MCM2/TOP2A combination (ProEx C), combinations are often superior to any single marker and should be tested. A critical deter‐ minant for the success of future investigations will be the standardization of sample prepara‐ tion and interpretation. Furthermore, before reaching the point of routine application, this ideal 'biomolecular' or 'immune-enhanced Papanicolaou test' needs to be evaluated in large prospective clinical trials with appropriate colposcopic, histological, and clinical endpoints as well as adequate follow-up [123].

#### **9.3. Screening methods for detect HPV infection**

the integration of HPV into the host cell and corresponding risk of CIN 2+ or cancer are in development, and may provide a useful way of screening women at risk for cervical cancer. Studies have shown that viral integrants are detected in 100% of HPV-18-positive and 70–80% of HPV-16-positive cases of cervical carcinoma [112, 113]. A smaller subset of HSIL (15%) and

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

Detection of p16(INK4a) correlates tightly with viral integration. In a normal cell, p16 blocks cyclindependent kinases (CDK) 4/6. Increased expression of the E6 and E7 oncogenes disrupt cell–cycle regulation, resulting in cell–cycle progression. In the normal cell, cell–cycle pro‐ gression is activated by CDK 4/6 and in part regulated by p16. Because in HPV-transformed cells, cell–cycle activation is caused by E7 and not by CDK 4/6, p16 has no effect on the cell– cycle activation. Increased expression of p16 in cells driven by viral oncogene-mediated cell-

Because the correlation between HPV mRNA and high-grade dysplasia is a biologically plausible biomarker of risk, HPV mRNA detection may improve the specificity in the evalu‐ ation of women with ASCUS and LSIL Pap smears [115]. Many women have lesions that will not progress to CIN3 or invasive cancer, and these women currently present a treatment dilemma. No reliable methods can identify those lesions that are likely to regress. As a result, these women are monitored with serial colposcopic examinations at great expense to patients and the healthcare community. Detection and quantification of mRNA transcripts in these women may further refine current broad-spectrum, high-risk HPV DNA typing by allowing clinicians to know whether or not the virus is actively replicating E6 and E7 oncogenes. Messenger RNA transcript assays show great promise for being able to stratify the risk of

The E6 strip test is also a biomarker that indicates viral integration. Schweizer et al. [116] evaluated the correlation of the HPV E6 test (Arbor Vita Corporation, Fremont, CA), which takes an hour to carry out and detects the HPV-E6 oncoprotein of HPV types 16,18 and 45,

Many genes are currently being evaluated as potential methylation biomarkers for cervical cancer, but assay reliability for these methylation markers is highly variable. Within the human genome, methylation of cytosines in the CpG dinucleotides (also known as CpG sites) clustered into islands associated with transcriptional promoters is an important cellular mechanism to regulate gene expression. Methylation of HPV DNA by infected cells may alter the expression patterns of viral genes that are relevant for infection and transformation [117]. Increased methylation of CpG sites within the HPV16 genome before diagnosis and at the time of diagnosis was associated with cervical precancer [118]. Some promising candidate genes

Another area of biomarker research is in the use of telomerase RNA component (TERC) identification by fluorescence in-situ hybridisation. Most cervical cancers have an extra copy of the long arm of chromosome 3, and consequently show amplification of TERC (present on

0% of LSIL contain transcriptionally active viral integrants [111].

cycle dysregulation can be detected through cellular immunostaining [114].

progression to high-grade dysplasia in women with abnormal cytology.

with detection of oncogenic HPV DNA in cytologic samples.

**9.2. Screening methods identifying epigenetic changes**

include DAPK1, CADM1, and RARB [119].

In order to facilitate the acceptance of novel HPV assays, mainly for cervical screening purposes, several recommendations have recently been published [70, 124, 125]. Meijer *et al.* proposed that before a new HPV assay can be used for cervical screening purposes, it should demonstrate at least similar if not better clinical characteristics (sensitivity, specificity, reproducibility, and so on) for the detection of CIN2+ as hc2 [125]. Other experts believe that large-scale clinical trials, with an assessment of prospective disease outcomes, are required to validate any proposed HPV screening test and that cross-sectional comparisons of new HPV assay to HC2 using several hundred specimens are not an acceptable form of assay validation [70]. Stoler *et al.* recently proposed that any novel HPV assay aiming to be used for cervical screening should have a clinical sensitivity of 92% ± 3% for CIN3+ to render a high NPV or the capacity to predict the future detection of a CIN3+ outcome that might occur during a recommended screening interval [124]. The HPV assay aiming to be used for cervical screening should also have a clinical specificity of at least 85% to achieve an adequate PPV for CIN3. The common idea behind all proposed recommendations is that a clinically useful HPV assay should achieve an optimal balance between clinical sensitivity and clinical specificity for detection of CIN2+/CIN3+ in order to minimize redundant or excessive follow-up procedures for hr-HPV-positive women with transient hr-HPV infections and/or without cervical lesions. Thus, as an example, a HPV assay with very high analytical sensitivity can yield a large number of clinically insignificant positive results, which will cause unnecessary clinical follow-up, unnecessary diagnostic procedures and unnecessary treatment of healthy women [126].

in false vaccine failures in vaccination trials. It should be noted that the HPV tests used in cervical cancer screening programs have different requirements for evaluation, since for that purpose, only HPV infections associated with high-grade cervical intraepithelial neoplasia or cancer and not those transient HPV infections that do not give rise to clinically meaningful disease are relevant. Since the latter are characterized by low viral loads, HPV screening assays

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 73

HPV serology is an essential technology for both HPV vaccinology and HPV epidemiology. Definitions of HPV-naïve subjects eligible for HPV vaccination trials include seronegativity for HPV. Immunogenicity of HPV vaccines has been used to bridge results from efficacy trials in adolescents to children and to evaluate different batches of HPV vaccines. Antibody measurements are also important in vaccinology research, e.g. for characterizing the immune response with respect to studies of seroconversion and antibody increases, cross-reactions, immune memory and immune persistence as well as kinetics of antibody responses and establishment of correlates of protection. Finally, HPV seroepidemiology is also useful for understanding the epidemiology of HPV infections in populations to be targeted by HPV vaccination programs. The lack of a standardized assay to measure HPV antibody levels has hindered both epidemiological studies of HPV infection and comparison of results from different HPV vaccine trials [134]. WHO Guidelines for HPV vaccines suggest that "initial assessment of immune responses to HPV VLP vaccines should be based on measurement of neutralizing antibodies in serum". The available data [134-136] suggest that neutralizing and ELISA antibody titres are usually highly correlated when the ELISA antigen target is confor‐ mationally intact VLPs. Due to the complexity and labour-intensiveness of neutralization assays, VLP-based ELISAs have been preferred in large epidemiological studies. E.g., a study of HPV seroprevalences was conducted by measuring HPV 16 antibodies with an HPV16 L1 VLP-based ELISA to estimate the public-health impact of HPV vaccination strategies [137].

WHO has been coordinating work to develop standard assays that will help in assessing vaccine quality and monitoring impact after vaccination [138]. In 2006, WHO established a global HPV laboratory network (LabNet) with a main focus being the harmonization and standardization of laboratory testing procedures to support consistent laboratory evaluation of regional disease burden and monitoring of the performance of HPV vaccines. At a WHO consultation in January 2008, a group of experts recommended that the HPV LabNet should develop or identify standardized assays for general use and that efforts towards standardiza‐ tion on VLP-ELISA should be a high priority of the WHO HPV LabNet [139]. Following the recommendation, the WHO HPV LabNet launched a serology standardization program encompassing: (i) an international collaborative study to evaluate and refine a direct HPV 16 VLP-ELISA suggested Standard Operating Procedure (SOP), (ii) an international request for donations of VLPs to be used as international reference reagents for serology, followed by characterization and selection of optimal reagents, and (iii) an international collaborative

do not have demands on analytical sensitivity that are as high [125].

**10. Measuring the immune response to vaccine**

In forthcoming years, self-sampling may become increasingly important in cervical screening since self-collection for HPV testing (HPV self-sampling) has shown to persuade a subset of non-attendees to participate [127-129]. Targeting non-attendees is important, because they are at higher risk of developing cervical cancer. Additionally, self-sampling may make cervical screening accessible to women in developing regions [130].

An accurate and internationally comparable HPV DNA detection and genotyping methodol‐ ogy is an essential component both in the evaluation of HPV vaccines and in the effective implementation and monitoring of HPV vaccination programs. Genotyping assays used today differ in their analytical performance with regard to type-specific sensitivity and specificity [131]. The evaluation of assay performance needs to be performed in a standardized manner, where different assay performances can be evaluated and results can be compared against a known and accepted standard over time.

In 2008, the WHO HPV LabNet conducted a proficiency study based on HPV DNA plasmids containing the genomes from 14 oncogenic HPV types and 2 benign HPV types and open for participation to laboratories worldwide [132]. This study demonstrated that it is possible to perform global proficiency studies with unitage traceable to ISs based on plasmid DNA and that such studies can provide an overview of the status of the HPV detection and typing methodology worldwide. More recently, based on a proficiency panel composed of the same HPV DNA plasmid material used in 2008, with the amount of DNA titrated in amounts traceable to the IS. The use of the same panel material allowed a reproducible, standardized evaluation of assay sensitivity over time. Specificity was defined as absence of incorrect typing. The sample preprocessing was evaluated with extraction controls of cervical cancer cell lines. The panel was distributed to 105 laboratories worldwide and analyzed using a range of HPV DNA typing assays in a blinded manner [133]. Among laboratories that used the same assay in both years, 27% were proficient in 2008, whereas 30% were proficient in 2010. They also saw a strong trend toward increased sensitivity of assays. For example, among the laboratories using the same assay in 2008 and 2010, 50 IU of HPV-16 could be detected by all (100%) laboratories in 2010, whereas 86% of laboratories could detect 50 IU of HPV-16 in 2008. However, for several laboratories, the increased sensitivity was accompanied by increased amounts of false-positive results, resulting in nonproficiency, suggesting that recommenda‐ tions for HPV laboratory testing include an increased emphasis on the use of negative controls in the assays.

The demands on sensitivity of HPV typing assays vary depending on the purpose of the testing. The WHO HPV LabNet proficiency panels are designed to evaluate the performance of HPV typing tests used in HPV vaccinology and HPV surveillance. In vaccinology, high analytical sensitivity is needed, as failure to detect prevalent infections at trial entry may result in false vaccine failures in vaccination trials. It should be noted that the HPV tests used in cervical cancer screening programs have different requirements for evaluation, since for that purpose, only HPV infections associated with high-grade cervical intraepithelial neoplasia or cancer and not those transient HPV infections that do not give rise to clinically meaningful disease are relevant. Since the latter are characterized by low viral loads, HPV screening assays do not have demands on analytical sensitivity that are as high [125].
