**6. HPV-DNA genotyping methods**

types; and lack of internal control to evaluate specimen adequacy or the presence of potentially interfering substances. In order to resolve the current problems of analytical inaccuracy and to improve HC2 throughput, a next-generation diagnostic system has been developed [54]. Significant improvements have been engineered into the next-generation hybrid-capture system, which builds on the current advantages of the FDA-approved HPV screening tech‐ nology, HC2. Central to the improved chemistry in the NextGen assay on the QIAensemble

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

TheAmplicorHPVTest(Amplicor;RocheMolecular Systems,Branchburg,NJ,USA),launched on the European market in 2004, is a qualitative PCR-based test designed to detect the same 13 HPV types as HC2: HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52,HPV-56,HPV-58,HPV-59andHPV-68.SimilarlytoHC2,Amplicorexpressestheresults of the tested group of hr-HPV types as positive or negative. Amplicor is based on standard PCR amplification and detection of PCR products on microwell plates [55]. The Cervista HPV HR Test (Cervista; Hologic) is another FDA-approved signal amplification-based qualitative test for the routine detection of 14 HPVs. In March 2009, the FDA approved Cervista for two indications: to screen patients with ASC-US cervical cytology results to determine the need for referral to colposcopy; and to be used adjunctively with cervical cytology to screen women 30 years of age andolderto assess thepresence or absence of hr-HPV types [56].In contrastto HC2, Cervista is based on the Invader chemistry, a signal amplification method for detecting specific nucleic acid sequences. With support from the Bill and Melinda Gates Foundation, a careHPV Test (Qiagen), based on simplified HC2 technology, has been recently developed to detect the 13HPVtypesincludedintheoriginalHC2plusHPV-66,inapproximately3h.Suchrapid,simple and affordable HPV tests, whereby results can be given to a patient within the same visit, are anticipated to have the greatest impact in countries in which cervical cancer screening pro‐ grams do not exist or in countries in which substantial loss to follow-up impairs the effective‐

**5. HPV-DNA screening methods with concurrent or reflex HPV-16 &**

Reflex HR HPV DNA testing is now recognized as a cost-effective strategy to refer women with an ASC-US cytology for colposcopy [58, 59]. High-risk HPV-DNA-based screening assays with individual or pooled HPV-16 and HPV-18 genotyping are a group of novel HPV assays in which qualitative detection of 13–14 HPV types is combined with concurrent or reflex HPV-16 and HPV-18 genotyping. The Abbott RealTi*me* High Risk HPV test (RealTi*me*; Abbott Molecular, Des Plaines, IL, USA) is a real-time PCR assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 other HPVs: 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. Amplification of human β-globin is used as an internal control. The assay was launched on the European market in January 2009. RealTi*me* is performed on the *m*2000rt real-time PCR instrument (Abbott Molecular) using a modified GP5+/GP6+ primer mix consisting of three forward and two reverse primers [60]. Analytical sensitivity of RealTime was comparable to

automated system is the new collection medium, DCM [54].

ness of cervical cancer screening programs [57].

**HPV-18 genotyping**

HPV DNA-based genotyping as says, which allow exact determination of several alpha-HPV types present in a clinical sample, are the largest group of currently available HPV commercial assays.IncontrasttothepreviouslydescribedtwogroupsofcommercialHPVassays,theclinical value of HPV DNA-based genotyping assays has still not been finally determined [70-72]. Currently, HPV genotyping methods are indispensable as research tools for the study of the natural history, transmission, pathogenesis and prevention of HPV infection. However, it is highly likely that genotyping methods will also have a role in clinical management in the near future [70, 73, 74]. As the use of prophylactic HPV vaccines becomes more widespread, surveillance for population-level effectiveness will become an increasingly important activity, which will require the use of a HPV genotyping method. If HPV genotyping is to be used for diagnostic applications and not just as a research tool, it will require standardized and validat‐ ed methods for the specific detection and identification of a defined spectrum of HPV types.

Although DNA sequencing is still considered to be the 'gold standard' for HPV genotyping, it is costly, time-consuming and difficult to apply in routine diagnostic settings. Thus, in daily practice, the majority of laboratories use nonsequencing based methods for HPV genotyping, However the next generation sequencing can be implemented in the HPV detection [75]. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of nextgeneration sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.

of 35 alpha-HPVs. The assay consists of pre-aliquoted, ready to use amplification mix tubes and allows amplification of a 450 bp region of the HPV *L1* gene together with an internal control template and the human *CFTR* gene using three different sets of biotinylated primers [84].

HPV Diagnosis in Vaccination Era http://dx.doi.org/10.5772/55818 67

The suspension array that uses bead-based xMAP or Luminex technology is based on the use of polystyrene beads with a diameter of 5.6 µm, which are internally dyed with various ratios of two spectrally distinct fluorophores (red and infrared). Different bead sets with specific absorption spectra can be mixed and used for the multiplexed detection of different analytes; currently, an array of 100 bead sets (bead mix) can be generated. For HPV genotyping purposes, each bead set in the bead mix is usually coupled to a single oligonucleotide probe specific for one HPV type. HPV genotyping is done by reverse hybridization technique using biotinylated PCR amplicons. After denaturation and hybridization of target HPV sequences to the beadbound probes, labeling of the hybridized biotinylated amplicons is done using R-phycoery‐ thrin-labeled streptavidin, serving as a reporter fluorophore. The bead sets are then read and analyzed on a Luminex analyzer; the HPV types are discerned according to the unique bead signature, whereas the presence of specific PCR amplicons is determined by R-phycoerythrin fluorescence. The Luminex readouts are expressed as the median fluorescent intensity of the reporter fluorescence for each HPV type. Positivity for the relevant types is calculated from the median fluorescent intensity over a defined threshold level, and can provide a semiquantitative numerical output. Several in-house genotyping protocols based on xMAP technology have been developed in the last 5 years [85-91]; some of them are considered to be the most sensitive HPV detection methods currently available [85]. In addition, at least two

Gel electrophoresis-based HPV genotyping assays utilize general primer-mediated PCR, typespecific or group-specific PCR, to screen for a broad spectrum of HPVs, followed by agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) is consequently applied to identify HPV type-specific restriction patterns. In addition to several in-house genotyping protocols based on RFLP, one commercial assay based on this technology is currently available. Yi et al [92] report the development of a highly sensitive, highly automated assay based on the MALDI-TOF-MS platform for the detection and individual genotyping of 14 different HR-HPV types. The use of the MassARRAY technique and combination of automated DNA extraction/ PCR pipetting procedures increased the detection throughput, which consequently decreased the cost per case of the assay. Now we can deal with 3,000 samples within 2 working days, and the current total cost per sample is about \$2 (US). The MS HPV genotyping assay is potentially suitable for routine HPV detection, especially in large-scale cervical cancer screening programs owing to its high throughput and low cost per case. Proper population-based clinical validation

is needed to establish the clinical relevance of this highly analytically sensitive assay.

Several recent studies have clearly shown that testing for HPV mRNA instead of HPV DNA can be clinically useful [70, 93, 94]. The most relevant transcripts for diagnostic purposes are those encoding viral oncoproteins E6 and E7. The detection of viral mRNA can be done by reverse

**7. HPV E6/E7 mRNA screening methods**

commercial assays based on this technology are available at present.

The most frequently used HPV genotyping assays today utilize the principle of reverse lineblot (RLB) hybridization. In these assays a fragment of the HPV genome is first PCR-amplified using biotinylated HPV-specific primers and the resulting amplicons are then denatured and hybridized with HPV-specific oligonucleotide probes immobilized as parallel lines on nylon or anitrocellulosemembranestrip.Afterhybridization,streptavidin-conjugatedalkalinephospha‐ tase or horseradish peroxidase is added, which binds to any biotinylated hybrid previously formed. Incubation with chromogenic substrates (e.g., BCIP/NBT for alkaline phosphatase) yields a colored precipitate at the probe positions where hybridization occurs. The genotyping strip is then read and interpreted visually by comparing the pattern of HPV-positive probes to the test reference guide for each of the targeted HPV types. In addition to the in-house GP5+/ GP6+ RLB Genotyping Assay which has been used in several important HPV trials [76, 77]. INNO-LiPA HPV Genotyping is one of the most widely used HPV genotyping tests. Several versions of this assay have been developed. In all versions, amplification of a 65 bp region of the HPV *L1* gene using biotinylated SPF10 primers is followed by hybridization of the resulting amplicons with 17–28 (depending on the assay version) HPV-specific oligonucleotide probes immobilized on a nitrocellulose strip [78]. An evaluation of INNO-LiPA *Extra,* performed on 70 HC2-positive samples, showed comparable genotyping results to the *digene* HPV Genotyping RHTestRUO(*digene*RHTest;Qiagen)andasignificantlyhigher sensitivityofINNO-LiPA*Extra* forthedetectionofmultiple infections [79].RecentlyisdescribedthismethodwithrealtimePCR [80]. The Linear Array HPV Genotyping Test (Linear Array) is one of the most commonly used HPV genotyping assays, which combines PCR amplification and reverse line-blot hybridiza‐ tion for the identification of 36 alpha-HPV types. Linear Array is based on the co-amplification of a 450 bp region of the HPV *L1* gene and a 268 bp region of the human β-globin gene, using biotinylated primer sets PGM09/PGMY11 and PC04/GH20 [81]. Steinau et al [82] compared the performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (RocheDiagnostics),INNO-LiPAHPVGenotypingExtra(LiPA)(Innogenetics),andthereverse hybridization assay (RH) (Qiagen). Although the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that LA has an advantage for internationally comparable genotyping studies.

Similar to reverse line-blot assays, microarray-based HPV genotyping assays also employ the principle ofreversehybridization. FollowingPCRamplificationof a fragment of a viral genome withHPV-specificprimers,theresultingampliconsaredenaturedandhybridizedwithanumber ofHPV-specificoligonucleotideprobesattachedonthesurfaceofaninsolublesupporterorDNA chip (also known as microchip, biochip and gene chip). DNA chips can consist of one to several DNA microarrays, thus enabling simultaneous analysis of multiple samples in a single experi‐ ment. After hybridization, fluorescence light from the bound PCR amplicon is detected by excitation with monochromatic light. Currently, laser scanners are used for the highly sensi‐ tive fluorescence microarray readout systems. The fluorescence label of the hybridizing amplicons can be introduced during PCR and/or during the hybridization step. Some of the microarray-based HPV genotyping tests utilize the principle of chromogenic precipitation insteadoffluorescencedetection.ThePapilloCheckHPV-ScreeningTest(PapilloCheck;Greiner Bio-OneGmbH,Frickenhausen,Germany)iscurrentlyoneofthetwomostfrequentlyusedPCRmicroarray-basedassays.Theassayallows identificationof24alpha-HPVtypes.[83].ClartHPV 2 – papillomavirus clinical arrays (Clart HPV 2; Genomica, Coslada, Spain) combine PCR amplification and an oligonucleotide microarray-based detection system for the identification of 35 alpha-HPVs. The assay consists of pre-aliquoted, ready to use amplification mix tubes and allows amplification of a 450 bp region of the HPV *L1* gene together with an internal control template and the human *CFTR* gene using three different sets of biotinylated primers [84].

The most frequently used HPV genotyping assays today utilize the principle of reverse lineblot (RLB) hybridization. In these assays a fragment of the HPV genome is first PCR-amplified using biotinylated HPV-specific primers and the resulting amplicons are then denatured and hybridized with HPV-specific oligonucleotide probes immobilized as parallel lines on nylon or anitrocellulosemembranestrip.Afterhybridization,streptavidin-conjugatedalkalinephospha‐ tase or horseradish peroxidase is added, which binds to any biotinylated hybrid previously formed. Incubation with chromogenic substrates (e.g., BCIP/NBT for alkaline phosphatase) yields a colored precipitate at the probe positions where hybridization occurs. The genotyping strip is then read and interpreted visually by comparing the pattern of HPV-positive probes to the test reference guide for each of the targeted HPV types. In addition to the in-house GP5+/ GP6+ RLB Genotyping Assay which has been used in several important HPV trials [76, 77]. INNO-LiPA HPV Genotyping is one of the most widely used HPV genotyping tests. Several versions of this assay have been developed. In all versions, amplification of a 65 bp region of the HPV *L1* gene using biotinylated SPF10 primers is followed by hybridization of the resulting amplicons with 17–28 (depending on the assay version) HPV-specific oligonucleotide probes immobilized on a nitrocellulose strip [78]. An evaluation of INNO-LiPA *Extra,* performed on 70 HC2-positive samples, showed comparable genotyping results to the *digene* HPV Genotyping RHTestRUO(*digene*RHTest;Qiagen)andasignificantlyhigher sensitivityofINNO-LiPA*Extra* forthedetectionofmultiple infections [79].RecentlyisdescribedthismethodwithrealtimePCR [80]. The Linear Array HPV Genotyping Test (Linear Array) is one of the most commonly used HPV genotyping assays, which combines PCR amplification and reverse line-blot hybridiza‐ tion for the identification of 36 alpha-HPV types. Linear Array is based on the co-amplification of a 450 bp region of the HPV *L1* gene and a 268 bp region of the human β-globin gene, using biotinylated primer sets PGM09/PGMY11 and PC04/GH20 [81]. Steinau et al [82] compared the performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (RocheDiagnostics),INNO-LiPAHPVGenotypingExtra(LiPA)(Innogenetics),andthereverse hybridization assay (RH) (Qiagen). Although the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that LA has an

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

advantage for internationally comparable genotyping studies.

Similar to reverse line-blot assays, microarray-based HPV genotyping assays also employ the principle ofreversehybridization. FollowingPCRamplificationof a fragment of a viral genome withHPV-specificprimers,theresultingampliconsaredenaturedandhybridizedwithanumber ofHPV-specificoligonucleotideprobesattachedonthesurfaceofaninsolublesupporterorDNA chip (also known as microchip, biochip and gene chip). DNA chips can consist of one to several DNA microarrays, thus enabling simultaneous analysis of multiple samples in a single experi‐ ment. After hybridization, fluorescence light from the bound PCR amplicon is detected by excitation with monochromatic light. Currently, laser scanners are used for the highly sensi‐ tive fluorescence microarray readout systems. The fluorescence label of the hybridizing amplicons can be introduced during PCR and/or during the hybridization step. Some of the microarray-based HPV genotyping tests utilize the principle of chromogenic precipitation insteadoffluorescencedetection.ThePapilloCheckHPV-ScreeningTest(PapilloCheck;Greiner Bio-OneGmbH,Frickenhausen,Germany)iscurrentlyoneofthetwomostfrequentlyusedPCRmicroarray-basedassays.Theassayallows identificationof24alpha-HPVtypes.[83].ClartHPV 2 – papillomavirus clinical arrays (Clart HPV 2; Genomica, Coslada, Spain) combine PCR amplification and an oligonucleotide microarray-based detection system for the identification The suspension array that uses bead-based xMAP or Luminex technology is based on the use of polystyrene beads with a diameter of 5.6 µm, which are internally dyed with various ratios of two spectrally distinct fluorophores (red and infrared). Different bead sets with specific absorption spectra can be mixed and used for the multiplexed detection of different analytes; currently, an array of 100 bead sets (bead mix) can be generated. For HPV genotyping purposes, each bead set in the bead mix is usually coupled to a single oligonucleotide probe specific for one HPV type. HPV genotyping is done by reverse hybridization technique using biotinylated PCR amplicons. After denaturation and hybridization of target HPV sequences to the beadbound probes, labeling of the hybridized biotinylated amplicons is done using R-phycoery‐ thrin-labeled streptavidin, serving as a reporter fluorophore. The bead sets are then read and analyzed on a Luminex analyzer; the HPV types are discerned according to the unique bead signature, whereas the presence of specific PCR amplicons is determined by R-phycoerythrin fluorescence. The Luminex readouts are expressed as the median fluorescent intensity of the reporter fluorescence for each HPV type. Positivity for the relevant types is calculated from the median fluorescent intensity over a defined threshold level, and can provide a semiquantitative numerical output. Several in-house genotyping protocols based on xMAP technology have been developed in the last 5 years [85-91]; some of them are considered to be the most sensitive HPV detection methods currently available [85]. In addition, at least two commercial assays based on this technology are available at present.

Gel electrophoresis-based HPV genotyping assays utilize general primer-mediated PCR, typespecific or group-specific PCR, to screen for a broad spectrum of HPVs, followed by agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) is consequently applied to identify HPV type-specific restriction patterns. In addition to several in-house genotyping protocols based on RFLP, one commercial assay based on this technology is currently available.

Yi et al [92] report the development of a highly sensitive, highly automated assay based on the MALDI-TOF-MS platform for the detection and individual genotyping of 14 different HR-HPV types. The use of the MassARRAY technique and combination of automated DNA extraction/ PCR pipetting procedures increased the detection throughput, which consequently decreased the cost per case of the assay. Now we can deal with 3,000 samples within 2 working days, and the current total cost per sample is about \$2 (US). The MS HPV genotyping assay is potentially suitable for routine HPV detection, especially in large-scale cervical cancer screening programs owing to its high throughput and low cost per case. Proper population-based clinical validation is needed to establish the clinical relevance of this highly analytically sensitive assay.
