Detection and Typing of Human Papillomavirus Nucleic Acids in Biological Fluids

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1 Public Health Genomics 2009;12: OI: / Published online: August 11, 2009 etection and Typing of Human Papillomavirus Nucleic Acids in Biological Fluids François Coutlée a c Marie-Hélène Mayrand a, b Michel Roger a, b Eduardo L. Franco c a épartements de Microbiologie et Immunologie et Gynécologie-Obstétrique, Université de Montréal, b épartements de Microbiologie-Infectiologie, Gynécologie-Obstétrique, Médecine Sociale et Préventive, et Centre de Recherche, Centre Hospitalier de l Université de Montréal, and c ivision of Cancer Epidemiology, McGill University, Montréal, Qué., Canada Key Words Cervical cancer screening etection, human papillomaviruses Nucleic acid amplification Signal amplification Typing, human papillomaviruses Abstract Human papillomaviruses (HPV) are the etiologic agents of cancer of the uterine cervix and several other neoplasias. etection of HPV infection will improve the sensitivity of primary and secondary screening of cervical cancer. The clinical indications for the use of HPV tests will have to consider the natural history of HPV infection and diseases, and the multiplicity of types involved. Signal amplification HPV NA tests detect several high-risk HPV types, are standardized, commercially available and approved for clinical use. Nucleic acid amplification techniques are ideal methods for epidemiologic purposes since they minimize misclassification of HPV infection status and allow detection of infection with low viral burden. They are currently under evaluation for clinical use. PCR is the most widespread method for HPV typing, especially with the use of consensus primers and typing with reverse hybridization techniques. Novel promising HPV detection strategies are now proposed, such as HPV mrna detection, and suspension or solid phase arrays. These novel techniques will have to be evaluated as stringently as actual assays in clinical studies. Although assays have been developed for the evaluation of viral load, viral integration and HPV polymorphism in molecular epidemiological studies, their role in clinical practice is not currently defined. Copyright 2009 S. Karger AG, Basel HPV is a necessary cause of cervical cancer [1]. ue to central role of HPV in anogenital carcinogenesis, HPV testing has received intense scrutiny as an adjunct or replacement to cervical cytology in cervical cancer screening. The potential role of HPV testing has been defined for 3 clinical applications: for the triage of equivocal cervical cytology results, in primary screening, as a standalone test or as an adjunct to cytology, and in the followup of treated high-grade cervical intraepithelial neoplasia (CIN-2,3) [2]. HPV testing is also essential in research: studies on the epidemiology and transmission of HPV infection, the natural history of anogenital precancerous lesions, and efficacy of vaccination also rely on the use of HPV NA detection and typing methods. Advances in molecular biology in the last 15 years have greatly contributed to improve our ability to detect and type HPV in biological fluids. The benefits and limitations of the var- Fax karger@karger.ch S. Karger AG, Basel /09/ $26.00/0 Accessible online at: François Coutlée, épartement de Microbiologie et Infectiologie Hôpital Notre-ame du Centre Hospitalier de l Université de Montréal 1560 Sherbrooke est, Montréal, Qué., H2L 4M1 (Canada) Tel ext , Fax francois.coutlee@ssss.gouv.qc.ca

2 Signal Signal Target NA Target NA a b Signal Signal Fig. 1. Assay formats for the detection and typing of HPV nucleic acids. a irect NA detection tests. b Signal amplification assays. c Genomic amplification assays. d Probe amplification assays. Signal = Intensity of signal emitted by the probes utilized in each assay format; = detector molecule used in probes or reagents for probe detection. c Target NA Target NA d ious assays available for HPV detection and typing have been recently discussed [3, 4]. Two strategies have been developed for the diagnosis of HPV infection. In the first strategy, a cocktail of reagents detects the presence of high-risk HPV type NA as a group (generic tests). In the second strategy, types are detected individually (typespecific tests). Irrespective of these strategies, 4 assay formats have been devised for the detection of microbial nucleic acid in specimens ( fig. 1 ). Each of these assay formats discussed below has potential applications and limitations ( table 1 ). HPV Testing evelopment for etection of Genital Infection The development and optimization of HPV detection tests have to consider some characteristics of HPV infection in the anogenital tract. The 7,900 base-pair HPV NA genome is divided into a non-coding regulatory region, an early region (E genes) involved in transformation, and a late region (L genes) coding for capsid proteins. Areas of homology between HPV types are located mainly in the E1, E6, and L1 genes. These conserved areas in the HPV genome explain the cross-hybridization reactions between types as well as the ability of consensus genomic amplification methods to detect different HPV types in one reaction. More than 100 HPV types have been characterized, 40 of which infect preferentially the epithelium of the anogenital tract [5]. Genital HPV types are also classified according to their association with preinvasive and invasive lesions. High-risk HPV types are associated with CIN-2,3 and invasive cancer [1, 6]. Although most sexually active individuals are exposed to HPV during their lifetime, most HPV infections resolve spontaneously. HPV can be found at relatively low copy numbers in women without lesion. Thus, increased analytical sensitivity of HPV detection tests may not necessarily translate into improved clinical utility. HPV NA detection methods are applied on samples collected from the anogenital tract. The quality of samples collected has a significant impact on the results obtained with molecular detection methods for any sexually transmitted disease [7]. Cervical HPV NA is detected more frequently in specimens collected with a cytobrush than with a swab [8]. Cervicovaginal lavages collect viral NA from the cervical and vaginal epithelia. Several studies have shown a good concordance for HPV NA detection between physician-obtained and self-administered samples [9]. For all samples, preservation of integrity of HPV NA or RNA is excellent when cells are resuspended in medium used to preserve samples for liquid cytology [10]. irect Probes for etection of HPV NA In these assays, sample NA is hybridized with labeled probes and the signal emitted by the probe is detected and interpreted as a positive reaction ( fig. 1 ). Unfortunately, these assays are only moderately sensitive etection and Typing of HPV Nucleic Acids in Biological Fluids Public Health Genomics 2009;12:

3 Table 1. Assay formats for HPV detection and typing Assay formats Assays Properties and applications Limitations irect detection Signal amplification In situ hybridization Correlation of HPV detection with histology Low sensitivity Hybrid capture-2 etection of presence of high-risk types as a group Test most evaluated for clinical use Not designed for typing Moderate analytical sensitivity but excellent clinical sensitivity for clinical triage and screening Some cross-reactivity with low-risk types Semiquantitative for viral load Gene amplification PCR etection of presence of high-risk types as a group or of specific types individually Several formats for detection and/or typing Consensus amplification of genital genotypes with individual typing Best analytical sensitivity Viral load evaluation Evaluation of HPV integration Evaluation of intra-type HPV polymorphism Applicable in a research setting Still under evaluation for clinical use Competition for amplification between types in mixed type infections Contamination and false-positive results (avoided by good lab practices) Analytical sensitivity needs to be optimized for useful clinical sensitivity Some cross-reactivity with low-risk types Typing is sometimes difficult with consensus assays for some HPV types due to constraints of probe selection or patents Transcriptionmediated amplification etection of presence of high-risk types as a group Not easy to develop in a research setting Excellent analytical sensitivity May allow discrimination between latent and active infection Quantitation possible Not designed for typing Test under evaluation for clinical use Clinical sensitivity needs to be determined Probe amplification Invader technology etection of presence of high-risk types as a group Not easy to develop in a research setting Excellent analytical sensitivity Not designed for typing Test under evaluation for clinical use Clinical sensitivity needs to be determined Not quantitative [11]. In situ hybridization permits localization of HPV infection within specific lesions ( table 1 ). Since in situ hybridization is positive only in the presence of more than 25 HPV NA copies per cell, high-grade lesions, which often contain lower amounts of HPV NA, can be falsely negative [12]. More sensitive in situ hybridization techniques are needed. Nucleic acid amplification technology (NAAT; see below) can be applied on NA extracted from biopsies. Although a good concordance between HPV results obtained on biopsies and exfoliated cells has been reported with these methods, multiple infections detected in exfoliated cells could be resolved to a single type in 50% of cases [13]. Moreover, a portion of biopsies submitted for HPV testing may not contain any residual lesion [13]. Attribution of cervical lesions to a single HPV type remains a difficult and tedious task with current methods. Signal Amplification NA-Based Assays: The Hybrid Capture-2 System Signal-amplification tests detect lower quantities of NA than direct methods. They amplify the detection signal emitted by the probe without modifying the initial amount of nucleic acids contained in samples ( fig. 1 ) [3, 14]. The detection of high-risk HPV NA in exfoliated cells with hybrid capture-2 (HC-2; Qiagen Inc., Germantown, Md., USA) is approved for clinical purposes by regulatory agencies from several countries. In this assay, processed sample NA is hybridized in solution with a mixture of non-isotopic single-stranded full-length RNA probes against 13 high-risk types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Hybridized products are transferred into microplate wells coated with an antibody that recognizes specifically NA-RNA hybrids. Bound 310 Public Health Genomics 2009;12: Coutlée /Mayrand /Roger /Franco

4 hybrids are detected with the addition of an alkaline phosphatase-labeled monoclonal antibody against NA- RNA hybrids and a chemiluminescent substrate. Results are expressed as the ratio of the specimen reactivity to the mean of the positive control. The HC-2 system has been well validated, detects several high-risk types in one reaction, uses a convenient format that avoids potential contamination encountered with gene amplification methods and is not impeded by the presence of interfering substances contained in samples [11]. Unlike cervical cytology, it does not require extensive training, is not subjective, and is reproducible with an intra-assay coefficient of variation below 5%. HC-2 can detect as few as 5,000 HPV NA copies per test [14]. HC-2 does not control for the quality of sample by measuring cell NA content. Several studies have evaluated the sensitivity and specificity of HC-2 against histology and/or PCR assays [11, 12, 14]. A good agreement, with values above 0.6, was found in several studies comparing HC-2 and PCR [8, 14]. There is some cross-reactivity between the high-risk probe cocktail and HPV types not included in the probe mix, such as types 6, 34, 42, 53, 54, 62, 66, 67, 70, 73 and 82, some of which are lowrisk types [8, 15, 16]. This cross-reactivity increases the overall sensitivity of the assay but contributes to lowering its specificity. HC-2-positive samples containing only low-risk genotypes are usually weakly reactive [17]. The clinical sensitivity of HC-2 to detect CIN-2,3 in various studies was above 90%, and the test had an excellent specificity, especially in women more than 30 years of age [2, 18]. HC-2 NA testing is available as an automatic high-throughput platform, the Rapid Capture System, which integrates all steps of sample processing and testing [19, 20]. In a prototype third-generation hybrid capture system (HC-3; not yet available commercially), sample NA is captured with biotin-labeled oligonucleotide probes, thus reducing the background noise that could be generated by endogenous NA-RNA hybrids. Blocker probes are also included to enhance the specificity of the test [21]. HC-2 in Clinical Management Only 10% of women with atypical cells of undetermined significance (ASC-US) Pap tests have CIN-2,3 [20]. Since up to 50% of women with ASC-US are not infected with high-risk HPV types and are thus unlikely to harbor CIN-2,3, the use of HPV testing avoids unnecessary diagnostic evaluation (colposcopy) [2]. Several NAAT assays described below are currently being evaluated for that purpose. Reflex HPV NA testing is the preferred option in the triage of women with ASC-US who provided samples for both cytology and HC-2 [2]. In the absence of lesion at colposcopy, HPV testing can be repeated in 12 months. Women testing positive for highrisk HPV at 12 months are referred for a second colposcopy, as the initial colposcopy may have been falsely negative [2]. Results from studies evaluating the sensitivity and specificity of HC-2 to detect high-grade lesions in women with ASC-US ranged from 89 to 96% and 74 to 85%, respectively [14, 22], with a negative predictive value around 98%. Results from the multicenter randomized clinical trial the Atypical Squamous Cells of Undetermined Significance/Low-grade Squamous Intra-epithelial Lesions Triage Study (ALTS) demonstrated that HPV NA testing had a sensitivity of 96% for detecting CIN3 while 56% of women were referred for colposcopy. When age was considered, HPV testing was very sensitive to detect CIN-3 in women at least 30 years old, and resulted in fewer referrals to colposcopy than repeat cytology [23]. The lower referral rate when using HC-2 has a positive impact on the cost-effectiveness of HPV NA testing [23]. The ALTS trial also showed that HPV testing by HC-2 does not help in the triage of women to colposcopy with a cytology result of low-grade squamous intra-epithelial lesion, since 83% of these women had HPV infection with high-risk types [24]. However, HC-2 is useful in the follow-up of women with such smears and negative colposcopic and histological findings [2]. In primary screening, studies conducted in developing countries reported a greater sensitivity for detecting CIN-2,3 with HPV NA testing (85 100%) over cytology (40 78%) [25]. In 2 studies conducted in Costa Rica and South Africa on 8,554 and 2,944 women, respectively, HC-2 detected 88% of high-grade lesions as a primary screening tool, with referral rates to colposcopy of 12 18%, while Pap smears detected 77% of high-grade lesions with a 7% referral rate [16, 26]. Similar results were reported in 2007 from a prospective randomized controlled trial in Canada, the CCCaST study, comparing HC-2 to conventional cytology for the primary screening of cervical cancer in women aged 30 years and above [27, 28]. Women testing positive in one or both tests as well as a random sample of women with negative results for both tests were evaluated by colposcopy. The sensitivity of HC-2 was significantly higher than that of cytology for high-grade squamous intra-epithelial lesion (95 vs. 55%). HPV testing could also be useful for quality control of cytology smear interpretation. For example, a Pap test etection and Typing of HPV Nucleic Acids in Biological Fluids Public Health Genomics 2009;12:

5 reporting negative while HC-2 is positive, could be automatically reviewed to eliminate false-negative cytology due to reading errors. HPV could also be used in the follow-up of women after treatment of cervical cancer precursors to identify those with residual/recurrent disease from those who are disease-free [2]. NAAT for HPV etection and Typing In these assays, the initial amount of HPV nucleic acids contained in samples is amplified and the products of this amplification are then detected with specific probes ( fig. 1 ). NAAT can detect 1 10 copies of HPV NA per test. However, ultra-sensitive assays do not necessarily imply a better performance for clinical use if the added sensitivity is offset by poor specificity [29]. NAAT assays can detect HPV types individually or as risk groups (generic tests). They can also be utilized for HPV viral load, integration and variant analysis, as shown in table 1. HPV typing could improve the specificity of HPV NA detection tests for clinical use, allow management of HPV-infected women according to risk of high-grade lesions, evaluate type-specific HPV prevalence, assess the geographic heterogeneity in HPV genotype distribution, assess the type-specific concordance between partners, monitor persistent infection in consecutive HPV-positive samples, study multiple type infections, monitor HPV infection in clinical trials on surgical treatments, and monitor the duration of protection in vaccinated populations and the potential shift in type distribution [29]. We will review in the next paragraphs the various NAAT assays for HPV detection and typing ( table 1 ). One of these techniques, the PCR is the most powerful tool for the epidemiological investigation of HPV infection or cervical cancer [12]. Consensus PCR assays have been devised to amplify in one reaction the majority of known, as well as novel, anogenital HPV genotypes. HPV typing is then accomplished by hybridization with typespecific oligonucleotide probes, by restriction fragment length polymorphism or NA sequencing [12]. The efficiency of consensus PCR assays will depend on the type of sample tested (fixed or fresh tissue), size of amplicon, use of degenerate versus non-degenerate primers, prevalence of multiple type infections, number of primers used in the mixture and sequence variation at the primer binding sites. The presence of multiple type infections can reduce the ability of consensus PCR to identify all types present in a sample because of competition for reagents [30]. Four consensus assays target conserved sequences in the HPV L1 gene. The MY09/MY11/HMB01 or PGMY09/ PGMY11 (yield a 450-bp amplicon), GP5+/GP6+ (150-bp amplicon) and SPF1/SPF2 (65-bp amplicon) consensus primer sets can amplify a wide spectrum of up to 36, 37, and 43 genital genotypes, respectively [19, 31 35]. After amplification with consensus primers, HPV amplicons are genotyped with reverse hybridization on line filters or on microtiter plates. The only consensus PCR test commercialized in North America is the Linear Array HPV genotyping test (Roche iagnostics, Laval, Canada) with PGMY primers. The SPF 10 Inno-LiPA HPV genotyping assay (Innogenetics NV, Ghent, Belgium) with SPF primers is utilized in Europe and is under evaluation by Health Canada. The degenerate pool of primers MY09/MY11/HMB01 amplifies a broad spectrum of HPV genotypes with varying levels of sensitivity [33] down to as few as 10 copies of viral NA from frequently encountered genital types. Since the insertion of nucleotide bases at positions of degeneracy is a random and irreproducible process, synthesis of degenerate primers does not ensure an equivalent representation of all degenerate primers. This variability could result in lot-to-lot variations in type-specific amplification efficiencies. The new PGMY09/PGMY11 set of consensus primers was designed to improve the sensitivity, specificity, and reproducibility of L1 consensus PCR [33, 36]. PGMY09/PGMY11 consensus primers comprise 18 different primers derived from the MY09- MY11 primer sites. Amplification of genital HPV types is improved over the MY primer pair, including types less efficiently amplified with MY09/MY11 such as types 35, 52 and 56 [33, 36, 37]. etection of high-risk HPV types with the Linear Array had a sensitivity of 92.3% for detecting CIN-3 and cancer in women with ASC-US in 1 study [38]. The risk for progressing to CIN-3 and cancer was the greatest for women positive with the Linear Array for HPV-16 [38]. The Linear Array could thus prove use - ful for genotype-specific risk stratification for clinical management. T he SPF 10 Inno-LiPA HPV genotyping assay (Innogenetics NV, Ghent, Belgium) uses a pool of 10 different primers containing inosine to optimize HPV amplification [32]. Amplicons are first tested in a microtiter plate general hybridization assay to detect HPV NA positivity. Subsequently, positive samples are analyzed by Inno- LiPA for 25 genotypes. In 1 publication on nearly 6,000 women, the crude agreement between the SPF 10 -LIPA system and HC-2 reached 88% with a value at 0.75 [30]. The overall agreement for detection of HPV NA with 312 Public Health Genomics 2009;12: Coutlée /Mayrand /Roger /Franco

6 both SPF and PGMY systems is also high [32, 39, 40]. The ease of use and the total run times were comparable between the Linear Array and Inno-LiPA [39]. When genotyping was considered, 1 96% concordance was found between the 2 assays. Types 42, 56 and 59 are more easily detected with PGMY primers, while types 31 and 52 are more easily amplified with SPF primers [32]. Typing for HPV-52 cannot be done with the Linear Array in the presence of HPV-33, 35 or 58, while HPVs 68 and 73 cannot be distinguished with the Inno-LiPA. GP5+/GP6+ primers detect types 43 and 44 more efficiently than MY09-MY11. The sensitivity of GP5+/GP6+ systems varies from 10 to 200 copies, depending on the type tested. Overall agreement between GP5+/GP6+ and HC-2 or Inno-LiPA is good [19, 34]. Overall, genotyping assays are moderately to highly complex and costly. Several NA detection methods have been used to type amplicons generated with consensus PCR for in-house HPV detection assays. The higher cost of commercialized assays is an issue for large-scale studies and favors in-house methods. However, the latter methods are not as well standardized and quality-controlled as commercialized assays. Typing assays utilizing membrane-based reverse line blot methods are restricted to a maximum of 40 probes per hybridization reaction. Array technologies permit the use of a much greater number of probes to detect a greater number of targets. The Luminex xmap suspension array technology can simultaneously detect several HPV genotypes by using 5.6- m polystyrene microspheres that are internally dyed with various ratios of 2 spectrally distinct fluorophors [41, 42]. An array of 100 bead sets with specific absorption spectra can be generated. The various HPV type-specific probes can be coupled to different bead sets [42]. A third fluorochrome incorporated into the amplicons allows detecting positive hybridization reactions on spheres when they pass by lasers. Although promising, HPV array assays need to be evaluated thoroughly in clinical settings and compared to standard detection assays. HPV typing can also be accomplished on solid-phase microarrays [43 46]. However, suspension arrays offer several advantages over microarrays, such as a favored hybridization kinetics resulting in shorter reaction times and fast instrument readout. In general, type-specific PCR tests are not a practical means for detecting HPV infection in clinical specimens because of the large number of types involved in genital diseases. However, because they are more sensitive than consensus assays (see below), they are useful for the evaluation of efficacy of vaccine against types included in HPV vaccine preparations. Identification of women infected with 1 of the highrisk types without genotype determination can be done by consensus PCR. The Amplicor HPV test (Roche iagnostics) detects the presence of the 13 high-risk genotypes also detected by HC-2 [47]. -globin is coamplified to assess the integrity of sample [48]. There was a 97.5% agreement in 1 study between results obtained with Amplicor and those obtained with Inno-LIPA [49]. Amplicor has also been evaluated and compared to HC-2 in a population of women with atypical squamous cells of undetermined significance [50]. Concordant results were obtained for 86.7% of samples and Amplicor had a negative predictive value of 97.4%. Some have suggested that a higher cutoff for positivity might increase specificity without affecting sensitivity [47]. Cross-reactivity of Amplicor was demonstrated with low-risk type 6, 11, 70, 67, 73 or 53 and with high-risk genotypes not included in the probe cocktail, such as types 66 and 82 [17]. Reactivity with low-risk types was often weak [17]. etection of the presence of infection with high-risk HPV types without genotype determination can also be accomplished by transcription-mediated amplification. Overexpression of the E6 and E7 HPV genes results from deregulation of these genes in higher-grade cervical disease. HPV E6/E7 mrna detection could thus be a more specific marker for the presence of CIN-2,3 than the detection of HPV NA [4]. In several investigations, fewer women were positive for HPV mrna than HPV NA in the absence of lesion [4]. Two NAATs detect HPV mrna: the PreTect TM HPV Proofer (NorChip/BioMérieux Inc.) and the Aptima HPV (Gen-Probe Inc., San iego, Calif., USA) assays. The Aptima HPV test is a transcription-mediated amplification assay for 15 high-risk genotypes. The assay is based on the capture of extracted mrna from samples with chimeric capture oligonucleotides [51] and selective isothermic amplification of HPV mrna sequences with primers and a reverse transcriptase. Transcription of amplicons that carry the T7 promoter added to the 5 end of 1 primer is accomplished with the T7 RNA polymerase. RNA amplicons are detected with acridinium ester-labeled probes in a protected hybridization assay. The initial evaluation of the assay revealed that fewer specimens tested positive for carcinogenic HPV E6E7 mrna than for high-risk HPV NA, especially for samples from women with low-grade disease [51]. Further studies will be needed to investigate the added clinical specificity of detecting mrna and also assess if a threshold mrna level could help to further increase this level of clinical specificity. etection and Typing of HPV Nucleic Acids in Biological Fluids Public Health Genomics 2009;12:

7 Probe Amplification or Modification Technologies The Invader technology (Third Wave Technologies, Madison, Wisc., USA) is under consideration for the generic detection of high-risk HPV genotypes [52]. The assay detects the same 13 genotypes detected with HC-2 plus HPV-66. It is based on the isothermic amplification of probes utilizing 3 probe pools and the cleavase enzyme. An -actin cellular control is included. On a limited number of samples, the Invader technology-based assay performed similarly to HC-2 [52]. This format has not been developed for individual typing. Table 2. Factors that influence the estimation of HPV NA load measured by real-time PCR Factors HPV quantitation by real-time PCR HPV-related factors HPV Underestimation by impaired amplification because of selection of reagents in variable sites of HPV polymorphism reduced binding of primers to HPV sequences reduced binding of probe to HPV sequences sequence data of the HPV gene that is targeted for quantitation from various isolates obtained in different geographic locations select primer and probe sequences in conserved areas of the HPV genome use SYBR Green instead of using a probe, but using primers that are very specific to their target sequences Conserved HPV sequences HPV disruption during integration Underestimation by competition for reagents during amplification cross-hybridization resulting in the binding of primers with types other than the targeted type, resulting in competition for reagents and/or for primers Overestimation by detection of related HPV types cross-hybridization of probe and primers with types related to the targeted type resulting in false-positive signal align sequences of related types and select primer and probe sequences in sequences specific for the type targeted in vitro testing to assess detection or competition of reagents with related HPV types Underestimation by absence of amplification disruption of the HPV sequence is located between primer binding sites Solution: select primers and probe outside the usual sites or genes disrupted during integration Sample-related factors Inhibitors of Underestimation by reduced efficiency of amplification PCR inhibitors of PCR are detected in anogenital secretions inhibitors are detected more frequently in lysates than in extracted NA the activity of inhibitors is sometimes dependent on primer sequence inhibitory activity of samples is often lost after dilution of sample or after keeping the sample for 48 h at 4 C screen inhibitory activity by quantitating a cellular gene screen inhibitory activity with the use of an internal control Cellularity of samples Quality of sample NA Underestimation by a reduced number of target cells HPV is detected in and outside cells; a poorly obtained sample with low cell quantity will alter the detection and quantitation of HPV NA standardize sampling procedures optimize sample collection (cytobrush or cervical lavage) Underestimation if sample NA degraded good sampling procedures collect cells in liquid-based cytology medium conserve extracted NA or lysates frozen quantitate a cellular gene to adjust for cellular quantity and NA quality of sample 314 Public Health Genomics 2009;12: Coutlée /Mayrand /Roger /Franco

8 Table 2 (continued) Factors HPV quantitation by real-time PCR Amplification-related factors Master mix components Underestimation of quantity of HPV NA and thermal profile calculate amplification efficiency, E = 10 1/s 1 (s being the slope of standard curve); if below 90%, optimize magnesium content and temperature profile; if above 100%, suspect primer dimer synthesis optimize hybridization temperature and master mix on titration curves of targeted HPV type mixed with cellular NA Competition with related HPV types High cellularity of samples Underestimation of quantity of HPV NA by competition for reagents as for HPV-related factors Underestimation of quantity of HPV NA too much sample NA (cellular or microbial) will interfere with primer and probe binding to target NA Solution: avoid high input of sample NA Viral Load Evaluation by PCR In the ALTS study, HPV viral load measured with HC-2 did not have any clinical utility due to the important overlap between groups [23]. Overall, the clinical significance of quantitative results obtained with HC-2 remains controversial. Quantitation of viral HPV NA in clinical specimens could improve the clinical utility of HPV detection methods, although this remains to be demonstrated due to the important overlap of viral load values between women without and with lesions [4]. Conventional and real-time PCR assays have been developed for type-specific HPV viral estimations, real-time PCR being more accurate and reproducible for viral quantitation in general [3]. Real-time PCR is also completed in a shorter period of time because amplification and detection are performed simultaneously. Real-time PCR also reduces the risk of contamination because amplification and detection are performed in the same reaction tube. Quantitative PCR assays require a control for amplification efficiency and sample inhibition. Several parameters of amplification have to be optimized to obtain reliable results ( table 2 ). Using these criteria, few truly quantitative PCR assays have been developed for HPV quantitation. Real-time PCR assays provide accurate measurements of the initial copy number of target NA contained in samples by continuously measuring the increments of fluorescence released during the amplification reaction. This monitoring of amplicon synthesis is next to impossible with conventional PCR. Real-time PCR combines amplification by rapid thermocycling with simultaneous fluorescence detection. Real-time PCR assays generate linear results over a wide range of target concentrations. Viral load is expressed as the number of HPV NA copies per cell or per microgram of cellular NA. Utility of Measuring HPV Integration in Human NA NA molecules from high-risk HPV types remain episomal in low-grade lesions but are mostly integrated in the human genome in high-grade lesions and invasive tumors. Integration disrupts the E2 gene, resulting in uncontrolled synthesis of E6 and E7 oncoproteins. Current assays to measure HPV integration are based on quantitation of HPV E6 relative to E2 NA [53]. etection of a greater quantity of HPV-16 E6 compared to HPV-16 E2 suggests the presence of integrated HPV-16 forms. HPV polymorphism could influence quantitation of integrated HPV NA, specifically from underestimation of E2 (episomal form) [54]. Confirmation of integration detected by real-time PCR has been accomplished by various techniques including in situ hybridization, Southern blot and PCR-sequencing methods that identify the presence of HPV-human NA junctions [55]. etection of transcriptionally active HPV genomes can also screen for the presence of integrated forms, but these forms may be silenced by episomal HPV forms when a mixture of episomal and integrated forms coexist in a lesion [56]. Further study is necessary to determine the value of measur- etection and Typing of HPV Nucleic Acids in Biological Fluids Public Health Genomics 2009;12:

9 ing HPV integration on the natural history of HPV infection as well as analysis of integration patterns for types other than type 16 [57]. HPV Variant Analysis There is genomic diversity within HPV types [58]. HPV variants are defined by mutations that generate less than 2% genetic variation in coding regions and less than 5% in non-coding regions, compared to the prototype isolate. HPV variants are now assessed mostly by PCR sequencing. Optimal classification of variants is carried out by analysis of the long control region where most variability is encountered. HPV polymorphism has been associated with persistence of HPV infection and the presence of high-grade lesions [58, 59]. Analysis of HPV polymorphism is relevant for research purpose but has not been defined for clinical purposes. Quality Assurance for HPV Tests The inter-laboratory reliability of hybrid capture has been evaluated [27, 60]. The accuracy of the various laboratories participating ranged from 83 to 92% and agreement ranged from 87 to 94% ( values from 0.61 to 0.83). Excellent intralaboratory and interlaboratory agreement was reported in an international proficiency study on the PGMY-line blot (Roche Molecular systems) [61]. Quality control panels and quality assurance programs are essential to maintain and monitor the quality of HPV testing. HPV NA standards are being developed by the World Health Organization as well as a proficiency panel that can be sent to laboratories around the world for evaluating the quality of HPV NA typing [62]. Conclusion The variety of assay formats for HPV NA detection and characterization will serve well the research and diagnostic community. The availability of several assays developed by various manufacturers as well as increased use of these tests by laboratories will likely lead to a drop in prices for genotyping assays. It is also possible that a genotype targeted against a limited number of clinically relevant types will reduce the overall cost and price of these tests for epidemiological and clinical purposes. Prospective cohort studies and clinical trials will further help define the optimal clinical algorithms in screening settings. Today the question is no longer will HPV testing be useful in clinical settings? but how and for whom will it be most useful?. HPV typing assays are mandatory in the era of vaccination to provide an evaluation of the efficacy of novel vaccine preparations. Ac k n ow l e d g m e n t s Research projects conducted by the authors are supported by the Réseau Fonds de Recherche en Santé du Québec (FRSQ)- SIA, and a team grant (No. CRN 83320) from the Canadian Institutes for Health Research (CIHR). References 1 Bosch FX, Lorincz A, Munoz N, Meijer CJLM, Shah KV: The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002; 55: Wright TCJ, Massad LS, unton CJ, Spitzer M, Wilkinson EJ, Solomon : 2006 consensus guidelines for the management of women with abnormal cervical screening tests. J Low Genit Tract is 2007; 11: Iftner T, Villa LL: Chapter 12: Human papillomavirus technologies. J Natl Cancer Inst Monogr 2003; 31: Gravitt PE, Coutlée F, Iftner T, Sellors J, Quint WGV: New Technologies in cervical cancer screening. Vaccine 2008; 26(suppl 10): K42 K52. 5 de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Classification of papillomaviruses. Virology 2004; 324: Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah K, Snidjers PJF, Meijer CJLM: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Eng J Med 2003; 348: Coutlée F, de Ladurantaye M, Tremblay C, Vincelette J, Labrecque L, Roger M: An important proportion of genital samples submitted for Chlamydia trachomatis detection by PCR contain small amounts of cellular NA as measured by beta-globin gene amplification. J Clin Microbiol 2000; 38: Peyton CL, Schiffman M, Lorincz AT, Hunt WC, Mielzynska I, Bratti C, Eaton S, Hildesheim A, Morera LA, Rodriguez AC, Herrero R, Sherman ME, Wheeler CM: Comparison of PCR- and hybrid capturebased human papillomavirus detection systems using multiple cervical specimen collection strategies. J Clin Microbiol 1998; 36: Petignat P, Faltin L, Bruchim I, Tramer MR, Franco EL, Coutlee F: Are self-collected samples comparable to physician-collected cervical specimens for human papillomavirus NA testing? A systematic review and meta-analysis. Gynecol Oncol 2007; 105: Public Health Genomics 2009;12: Coutlée /Mayrand /Roger /Franco

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